技术领域technical field
本发明属于生物工程领域中的转基因细胞系，特别是涉及一种CPS1报告基因干细胞体系及其构建方法与应用。The invention belongs to a transgenic cell line in the field of bioengineering, in particular to a CPS1 reporter gene stem cell system and its construction method and application.
背景技术Background technique
肝脏是身体内以代谢功能为主的器官，在身体里具有分泌白蛋白、合成储存糖原、清除体内有毒氨等功能，也是最主要的药物代谢器官。肝脏生命活动主要依赖肝实质细胞完成，其数量约占全部肝脏细胞的65％及总肝量的70％-80％。1969年Berry等首次发明了两步灌流法分离原代肝细胞(Berry et al.J Cell Biol.1969,43(3):506-520.)。随着分子生物学、细胞生物学、材料学等学科的发展，分离肝细胞的方法在陆续改进和完善。目前，获得高纯度和高活性的原代肝细胞可应用两步灌流法联合密度梯度离心法(Dalet etal.Anal Biochem.1982,122(1):119-123.)或磁珠分离法(Gailhouste L.Methods MolBiol.2012,826:33-47.)。从尸体肝脏分离人原代肝细胞受到伦理道德的限制，来源非常困难，分离获得的原代肝细胞仅能在培养初期维持较好的形态和功能，持续培养中一些与肝功能相关的重要酶类活性将大量丢失，且原代肝细胞是终末分化细胞，体外很难扩增和传代培养(Yamamoto et al.Hepatol Res.2006,35(3):169-177.)。这些特性极大限制了原代肝细胞的应用，但也促进了应用各种策略获得成熟肝细胞的研究。The liver is the organ with the main metabolic function in the body. It has the functions of secreting albumin, synthesizing and storing glycogen, and removing toxic ammonia from the body. It is also the most important drug metabolism organ. Liver life activities mainly depend on hepatic parenchymal cells, which account for 65% of all liver cells and 70%-80% of the total liver volume. In 1969, Berry et al first invented the two-step perfusion method to separate primary hepatocytes (Berry et al. J Cell Biol. 1969, 43(3): 506-520.). With the development of molecular biology, cell biology, materials science and other disciplines, the methods of isolating hepatocytes are gradually improved and perfected. Currently, two-step perfusion combined with density gradient centrifugation (Dalet et al. Anal Biochem. 1982, 122 (1): 119-123.) or magnetic bead separation (Gailhouste L. Methods Mol Biol. 2012, 826:33-47.). The isolation of primary human hepatocytes from cadaveric livers is restricted by ethics and morality, and the source is very difficult. The isolated primary hepatocytes can only maintain a good shape and function in the initial stage of culture, and some important enzymes related to liver function in continuous culture A large number of similar activities will be lost, and primary hepatocytes are terminally differentiated cells, which are difficult to expand and subculture in vitro (Yamamoto et al. Hepatol Res. 2006, 35(3): 169-177.). These characteristics greatly limit the application of primary hepatocytes, but also promote the application of various strategies to obtain mature hepatocytes.
目前，可从肝脏干/祖细胞、胚胎干细胞、诱导多能性干细胞、多种成体干/祖细胞诱导分化以及通过谱系重编程从体细胞获得肝细胞。人的胚胎干细胞(hESCs)是从早期胚胎中发现、能在体外培养的一种高度未分化细胞，具有胚胎发育的全能性即具有向3个胚层的组织和细胞分化的特性，在特定条件下可被诱导分化为肝细胞。如何将hESCs向肝细胞诱导分化，并获得具有成熟肝细胞功能的细胞是近年来干细胞研究领域关注的重点。通过序贯加入不同细胞因子和/或小分子组合，不同种属的胚胎干细胞在体外可成功被诱导分化为与成熟肝细胞类似的肝细胞样细胞(Baharvand et al.Int J Dev Biol.2006,50(7):645-652；Cai et al.Hepatol.2007,45(5):1229-1239；Hay et al.Stem Cells.2008,26(4):894-902.)，而人诱导多能性干细胞也可以被相似的方法诱导分化为肝细胞样细胞(Si-Tayeb et al.Hepatology.2010,51(1):297-305；Sullivan et al.Hepatology.2010,51(1):329-335.)。尽管不同的研究团队使用不同的细胞因子和/或小分子组合，不同方案的相同之处都在于采用多步法。因而在各阶段特别是早期阶段，细胞分化方向发生的偏离都会在后续各阶段逐渐累积，并剧烈放大，从而造成诱导效率低下和诱导获得的细胞呈现异质化。这种异质化在干细胞肝向诱导分化中表现为：最终获得的细胞，不仅包括成熟肝细胞，同时也包括肝祖细胞、肝干细胞以及其它中间态细胞。由于中间态细胞不具备肝细胞特有的功能或是肝细胞功能低下，从而使得诱导获得的细胞整体功能低下。如何有效地监测、优化干细胞肝向诱导分化，获得功能较强的诱导肝细胞，是干细胞来源肝细胞应用于研究和临床的重大挑战。Currently, hepatocytes can be derived from hepatic stem/progenitor cells, embryonic stem cells, induced pluripotent stem cells, induced differentiation of various adult stem/progenitor cells, and from somatic cells through lineage reprogramming. Human embryonic stem cells (hESCs) are highly undifferentiated cells found in early embryos and can be cultured in vitro. They have the totipotency of embryonic development, that is, they have the characteristics of differentiating to the tissues and cells of the three germ layers. Under certain conditions Can be induced to differentiate into hepatocytes. How to induce hESCs to differentiate into hepatocytes and obtain cells with mature hepatocyte function is the focus of stem cell research in recent years. By sequentially adding different cytokines and/or small molecule combinations, embryonic stem cells of different species can be successfully induced to differentiate into hepatocyte-like cells similar to mature hepatocytes in vitro (Baharvand et al.Int J Dev Biol.2006, 50(7):645-652; Cai et al.Hepatol.2007,45(5):1229-1239; Hay et al.Stem Cells.2008,26(4):894-902.), while human induced more Competent stem cells can also be induced to differentiate into hepatocyte-like cells by similar methods (Si-Tayeb et al.Hepatology.2010,51(1):297-305; Sullivan et al.Hepatology.2010,51(1):329 -335.). Although different research groups use different combinations of cytokines and/or small molecules, the different protocols share a multi-step approach. Therefore, in each stage, especially in the early stage, the deviation of the direction of cell differentiation will gradually accumulate in the subsequent stages and be greatly amplified, resulting in low induction efficiency and heterogeneity of the induced cells. This heterogeneity is manifested in the hepatic differentiation of stem cells: the finally obtained cells include not only mature hepatocytes, but also hepatic progenitor cells, hepatic stem cells and other intermediate cells. Since the intermediate cells do not have the specific functions of the liver cells or the functions of the liver cells are low, the overall function of the induced cells is low. How to effectively monitor and optimize the hepatic differentiation of stem cells and obtain highly functional induced hepatocytes is a major challenge for the application of stem cell-derived hepatocytes in research and clinical practice.
发明内容Contents of the invention
本发明的目的是提供一种能肝向诱导分化并获得均质化的诱导肝细胞的氨甲酰基磷酸合成酶1(CPS1)报告基因干细胞及其构建方法。The object of the present invention is to provide a carbamoyl phosphate synthase 1 (CPS1) reporter gene stem cell capable of inducing differentiation to the liver and obtaining homogenized induced hepatocytes and a construction method thereof.
本发明所提供的氨甲酰基磷酸合成酶1报告基因干细胞(CPS1报告基因干细胞)的构建方法，包括以下步骤：The method for constructing carbamoyl phosphate synthase 1 reporter gene stem cells (CPS1 reporter gene stem cells) provided by the present invention comprises the following steps:
1)构建携带CPS1基因的向导RNA(sgRNA)编码序列的骨架载体，简称sgCPS1；1) Construct a backbone vector carrying the coding sequence of the guide RNA (sgRNA) of the CPS1 gene, referred to as sgCPS1;
2)构建含有荧光蛋白的CPS1基因的打靶载体；2) Construct the targeting vector of CPS1 gene containing fluorescent protein;
3)将骨架载体sgCPS1和CPS1基因的打靶载体转染到多潜能干细胞系中，经筛选获得稳定的荧光标记CPS1的转基因干细胞。3) Transfect the backbone vector sgCPS1 and the targeting vector of the CPS1 gene into a pluripotent stem cell line, and obtain stable fluorescently labeled CPS1 transgenic stem cells through screening.
在上述构建方法中，所述步骤1)中构建骨架载体的工具载体包括px458、px330、px461和px333，优选为pX458；用pX458工具载体构建的携带CPS1基因的sgRNA的骨架载体命名为pX458-sgCPS1，其核苷酸序列如序列表中序列1所示。In the above construction method, the tool vectors for constructing the backbone vector in step 1) include px458, px330, px461 and px333, preferably pX458; the backbone vector of the sgRNA carrying the CPS1 gene constructed with the pX458 tool vector is named pX458-sgCPS1 , the nucleotide sequence of which is shown in sequence 1 in the sequence listing.
构建骨架载体pX458-sgCPS1包括以下步骤：Construction of the backbone vector pX458-sgCPS1 includes the following steps:
1.1)设计靶向CPS1基因(GenBank号：1373)的sgRNA，根据CPS1 sgRNA序列获得CPS1基因的靶标序列为：AGCTGTGCAGAAATCTCGCA(位于CPS1基因自5’端第4759-4778位碱基)，根据CPS1基因的靶标序列获得CPS1 sgRNA编码序列，在CPS1 sgRNA编码序列两端加上BbsI酶切序列，最终获得包含CPS1 sgRNA编码序列的寡核苷酸序列：1.1) Design the sgRNA targeting the CPS1 gene (GenBank No.: 1373), and obtain the target sequence of the CPS1 gene according to the CPS1 sgRNA sequence: AGCTGTGCAGAAATCTCGCA (located at bases 4759-4778 from the 5' end of the CPS1 gene), according to the CPS1 gene Obtain the coding sequence of CPS1 sgRNA for the target sequence, add the BbsI digestion sequence at both ends of the coding sequence of CPS1 sgRNA, and finally obtain the oligonucleotide sequence containing the coding sequence of CPS1 sgRNA:
oligo1:5’-CACCAGCTGTGCAGAAATCTCGCA-3’(下划线部分为BbsI酶切序列)，oligo1:5'-CACC AGCTGTGCAGAAATCTCGCA-3' (the underlined part is the BbsI digestion sequence),
oligo2:5’-TTTGACGCTCTAAAGACGTGTCGA-3’(下划线部分为BbsI酶切序列)；oligo2: 5'-TTTG ACGCTCTAAAGACGTGTCGA-3' (the underlined part is the BbsI digestion sequence);
1.2)将合成的包含CPS1 sgRNA编码序列的寡核苷酸oligo1和oligo2进行退火并磷酸化；1.2) Anneal and phosphorylate the synthetic oligonucleotides oligo1 and oligo2 comprising the CPS1 sgRNA coding sequence;
1.3)将工具载体pX458用BbsI进行酶切，回收、纯化酶切工具载体pX458；1.3) Digest the tool vector pX458 with BbsI, recover and purify the enzyme-cut tool vector pX458;
1.4)将退火及磷酸化的包含CPS1 sgRNA编码序列的寡核苷酸oligo1和oligo2连入经酶切的工具载体pX458中；1.4) Ligate the annealed and phosphorylated oligonucleotides oligo1 and oligo2 comprising the CPS1 sgRNA coding sequence into the enzyme-cut tool vector pX458;
1.5)将连接产物转化DH5α感受态细菌，涂布于Amp+培养平板，37℃孵育过夜(16-20小时)；1.5) Transform the ligation product into DH5α-competent bacteria, spread it on the Amp+ culture plate, and incubate at 37°C overnight (16-20 hours);
1.6)从生长出的单克隆细菌中提取重组质粒，得到携带CPS1 sgRNA编码序列的重组骨架载体pX458-sgCPS1。重组骨架载体pX458-sgCPS1的核苷酸序列如序列表中序列1所示。1.6) Extract the recombinant plasmid from the grown monoclonal bacteria to obtain the recombinant backbone vector pX458-sgCPS1 carrying the CPS1 sgRNA coding sequence. The nucleotide sequence of the recombinant backbone vector pX458-sgCPS1 is shown as sequence 1 in the sequence listing.
所述步骤2)中用于构建CPS1基因打靶载体的出发载体为包含荧光蛋白(如tdTomato但不限于tdTomato，还可以包括GFP、EGFP、YFP等多种荧光报告基因)的原核表达载体，包括pET-tdtomato、pQE-tdtomato、pUC-tdtomato等，优选为pET-tdtomat。以pET32-tdTomato为出发载体构建的CPS1基因打靶载体命名为pET32-CPSLR-tdTomato，构建过程包括以下步骤：The starting vector used to construct the CPS1 gene targeting vector in the step 2) is a prokaryotic expression vector containing a fluorescent protein (such as tdTomato but not limited to tdTomato, and can also include various fluorescent reporter genes such as GFP, EGFP, YFP, etc.), including pET - tdtomato, pQE-tdtomato, pUC-tdtomato, etc., preferably pET-tdtomat. The CPS1 gene targeting vector constructed with pET32-tdTomato as the starting vector is named pET32-CPSLR-tdTomato. The construction process includes the following steps:
2.1)获得CPS1基因的左臂插入片段(CPSL)和右臂插入片段(CPSR)，左臂插入片段(CPSL)的核苷酸序列如序列表中序列2所示，右臂插入片段(CPSR)的核苷酸序列如序列表中序列3所示；2.1) Obtain the left arm insertion segment (CPSL) and the right arm insertion segment (CPSR) of the CPS1 gene, the nucleotide sequence of the left arm insertion segment (CPSL) is as shown in sequence 2 in the sequence table, the right arm insertion segment (CPSR) The nucleotide sequence is shown in sequence 3 in the sequence listing;
2.2)用Kpn I和EcoR V对左臂插入片段(CPSL)及pET32-tdTomato载体进行双酶切，将两种双酶切产物进行连接，将连接产物转化DH5α感受态细菌，涂布于Amp+LB培养平板，37℃孵育过夜(16-20小时)，挑选单克隆生长细菌提取重组质粒，得到携带左臂插入片段(CPSL)的重组载体，命名为pET32-CPSL-tdTomato；2.2) Use Kpn I and EcoR V to double-digest the left-arm insert (CPSL) and the pET32-tdTomato vector, connect the two double-digested products, transform the ligated product into DH5α competent bacteria, and spread it on Amp+ LB culture plate, incubate overnight (16-20 hours) at 37°C, select single-clonal growing bacteria to extract the recombinant plasmid, and obtain the recombinant vector carrying the left arm insert (CPSL), named pET32-CPSL-tdTomato;
2.3)用Hind III和Sal I对右臂插入片段(CPSR)及pET32-CPSL-tdTomato载体进行双酶切，将两种双酶切产物进行连接，将连接产物转化DH5α感受态细菌，涂布于Amp+LB培养平板，37℃孵育过夜(16-20小时)，挑选单克隆生长细菌提取重组质粒，得到携带左臂插入片段(CPSL)和右臂插入片段(CPSR)的重组载体，命名为pET32-CPSLR-tdTomato(简称CPSLR-2A-tdTomato)，其核苷酸序列如序列表中序列4所示。2.3) Use Hind III and Sal I to double digest the right arm insert (CPSR) and the pET32-CPSL-tdTomato vector, connect the two double digested products, transform the ligated products into DH5α competent bacteria, and spread them on Amp+LB culture plate, incubate overnight at 37°C (16-20 hours), select single-clonal growing bacteria to extract the recombinant plasmid, and obtain a recombinant vector carrying a left-arm insert (CPSL) and a right-arm insert (CPSR), named pET32 - CPSLR-tdTomato (CPSLR-2A-tdTomato for short), the nucleotide sequence of which is shown in sequence 4 in the sequence listing.
所述步骤3)中的多潜能干细胞为胚胎干细胞、诱导形成的多潜能干细胞或通过谱系重编程获得的多潜能干细胞，优选为哺乳动物细胞，更优选为小鼠或人细胞，最优选为人细胞；其中，所述胚胎干细胞可为下述任一种NIH编号的细胞系：BG01、BG02、BG03、BG04、SA01、SA02、SA03、ES01、ES02、ES03、ES04、ES05、ES06、TE03、TE32、TE33、TE04、TE06、TE62、TE07、TE72、UC01、UC06、WA01、WA07、WA09、WA13和WA14。The pluripotent stem cells in step 3) are embryonic stem cells, induced pluripotent stem cells or pluripotent stem cells obtained through lineage reprogramming, preferably mammalian cells, more preferably mouse or human cells, most preferably human cells wherein, the embryonic stem cells can be any of the following NIH numbered cell lines: BG01, BG02, BG03, BG04, SA01, SA02, SA03, ES01, ES02, ES03, ES04, ES05, ES06, TE03, TE32, TE33, TE04, TE06, TE62, TE07, TE72, UC01, UC06, WA01, WA07, WA09, WA13 and WA14.
步骤3)中多潜能干细胞为人胚胎干细胞(hESCs)，细胞转染及筛选方法为：将pX458-sgCPS1质粒和pET32-CPSLR-tdTomato质粒(以此两种质粒为例但不限于这两种)按1:3(质量比)比例混合，每106人胚胎干细胞与2μg混合质粒混匀，1050V30pulse电击两次，将细胞重悬于2mL包含10μM Rho相关蛋白激酶抑制剂Y27632(增强细胞贴附和存活)的E8培养基中，24小时后加入200μg/mL G418(Geneticin,遗传霉素)进行筛选(筛选标准为具有G418抗性且呈正常胚胎干细胞克隆化生长)，15天后，获得G418抗性克隆，为CPS1-tdtomato人胚胎干细胞。The pluripotent stem cells in step 3) are human embryonic stem cells (hESCs), and the cell transfection and screening methods are as follows: pX458-sgCPS1 plasmid and pET32-CPSLR-tdTomato plasmid (this two plasmids are taken as an example but not limited to these two) by Mix at a ratio of 1:3 (mass ratio), mix each 106 human embryonic stem cells with 2 μg of the mixed plasmid, shock twice with 1050V30pulse, and resuspend the cells in 2 mL containing 10 μM Rho-associated protein kinase inhibitor Y27632 (to enhance cell attachment and survival) In the E8 medium, 200 μg/mL G418 (Geneticin, Geneticin) was added after 24 hours for screening (screening criteria were G418 resistance and normal embryonic stem cell cloning growth). After 15 days, G418-resistant clones were obtained. for CPS1-tdtomato human embryonic stem cells.
用上述方法获得的氨甲酰基磷酸合成酶1(CPS1)报告基因干细胞，特别是实施例中具体得到的保藏编号为CGMCC No.13809名称为CPS1-tdTomato-hESC的CPS1-tdtomato报告基因人胚胎干细胞(Human Embryonic Stem Cells，hESC)也属于本发明。The carbamoyl phosphate synthase 1 (CPS1) reporter gene stem cells obtained by the above method, especially the CPS1-tdtomato reporter human embryonic stem cells with the preservation number CGMCC No.13809 and the name CPS1-tdTomato-hESC specifically obtained in the examples (Human Embryonic Stem Cells, hESC) also belong to the present invention.
命名为CPS1-tdTomato-hESC的CPS1-tdtomato报告基因人胚胎干细胞(HumanEmbryonic Stem Cells，hESC)已于2017年03月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，邮政编码：100101)，保藏编号为CGMCC No.13809。The CPS1-tdtomato reporter gene human embryonic stem cells (Human Embryonic Stem Cells, hESC) named CPS1-tdTomato-hESC have been preserved in the General Microbiology Center of the China Committee for the Collection of Microorganisms on March 15, 2017 (Address: Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Zip Code: 100101), and the deposit number is CGMCC No.13809.
由氨甲酰基磷酸合成酶1报告基因干细胞经诱导分化得到的定型内胚层细胞(definitive endoderm，DE)、肝干细胞(hepatic stem cell，HS)、肝祖细胞(hepatoblast，HB)或肝细胞(hepatocyte，HEP)，特别是肝向诱导分化获得的诱导肝细胞(肝样细胞)也属于本发明。Definitive endoderm (DE), hepatic stem cell (HS), hepatoblast (HB) or hepatocytes derived from carbamoyl phosphate synthase 1 reporter stem cells , HEP), especially the induced hepatocytes (hepatoid cells) obtained by inducing differentiation to the liver also belong to the present invention.
氨甲酰基磷酸合成酶1报告基因干细胞(特别是CPS1-tdTomato-hESC人胚胎干细胞)在筛选肝向诱导分化体系中诱导剂中的应用也属于本发明。The application of carbamoyl phosphate synthase 1 reporter gene stem cells (especially CPS1-tdTomato-hESC human embryonic stem cells) in screening inducers in the liver-oriented differentiation system also belongs to the present invention.
氨甲酰基磷酸合成酶1报告基因干细胞(特别是CPS1-tdTomato-hESC人胚胎干细胞)肝向诱导分化后在肝药物代谢研究、肝毒性化合物筛选中的应用也属于本发明。The application of carbamoyl phosphate synthetase 1 reporter gene stem cells (especially CPS1-tdTomato-hESC human embryonic stem cells) to hepatic induction and differentiation in the study of liver drug metabolism and the screening of hepatotoxic compounds also belongs to the present invention.
由氨甲酰基磷酸合成酶1报告基因干细胞肝向诱导分化获得的诱导肝细胞在肝毒性化合物筛选中的应用也属于本发明。The application of induced hepatocytes obtained from carbamoyl phosphate synthetase 1 reporter gene stem cell liver-induced differentiation in the screening of hepatotoxic compounds also belongs to the present invention.
本发明提供了一种氨甲酰基磷酸合成酶1(CPS1)报告基因干细胞及其构建方法与应用。本发明的氨甲酰基磷酸合成酶1(CPS1)报告基因干细胞可在诱导剂诱导体系中向肝细胞诱导分化，可获得具有较以往更高氨代谢能力的肝细胞样细胞，获得的肝细胞样细胞纯度更高、且能呈现均质化；细胞中的报告标签(荧光标签)使本发明氨甲酰基磷酸合成酶1(CPS1)报告基因干细胞在诱导分化肝细胞样细胞中能对CPS1蛋白进行示踪，能够实时监测到诱导剂对肝细胞的诱导状态，因此本发明的CPS1报告基因干细胞可用于分化诱导体系中诱导剂的筛选，并进一步可利用诱导得到的肝样细胞报告平台实现肝药物代谢研究、肝药物毒性评价。The invention provides a carbamoyl phosphate synthase 1 (CPS1) reporter gene stem cell, a construction method and application thereof. The carbamoyl phosphate synthase 1 (CPS1) reporter gene stem cells of the present invention can be induced to differentiate into hepatocytes in an inducer induction system, and hepatocyte-like cells with higher ammonia metabolism ability can be obtained than before, and the obtained hepatocyte-like The cells are more pure and can be homogenized; the reporter label (fluorescent label) in the cell enables the carbamoyl phosphate synthase 1 (CPS1) reporter gene stem cells of the present invention to detect the CPS1 protein in induced differentiation hepatocyte-like cells. Tracing can monitor the induction state of the inducer on the liver cells in real time, so the CPS1 reporter gene stem cells of the present invention can be used for the screening of the inducer in the differentiation induction system, and further can use the induced liver-like cell reporter platform to realize liver medicine Metabolism studies, liver drug toxicity evaluation.
附图说明Description of drawings
图1为CPS1基因打靶策略示意图；Figure 1 is a schematic diagram of the CPS1 gene targeting strategy;
图2为CPS1基因打靶载体pET32-CPSLR-tdTomato构建过程的示意图；Figure 2 is a schematic diagram of the construction process of the CPS1 gene targeting vector pET32-CPSLR-tdTomato;
图3为CPS1基因打靶载体pET32-CPSLR-tdTomato的酶切鉴定结果；Figure 3 is the result of enzyme digestion identification of the CPS1 gene targeting vector pET32-CPSLR-tdTomato;
图4为CPS1-tdtomato报告基因人胚胎干细胞经肝素诱导分化至30天诱导分化得到的诱导肝细胞的形态及荧光蛋白表达情况；Figure 4 shows the morphology and fluorescent protein expression of induced hepatocytes obtained from CPS1-tdtomato reporter human embryonic stem cells differentiated by heparin induction for 30 days;
图5为CPS1-tdtomato报告基因人胚胎干细胞诱导分化为肝细胞样细胞的形态及荧光蛋白表达情况(左幅为诱导获得肝样细胞的相差影像，右幅为诱导获得肝样细胞的荧光影像；上排为肝素处理组，下排为对照组)；Figure 5 shows the morphology and fluorescent protein expression of CPS1-tdtomato reporter human embryonic stem cells induced to differentiate into hepatocyte-like cells (the left panel is a phase-contrast image of induced liver-like cells, and the right panel is a fluorescent image of induced liver-like cells; The upper row is the heparin treatment group, the lower row is the control group);
图6为CPS1-tdtomato报告人胚胎干细胞经诱导分化为肝细胞的白蛋白和尿素含量(A幅为白蛋白，B幅为尿素)；Figure 6 is the albumin and urea content of CPS1-tdtomato reporter human embryonic stem cells induced to differentiate into hepatocytes (A panel is albumin, B panel is urea);
图7为CPS1-tdtomato报告人胚胎干细胞诱导中加入睾酮孵育后对剩余睾酮和产生的6-羟基睾酮定量检测结果(A幅为睾酮，B幅为6-羟基睾酮)。Figure 7 shows the results of quantitative detection of remaining testosterone and 6-hydroxytestosterone produced by CPS1-tdtomato reporter human embryonic stem cell induction after incubation with testosterone (panel A is testosterone, panel B is 6-hydroxytestosterone).
具体实施方式Detailed ways
本发明要提供一种能够向肝细胞样细胞诱导分化的CPS1报告基因多潜能干细胞体系。The present invention provides a CPS1 reporter gene pluripotent stem cell system capable of inducing differentiation to hepatocyte-like cells.
本发明CPS1基因打靶策略如图1所示，发明人在基于以下几方面的研究、分析后具体实施完成本发明：The CPS1 gene targeting strategy of the present invention is shown in Figure 1, and the inventors completed the present invention after research and analysis based on the following aspects:
氨基酰基磷酸合成酶1(CPS1)及其基因Aminoacylphosphate synthase 1 (CPS1) and its gene
体内过量氨的代谢主要通过肝细胞内尿素循环完成。整个尿素循化的实现需要5种酶：氨基甲酰磷酸合成酶1、鸟氨酸氨甲酰基转移酶，精氨酸琥珀酸盐合成酶、精氨酸琥珀酸裂解酶、和精氨酸酶。氨基甲酰磷酸合成酶1(CPS1)是肝细胞尿素循环的第一个酶，也是整个解氨过程的限速酶。尿素循环的功能是将有毒的氨合成无毒的尿素，从而避免高氨血症的发生。研究显示CPS1缺失的小鼠出生后不久就会死于高血氨症，显示出CPS1基因和功能完整在肝细胞发挥解氨功能的重要作用。肝细胞的发育进入祖细胞阶段逐渐表达CPS1基因以避免氨的损伤，CPS1的表达水平与细胞的成熟程度、功能状态以及解氨活性密切相关，因而CPS1基因和功能完整是肝细胞发挥解氨功能的决定性因素之一。本发明在实施例中以CPS1为靶标基因，通过在基因组水平对CPS1进行标记，筛选分离获得CPS1不同表达强度的克隆株，在肝细胞诱导中用以指示肝细胞成熟状态，获得成熟肝细胞的比例，指示肝细胞功能与CPS1表达水平以及细胞荧光强度的相关性，并以此报告平台为基础建立高通量的小分子筛选平台，进而可获得用于能够提高肝细胞功能或抑制肝细胞功能的小分子，并用以评价诱导分化效率和优化诱导方案。The metabolism of excess ammonia in the body is mainly completed through the urea cycle in the liver cells. The realization of the entire urea cycle requires 5 enzymes: carbamoyl phosphate synthase 1, ornithine carbamoyltransferase, arginine succinate synthase, arginine succinate lyase, and arginase . Carbamoyl phosphate synthase 1 (CPS1) is the first enzyme of the urea cycle in hepatocytes and the rate-limiting enzyme for the entire ammonia-lysis process. The function of the urea cycle is to synthesize non-toxic urea from toxic ammonia, thereby avoiding the occurrence of hyperammonemia. Studies have shown that CPS1-deficient mice will die of hyperammonia shortly after birth, showing that the CPS1 gene and function integrity play an important role in the ammonia-lysis function of liver cells. The development of hepatocytes enters the stage of progenitor cells and gradually expresses the CPS1 gene to avoid ammonia damage. The expression level of CPS1 is closely related to the maturity, functional state and ammonia-lysis activity of the cells. Therefore, the integrity of the CPS1 gene and function is the key to the liver cells' ammonia-lysis function. one of the decisive factors. In the embodiment of the present invention, CPS1 is used as the target gene, and by marking CPS1 at the genome level, screening and separating clones with different expression intensities of CPS1, which are used to indicate the maturation state of hepatocytes in the induction of hepatocytes, and to obtain mature hepatocytes Ratio, indicating the correlation between liver cell function and CPS1 expression level and cell fluorescence intensity, and based on this reporting platform, a high-throughput small molecule screening platform can be obtained, which can be used to improve liver cell function or inhibit liver cell function small molecules, and used to evaluate the efficiency of inducing differentiation and optimize the induction scheme.
另一方面，具有活性的氨基甲酰磷酸合成酶1(CPS1)在线粒体中发挥功能，因此当肝细胞受到损伤，特别是线粒体毒性损伤时，线粒体形态的改变能够通过CPS1蛋白在线粒体内的聚集状态显示出来。因而，利用荧光标签对CPS1蛋白进行示踪时，能够实时监测到化合物的肝细胞毒性，即细胞荧光从定位于细胞浆至全细胞,且呈高度聚集，强度急剧增强，表明化合物的肝细胞毒性大，利用CPS1报告平台指示获得的成熟肝细胞(肝样细胞)能进一步实现肝药物代谢研究、肝药物毒性评价。On the other hand, active carbamoyl phosphate synthase 1 (CPS1) functions in mitochondria, so when liver cells are damaged, especially mitochondrial toxic damage, changes in mitochondrial morphology can be achieved through the accumulation of CPS1 protein in mitochondria The status is displayed. Therefore, when the fluorescent label is used to trace the CPS1 protein, the hepatotoxicity of the compound can be monitored in real time, that is, the cell fluorescence is located in the cytoplasm to the whole cell, and it is highly aggregated, and the intensity is sharply enhanced, indicating the hepatotoxicity of the compound. Large, using the CPS1 reporter platform to indicate that the obtained mature hepatocytes (hepatoid cells) can further realize the study of liver drug metabolism and the evaluation of liver drug toxicity.
氨基甲酰磷酸合成酶1是由位于基因组2q35的氨基甲酰磷酸合成酶1编码。DNA基因组中共120kb，其中有38个外显子和37个内含子，编码一个含1500个氨基酸的多肽(Summar ML,et al.Gene 2003；311:51-57.Takakusa H,et al.Biochem Biophys ResCommun 2012；420:54-60.)。165kDa的氨基甲酰磷酸合成酶1在细胞浆中产生，然后转移到线粒体修饰为成熟的160kDa大小的形式。成熟的氨基甲酰磷酸合成酶1与其辅因子N-乙酰谷氨酸、并消耗2分子ATP，催化氨和碳酸氢盐形成氨基甲酰磷酸盐。氨基甲酰磷酸盐的合成主要分三步：在ATP作用下磷酸化碳酸氢盐、由碳酸磷酸酐和氨合成氨基甲酸酯，最后在ATP作用下通过氨基甲酸酯的磷酸化作用形成。Carbamoyl phosphate synthase 1 is encoded by carbamoyl phosphate synthase 1 located at genome 2q35. The DNA genome has a total of 120kb, including 38 exons and 37 introns, encoding a polypeptide containing 1500 amino acids (Summar ML, et al. Gene 2003; 311:51-57. Takakusa H, et al. Biochem Biophys ResCommun 2012;420:54-60.). The 165 kDa carbamoyl phosphate synthase 1 is produced in the cytoplasm and then translocated to the mitochondria for modification to the mature 160 kDa sized form. Mature carbamoyl phosphate synthase 1, together with its cofactor N-acetylglutamate, consumes 2 molecules of ATP to catalyze the formation of carbamoyl phosphate from ammonia and bicarbonate. The synthesis of carbamoyl phosphate is mainly divided into three steps: phosphorylation of bicarbonate under the action of ATP, synthesis of carbamate from phosphoric acid anhydride and ammonia, and finally formation of carbamoyl phosphorylation under the action of ATP.
报告基因reporter gene
本发明中使用荧光蛋白作为报告基团对CPS1示踪，可用的荧光报告基因如tdTomato、GFP、EGFP、YFP等多种。以tdTomato为例，本发明中可使用包含有tdTomato的原核表达载体，如pET-tdtomato、pQE-tdtomato、pUC-tdtomato等。In the present invention, fluorescent protein is used as a reporter group to trace CPS1, and various fluorescent reporter genes such as tdTomato, GFP, EGFP, YFP, etc. are available. Taking tdTomato as an example, prokaryotic expression vectors containing tdTomato, such as pET-tdtomato, pQE-tdtomato, pUC-tdtomato, etc., can be used in the present invention.
以下以具体实施例详述本发明。实施例中所用方法如无特别说明均为常规方法，具体步骤可参见：《Molecular Cloning:A Laboratory Manual》(Sambrook，J.，Russell，David W.，Molecular Cloning:A Laboratory Manual，3rd edition，2001，NY，ColdSpring Harbor)。The present invention is described in detail below with specific examples. The method used in the embodiment is conventional method unless otherwise specified, and concrete steps can be referred to: " Molecular Cloning: A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001 , NY, Cold Spring Harbor).
所述百分比浓度如无特别说明均为质量/质量(W/W，单位g/100g)百分比浓度、质量/体积(W/V，单位g/100mL)百分比浓度或体积/体积(V/V，单位mL/100mL)百分比浓度。Said percentage concentration is mass/mass (W/W, unit g/100g) percentage concentration, mass/volume (W/V, unit g/100mL) percentage concentration or volume/volume (V/V, unit g/100mL) percentage concentration unless otherwise specified. Unit mL/100mL) percentage concentration.
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。The acquisition methods of various biological materials described in the examples are only to provide an experimental acquisition method to achieve the purpose of specific disclosure, and should not be a limitation on the source of the biological materials in the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and ethics can be replaced according to the tips in the examples.
所用引物和DNA片段由中美泰和公司和奥科公司合成。The primers and DNA fragments used were synthesized by Zhongmei Taihe Company and Aoke Company.
实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，实施例将有助于理解本发明，但是本发明的保护范围不限于下述的实施例。Embodiment is carried out under the premise of technical solution of the present invention, has provided detailed embodiment and specific operation process, embodiment will help to understand the present invention, but protection scope of the present invention is not limited to following embodiment .
实施例1、设计氨甲酰基磷酸合成酶1(CPS1)基因的单链向导RNA(sgRNA)和骨架载体构建Embodiment 1, design the single-stranded guide RNA (sgRNA) of carbamoyl phosphate synthetase 1 (CPS1) gene and backbone vector construction
1)设计氨甲酰基磷酸合成酶1(CPS1)基因的单链向导RNA(sgRNA)利用MIT张锋实验室网站(网址：crispr.mit.edu)sgRNA target设计软件明确CPS1基因(GenBank号：1737)的sgRNA识别位点，获得CPS1基因的靶标序列为：AGCTGTGCAGAAATCTCGCA(位于CPS1基因自5’端第4759-4778位碱基)，并在此序列上加上可与Bbs I酶切产生的粘性末端形成配对的核苷酸，具体序列如下：1) Design the single-stranded guide RNA (sgRNA) of the carbamoyl phosphate synthase 1 (CPS1) gene by using the sgRNA target design software on the website of MIT Zhang Feng Laboratory (URL: crispr.mit.edu) to identify the CPS1 gene (GenBank number: 1737 ), the target sequence of the CPS1 gene is: AGCTGTGCAGAAATCTCGCA (located at 4759-4778 bases from the 5' end of the CPS1 gene), and a cohesive end that can be digested with Bbs I is added to this sequence The nucleotides that form a pair, the specific sequence is as follows:
oligo1:5’-CACCAGCTGTGCAGAAATCTCGCA-3’，oligo1:5'-CACC -AGCTGTGCAGAAATCTCGCA-3',
oligo2:5’-TTTGACGCTCTAAAGACGTGTCGA-3’；oligo2:5'-TTTG -ACGCTCTAAAGACGTGTCGA-3';
下划线标注序列可与Bbs I酶切末端互补，Oligo1和Oligo 2包含引导序列sgRNA,退火后可与工具载体pX458质粒包含的骨架序列形成sgRNA。The underlined sequence can be complementary to the end of Bbs I digestion. Oligo1 and Oligo 2 contain guide sequence sgRNA, which can form sgRNA with the backbone sequence contained in the tool vector pX458 plasmid after annealing.
2)将合成的包含CPS1gRNA编码序列的寡核苷酸序列按以下反应体系及反应条件退火并进行磷酸化2) Anneal and phosphorylate the synthesized oligonucleotide sequence containing the CPS1gRNA coding sequence according to the following reaction system and reaction conditions
反应体系如下：The reaction system is as follows:
反应条件为：37℃30分钟；95℃5分钟，并以每分钟5℃逐渐降低至25℃。The reaction conditions are: 37°C for 30 minutes; 95°C for 5 minutes, and gradually decrease to 25°C at 5°C per minute.
3)pX458载体的BbsI酶切与纯化3) BbsI digestion and purification of pX458 vector
酶切体系如下：The enzyme digestion system is as follows:
酶切条件为：37℃3小时。The digestion conditions are: 37°C for 3 hours.
用胶纯化试剂盒(购自北京天根公司)回收、纯化经BbsI酶切的载体pX458。The vector pX458 digested with BbsI was recovered and purified with a gel purification kit (purchased from Beijing Tiangen Company).
4)将退火及磷酸化的包含CPS1 sgRNA编码序列的寡核苷酸连接入BbsI酶切载体pX4584) Ligate the annealed and phosphorylated oligonucleotides containing the CPS1 sgRNA coding sequence into the BbsI restriction vector pX458
连接体系如下：The connection system is as follows:
连接条件为：室温连接10分钟。The connection conditions are: connection at room temperature for 10 minutes.
5)转化5) Conversion
将连接产物转化DH5α感受态细菌，涂布于Amp+(浓度100mg/mL)LB培养平板，37℃孵育过夜(16-20小时)Transform the ligation product into DH5α competent bacteria, spread on Amp+ (concentration 100mg/mL) LB culture plate, and incubate at 37°C overnight (16-20 hours)
6)测序鉴定6) Sequencing identification
从生长出的单克隆细菌提取重组质粒进行测序，核苷酸序列如序列表中序列1所示，测序结果表明获得了序列正确的携带CPS1 sgRNA编码序列的重组载体，命名为pX458-sgCPS1(CPS1基因的骨架载体)。The recombinant plasmid was extracted from the grown monoclonal bacteria for sequencing. The nucleotide sequence was shown in sequence 1 in the sequence table. The sequencing results showed that a recombinant vector carrying the CPS1 sgRNA coding sequence with the correct sequence was obtained, named pX458-sgCPS1 (CPS1 gene backbone vector).
该实施例中，步骤3)中用于构建携带CPS1基因的向导RNA(gRNA)的工具载体包括但不限于px458、px330、px461和px333，本实施例以pX458为例进行说明。也可选用其它工具载体以相同的过程得到CPS1基因的骨架载体，不再一一赘述。In this embodiment, the tool vectors used to construct the guide RNA (gRNA) carrying the CPS1 gene in step 3) include but are not limited to px458, px330, px461 and px333, and this embodiment uses pX458 as an example for illustration. Other tool vectors can also be used to obtain the backbone vector of the CPS1 gene in the same process, which will not be repeated one by one.
实施例2、构建CPS1基因的打靶载体pET32-CPSLR-tdTomatoExample 2. Construction of the targeting vector pET32-CPSLR-tdTomato of the CPS1 gene
如图2所示，本实施例以引入荧光基团tdTomato为例，说明CPS1基因的打靶载体pET32-CPSLR-tdTomato的构建过程。包括以下步骤：As shown in FIG. 2 , this embodiment takes the introduction of the fluorescent group tdTomato as an example to illustrate the construction process of the targeting vector pET32-CPSLR-tdTomato of the CPS1 gene. Include the following steps:
1)设计CPS1基因左臂插入片段(CPSL)和右臂插入片段(CPSR)扩增引物，引物序列如下(请提供下述引物序列)：1) Design primers for amplification of the CPS1 gene left arm insert (CPSL) and right arm insert (CPSR). The primer sequences are as follows (please provide the following primer sequences):
CPS1基因左臂插入片段(CPSL)扩增引物：CPS1 Gene Left Arm Insert Segment (CPSL) Amplification Primers:
正向引物(CPSL-F)：5‘-GAAGATCTTTGTGTGAATCTTCAGGAATA-3’Forward primer (CPSL-F): 5'-GAAGATCTTTGTGTGAATCTTCAGGAATA-3'
反向引物(CPSL-R)：5’-CGGATATCTGCTGCTTTTCCAGCACTGT-3’；Reverse primer (CPSL-R): 5'-CGGATATCTGCTGCTTTTCCAGCACTGT-3';
CPS1基因右臂插入片段(CPSR)扩增引物：CPS1 Gene Right Arm Insert Segment (CPSR) Amplification Primers:
正向引物(CPSR-F)：5’-ACGCGTCGACAGATGCAGACACCCCAGCC-3’Forward primer (CPSR-F): 5'-ACGCGTCGACAGATGCAGACACCCCAGCC-3'
反向引物(CPSR-R)：5’-CCCAAGCTTGAAGTAATGAAAGTCTTGAC-3’。Reverse primer (CPSR-R): 5'-CCCAAGCTTGAAGTAATGAAAGTCTTGAC-3'.
2)PCR扩增CPS1基因左臂插入片段(CPSL)和右臂插入片段(CPSR)2) PCR amplification of CPS1 gene left arm insert (CPSL) and right arm insert (CPSR)
PCR反应体系为：The PCR reaction system is:
12.5μL 2×primerstar buffer，12.5μL 2×primerstar buffer,
PCR反应条件为：98℃1min，30×(98℃15s，68℃2min30s)，68℃7min。The PCR reaction conditions were: 98° C. for 1 min, 30× (98° C. for 15 s, 68° C. for 2 min and 30 s), and 68° C. for 7 min.
反应结束后，对PCR扩增产物进行1％琼脂糖凝胶电泳检测，分别回收并纯化2300bp的左臂插入片段(CPSL)和2000bp的右臂插入片段(CPSR)。After the reaction, the PCR amplification products were detected by 1% agarose gel electrophoresis, and the 2300bp left arm insert (CPSL) and the 2000bp right arm insert (CPSR) were recovered and purified respectively.
3)将左臂插入片段(CPSL)和右臂插入片段(CPSR)分别连接入pZERO克隆载体(北京全式金公司)中，3) Ligate the left arm insert (CPSL) and the right arm insert (CPSR) into the pZERO cloning vector (Beijing Quanshijin Company), respectively,
连接体系为：The connection system is:
连接条件为：室温10min。The connection conditions are: 10 min at room temperature.
4)转化4) Conversion
将步骤3)连接产物转化DH5α感受态细菌，涂布于Amp+(100mg/mL)LB培养平板，37℃孵育过夜(16-20小时)。The ligation product of step 3) was transformed into DH5α competent bacteria, spread on Amp+ (100 mg/mL) LB culture plate, and incubated overnight (16-20 hours) at 37°C.
5)测序鉴定5) Sequencing identification
从生长出的单克隆细菌提取重组质粒进行测序，挑选阳性克隆进行测序，测序结果表明获得了序列正确的左臂插入片段(CPSL)和右臂插入片段(CPSR)，左臂插入片段(CPSL)的核苷酸序列如序列表中序列2所示，右臂插入片段(CPSR)的核苷酸序列如序列表中序列3所示。The recombinant plasmids were extracted from the grown monoclonal bacteria for sequencing, and the positive clones were selected for sequencing. The sequencing results showed that the correct sequence of the left-arm insert (CPSL) and right-arm insert (CPSR), and the left-arm insert (CPSL) were obtained. The nucleotide sequence of the CPSR is shown in sequence 2 in the sequence listing, and the nucleotide sequence of the right arm insertion fragment (CPSR) is shown in sequence 3 in the sequence listing.
6)用KpnI和EcoRV对左臂插入片段(CPSL)及pET32-tdTomato载体(pET32载体为商购，tdtomato编码红色荧光蛋白，作用是提供可示踪的报告标签)进行双酶切，6) Use KpnI and EcoRV to double-enzyme digest the left arm insert (CPSL) and the pET32-tdTomato vector (the pET32 vector is commercially available, and tdtomato encodes red fluorescent protein, which serves to provide a traceable reporter label),
酶切体系为：The enzyme digestion system is:
补水至50μL。Make up to 50 μL.
酶切条件为：37℃3小时。The digestion conditions are: 37°C for 3 hours.
将两种双酶切产物回收后进行连接，After recovering the two double-digested products, they were ligated,
连接体系为：The connection system is:
连接条件为：室温10minThe connection conditions are: 10min at room temperature
7)转化7) Conversion
将步骤6)连接产物转化DH5α感受态细菌，涂布于Amp+(浓度100mg/mL)LB培养平板，37℃孵育过夜(16-20小时)，挑选单克隆生长细菌进行PCR和KpnI/EcoRV酶切鉴定，命名为pET32-CPSL-tdTomato。Transform the ligation product of step 6) into DH5α competent bacteria, spread it on Amp+ (concentration 100mg/mL) LB culture plate, incubate overnight (16-20 hours) at 37°C, and select monoclonal growth bacteria for PCR and KpnI/EcoRV enzyme Cut identification, named pET32-CPSL-tdTomato.
8)用HindIII和SalI对右臂插入片段(CPSR)及pET32-CPSL-tdTomato载体进行双酶切8) Use HindIII and SalI to double digest the right arm insert (CPSR) and pET32-CPSL-tdTomato vector
酶切体系为：The enzyme digestion system is:
酶切条件为：37℃3小时。The digestion conditions are: 37°C for 3 hours.
将两种双酶切产物回收后进行连接，After recovering the two double-digested products, they were ligated,
连接体系为：The connection system is:
连接条件为：室温10min。The connection conditions are: 10 min at room temperature.
9)将步骤8)连接产物转化DH5α感受态细菌，涂布于Amp+(浓度100mg/mL)LB培养平板，37℃孵育过夜(16-20小时)，挑选单克隆生长细菌进行PCR和酶切鉴定。9) Transform the ligation product from step 8) into DH5α competent bacteria, spread it on Amp+ (concentration 100mg/mL) LB culture plate, incubate overnight (16-20 hours) at 37°C, and select monoclonal growth bacteria for PCR and enzyme digestion Identification.
酶切鉴定结果如图3所示(第1泳道：核糖核酸标志物DL15000，第2泳道：CPSLR-tdTomato(即pET32-CPSL-tdTomato)，第3泳道：CPSLR-tdTomato Kpn I/EcoR V，第4泳道：CPSLR-tdTomato Kpn I/Hind III，第5泳道：CPSLR-tdTomato EcoRV/Hind III，第6泳道：CPSLR-tdTomato Sal I/Hind III)，酶切鉴定结果表明载体构建成功。再进行测序鉴定，核苷酸序列如序列表中序列4所示，测序结果表明获得了序列正确的携带左臂插入片段(CPSL)和右臂插入片段(CPSR)的重组载体，命名为pET32-CPSLR-tdTomato(CPS1基因的打靶载体)。The results of enzyme digestion identification are shown in Figure 3 (the first lane: ribonucleic acid marker DL15000, the second lane: CPSLR-tdTomato (ie pET32-CPSL-tdTomato), the third lane: CPSLR-tdTomato Kpn I/EcoR V, the third lane: Lane 4: CPSLR-tdTomato Kpn I/Hind III, Lane 5: CPSLR-tdTomato EcoRV/Hind III, Lane 6: CPSLR-tdTomato Sal I/Hind III), the results of enzyme digestion and identification indicated that the vector was constructed successfully. Sequencing was carried out again, and the nucleotide sequence was shown in sequence 4 in the sequence table. The sequencing results showed that a recombinant vector carrying a left arm insert (CPSL) and a right arm insert (CPSR) with the correct sequence was obtained, named pET32- CPSLR-tdTomato (targeting vector for CPS1 gene).
本实施例显示了以pET32-tdtomat为出发载体构建CPS1基因打靶载体pET32-CPSLR-tdTomatop的过程，也可选用其它出发载体(载体不仅限于PET载体系列，只要包括tdtomato的载体都能用，如pQE-tdTomato、pUC-tdTomato等)以相同的过程得到CPS1基因的打靶载体，不再一一赘述。This example shows the process of constructing the CPS1 gene targeting vector pET32-CPSLR-tdTomatop using pET32-tdtomat as the starting vector, and other starting vectors can also be used (carriers are not limited to PET vector series, as long as the vectors including tdtomato can be used, such as pQE -tdTomato, pUC-tdTomato, etc.) to obtain the targeting vector of the CPS1 gene through the same process, and will not repeat them one by one.
另外，荧光报告基团也不限于tdTomato，例如也可选用GFP、EGFP、YFP等荧光基团，在此也不一一赘述。In addition, the fluorescent reporter group is not limited to tdTomato. For example, fluorescent groups such as GFP, EGFP, and YFP can also be used, which will not be repeated here.
实施例3、构建CPS1-tdtomato报告基因人胚胎干细胞Embodiment 3, construction CPS1-tdtomato reporter gene human embryonic stem cell
将pX458-sgCPS1(CPS1基因的骨架载体)和pET32-CPSLR-2A-tdTomato(CPS1基因的打靶载体)按1:3比例(质量比)混合，每106人胚胎干细胞(hESCs)细胞(购自WiCell研究所，NIH的编号WA09)与2μg混合质粒混匀，1050V 30pulse电击两次(Life Technology公司)，将细胞重悬于2mL包含10μM Rho相关蛋白激酶抑制剂Y27632(增强细胞贴附和存活)的E8培养基(购自Stemcell公司)中，24小时后加入200μg/mL G418(新霉素，Gibco公司，货号10131035)进行筛选(筛选标准为具有G418抗性且呈正常胚胎干细胞克隆化生长)。15天后，获得10个G418抗性克隆，提取其基因组DNA，提取其基因组DNA，用来源于CPS1DNA序列和tdtomato基因序列的寡核苷酸作为引物(序列：5‘-GAAGATCT TTGTGTGAATCTTCAGGAATA-3’和5’-CCATGTTGTTGTCCTCGGAGGA-3’)进行PCR扩增并对PCR扩增产物进行测序，扩增产物CRISPR/CAS剪切位点PCR鉴定序列为:TGCGAGATTTCTGCACAGCT,确认报告基因插入位点正确，表明CPS1-tdtomato报告基因人胚胎干细胞构建成功。Mix pX458-sgCPS1 (skeleton carrier of CPS1 gene) and pET32-CPSLR-2A-tdTomato (targeting carrier of CPS1 gene) at a ratio of 1:3 (mass ratio), and each 106 human embryonic stem cells (hESCs) cells (purchased from WiCell Research Institute, NIH No. WA09) was mixed with 2 μg of mixed plasmids, 1050V 30pulse electric shock twice (Life Technology Company), and the cells were resuspended in 2 mL containing 10 μM Rho-related protein kinase inhibitor Y27632 (enhancing cell attachment and survival) In E8 medium (purchased from Stemcell), 24 hours later, 200 μg/mL G418 (neomycin, Gibco, Cat. No. 10131035) was added for selection (screening criteria were G418 resistance and normal embryonic stem cell clonal growth). After 15 days, 10 G418-resistant clones were obtained, and their genomic DNA was extracted, and oligonucleotides derived from the CPS1 DNA sequence and the tdtomato gene sequence were used as primers (sequence: 5'-GAAGATCT TTGTGTGAATCTTCAGGAATA-3' and 5 '-CCATGTTGTTGTCCTCGGAGGA-3') for PCR amplification and sequencing of the PCR amplification product, the amplified product CRISPR/CAS cleavage site PCR identification sequence is: TGCGAGATTTCTGCACAGCT, confirming that the reporter gene insertion site is correct, indicating that the CPS1-tdtomato reporter Genetic human embryonic stem cells were successfully constructed.
本实施例表明了将pX458-sgCPS1和pET32-CPSLR-2A-tdtomato转染到人胚胎干细胞中，经筛选获得稳定的CPS1-tdtomato报告基因人胚胎干细胞。This example shows that pX458-sgCPS1 and pET32-CPSLR-2A-tdtomato were transfected into human embryonic stem cells, and stable CPS1-tdtomato reporter gene human embryonic stem cells were obtained after screening.
该CPS1-tdtomato报告基因人胚胎干细胞(Human Embryonic Stem Cells，hESC)命名为CPS1-tdTomato-hESC，已于2017年03月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，邮政编码：100101)，保藏编号为CGMCC No.13809。The CPS1-tdtomato reporter gene Human Embryonic Stem Cells (Human Embryonic Stem Cells, hESC) named CPS1-tdTomato-hESC, has been preserved on March 15, 2017 in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures (Address: Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 1, Courtyard 1, Beichen West Road, Chaoyang District, Zip Code: 100101), and the deposit number is CGMCC No.13809.
本发明中，胚胎干细胞可为从商业途径获得的人胚胎干细胞，如下述任一种细胞系：BG01、BG02、BG03、BG04、SA01、SA02、SA03、ES01、ES02、ES03、ES04、ES05、ES06、TE03、TE32、TE33、TE04、TE06、TE62、TE07、TE72、UC01、UC06、WA01、WA07、WA09、WA13和WA14，所述编号为NIH的编号。不同编号的细胞可以上述相同的过程进行实施，不再一一赘述。In the present invention, embryonic stem cells can be human embryonic stem cells obtained from commercial sources, such as any of the following cell lines: BG01, BG02, BG03, BG04, SA01, SA02, SA03, ES01, ES02, ES03, ES04, ES05, ES06 , TE03, TE32, TE33, TE04, TE06, TE62, TE07, TE72, UC01, UC06, WA01, WA07, WA09, WA13 and WA14, and the numbers are NIH numbers. Cells with different numbers can be implemented in the same process as above, which will not be repeated one by one.
另外，可选用的为多潜能干细胞，其不限于胚胎干细胞，也可以为诱导形成的多潜能干细胞或通过谱系重编程获得的多潜能干细胞。这些干细胞为哺乳动物细胞，优选为小鼠细胞或人细胞。In addition, pluripotent stem cells can be used, which are not limited to embryonic stem cells, and can also be induced pluripotent stem cells or pluripotent stem cells obtained through lineage reprogramming. These stem cells are mammalian cells, preferably mouse cells or human cells.
实施例4、CPS1-tdtomato报告基因人胚胎干细胞向肝细胞的诱导分化Example 4, CPS1-tdtomato reporter gene human embryonic stem cells induced differentiation to hepatocytes
将实施例3获得的CPS1-tdtomato报告基因人胚胎干细胞分四个阶段诱导ESCs向成熟肝细胞分化，分别是定型内胚层(definitive endoderm，DE)、肝干细胞(hepatic stemcell，HS)、肝祖细胞(hepatoblast，HB)、肝细胞(hepatocyte，HEP)，各阶段诱导条件分别为：The CPS1-tdtomato reporter human embryonic stem cells obtained in Example 3 were divided into four stages to induce ESCs to differentiate into mature hepatocytes, namely definitive endoderm (definitive endoderm, DE), hepatic stem cells (hepatic stem cells, HS), and hepatic progenitor cells. (hepatoblast, HB), hepatocytes (hepatocyte, HEP), the induction conditions of each stage are as follows:
DE阶段：杜氏改良Eagle培养基(DMEM/F-12，购自Thermo fisher公司)+活化素A(Activin A，AA，100ng/ml)+Wnt3a(50ng/ml)作用时间3-6天，得到定型内胚层细胞(definitive endoderm，DE)；DE stage: Duchenne's modified Eagle medium (DMEM/F-12, purchased from Thermo fisher company) + activin A (Activin A, AA, 100ng/ml) + Wnt3a (50ng/ml) for 3-6 days to obtain Definitive endoderm cells (definitive endoderm, DE);
HS阶段：肝细胞培养基(Hepatocyte medium,购自Sciencell公司，Catalog#5201)+Wnt3a(50ng/ml)+成纤维生长因子4(Fibroblast growth factor 4,FGF4，25ng/ml)+视黄酸(Retinoic acid,RA，0.1μM)，作用时间3-6天，得到肝干细胞(hepatic stem cell，HS)；HS stage: Hepatocyte medium (Hepatocyte medium, purchased from Sciencell, Catalog#5201)+Wnt3a (50ng/ml)+fibroblast growth factor 4 (Fibroblast growth factor 4, FGF4, 25ng/ml)+retinoic acid ( Retinoic acid, RA, 0.1μM), the action time is 3-6 days, and hepatic stem cells (HS) are obtained;
HB阶段：肝细胞培养基+成纤维生长因子4(25ng/ml)+骨形成蛋白4(Bonemorphogenetic protein 4,BMP4，25ng/ml)+肝细胞生长因子(Hepatocyte growthfactor,HGF，25ng/ml)，作用时间为6天，得到肝祖细胞(hepatoblast，HB)；HB stage: hepatocyte culture medium + fibroblast growth factor 4 (25ng/ml) + bone morphogenic protein 4 (Bonemorphogenetic protein 4, BMP4, 25ng/ml) + hepatocyte growth factor (Hepatocyte growth factor, HGF, 25ng/ml), The action time is 6 days, and the hepatic progenitor cells (hepatoblast, HB) are obtained;
最后为HEP阶段：肝细胞培养基+肝细胞生长因子+抑瘤素M(oncostatin M,OSM,10ng/ml)+地塞米松(dexamethasone,Dex,1μM)，作用时间7-15天，得到肝细胞(hepatocyte，HEP)。The last is the HEP stage: hepatocyte culture medium + hepatocyte growth factor + oncostatin M (oncostatin M, OSM, 10ng/ml) + dexamethasone (Dex, 1μM), the action time is 7-15 days, and the liver Cells (hepatocyte, HEP).
活化素A，Wnt3a,成纤维生长因子4，骨形成蛋白4，肝细胞生长因子和抑瘤素M购自R&D公司，地塞米松购自Sigma-Aldrich公司。Activin A, Wnt3a, fibroblast growth factor 4, bone morphogenic protein 4, hepatocyte growth factor and oncostatin M were purchased from R&D Company, and dexamethasone was purchased from Sigma-Aldrich Company.
诱导分化30天的CGMCC No.13809人胚胎干细胞CPS1-tdTomato-hESC的细胞表型和荧光蛋白表达情况如图4所示(左图为诱导获得肝样细胞的相差影像，右图为诱导获得肝样细胞的荧光影像)，显示诱导分化至30天的细胞与原代分离的成熟肝实质细胞具有相似的形态，大量的细胞表达红色荧光蛋白，这表明CPS1-tdtomato报告基因人胚胎干细胞已成功被诱导分化为肝样细胞。The cell phenotype and fluorescent protein expression of CGMCC No.13809 human embryonic stem cells CPS1-tdTomato-hESC induced and differentiated for 30 days are shown in Figure 4 (the left picture is the phase contrast image of induced liver-like cells, and the right picture is the induced liver-like cells). Fluorescent images of cells-like cells), showing that cells induced to differentiate for 30 days have similar morphology to primary isolated mature hepatic parenchymal cells, and a large number of cells express red fluorescent protein, which indicates that human embryonic stem cells with CPS1-tdtomato reporter gene have been successfully detected Induces differentiation into hepatic-like cells.
实施例5、CPS1-tdtomato报告基因人胚胎干细胞的应用Example 5, Application of CPS1-tdtomato reporter gene human embryonic stem cells
一、筛选肝向诱导分化中的诱导剂1. Screening of inducers for hepatic differentiation
CPS1-tdtomato报告基因人胚胎干细胞可用于筛选肝向诱导分化中的诱导剂。本实施例利用不同诱导方案诱导CPS1-tdtomato报告基因人胚胎干细胞，并检测获得肝样细胞功能的功能(包括分泌白蛋白和产生尿素)。CPS1-tdtomato reporter human embryonic stem cells can be used to screen inducers for hepatic differentiation. In this example, different induction schemes were used to induce CPS1-tdtomato reporter human embryonic stem cells, and the function of acquiring hepatic-like cell functions (including secretion of albumin and production of urea) was detected.
CPS1-tdtomato报告人胚胎干细胞(hESCs)经不同诱导方案DE阶段-DE培养基3-6天；HS、HB以及HEP阶段分别在上述实施例4诱导体系中添加0.2μg/ml肝素(购自英国Iduron公司，货号：HO20)，以实施例4中不添加肝素的方案为对照，诱导至30天，CGMCC No.13809人胚胎干细胞CPS1-tdTomato-hESC诱导分化为肝细胞样细胞的形态及荧光蛋白表达情况如图5所示(左图为诱导获得肝样细胞的相差影像，右图为诱导获得肝样细胞的荧光影像；上排为肝素处理组，下排为对照组)，可以看出从HS阶段开始添加肝素能够有效增加荧光细胞的数量和比例，增强hESCs肝向的诱导效率。同时，添加肝素的诱导方案将细胞诱导分化至30天时，获得的肝样细胞呈现均质化的形态，鲜有中间态细胞出现，表明本发明CPS1-tdtomato报告人胚胎干细胞可用于指示肝细胞分化效率和诱导方案的筛选。CPS1-tdtomato reporter human embryonic stem cells (hESCs) undergo different induction schemes DE stage-DE medium for 3-6 days; HS, HB, and HEP stages were added with 0.2 μg/ml heparin (purchased from UK Iduron company, article number: HO20), taking the scheme without adding heparin in Example 4 as a control, induction to 30 days, CPS1-tdTomato-hESC induction of CGMCC No.13809 human embryonic stem cells differentiated into hepatocyte-like cell morphology and fluorescent protein The expression situation is shown in Figure 5 (the left picture is the phase-contrast image of induced liver-like cells, and the right picture is the fluorescence image of induced liver-like cells; the upper row is the heparin treatment group, and the lower row is the control group), it can be seen from Adding heparin at the beginning of the HS stage can effectively increase the number and proportion of fluorescent cells, and enhance the induction efficiency of hESCs hepatic orientation. At the same time, when the cells were induced to differentiate by adding heparin for 30 days, the obtained hepatic cells showed a homogeneous morphology, and few intermediate cells appeared, indicating that the CPS1-tdtomato reporter human embryonic stem cells of the present invention can be used to indicate the differentiation of hepatocytes Screening for efficiency and induction regimens.
细胞诱导至29天，每孔细胞中加入含10mM NH4Cl的无酚红HM培养基(购自Gibco公司)，放入细胞培养箱，37℃、5％CO2孵育24小时，收集上清，利用白蛋白酶联免疫试剂盒(Bethyl Albumin ELISA Kit)和尿素检测试剂盒(QuantichromTM Urea Assay Kit DIUR-048)检测上清中的白蛋白和尿素含量，CPS1-tdtomato报告人胚胎干细胞(hESCs)经不同诱导方案诱导分化为肝细胞样细胞的白蛋白和尿素，结果如图6所示(A幅为白蛋白，B幅为尿素)，可以看出添加肝素的诱导方案获得的肝样细胞分泌更多的白蛋白和尿素，表明肝素能够有效地增加诱导分化肝样细胞的成熟程度。After the cells were induced to 29 days, add phenol red-free HM medium (purchased from Gibco) containing 10mM NH4 Cl to each well of the cells, put them in a cell culture incubator, incubate at 37°C and 5% CO2 for 24 hours, and collect the supernatant , using albumin ELISA kit (Bethyl Albumin ELISA Kit) and urea detection kit (QuantichromTM Urea Assay Kit DIUR-048) to detect the content of albumin and urea in the supernatant, CPS1-tdtomato reporter human embryonic stem cells (hESCs) The albumin and urea that differentiated into hepatocyte-like cells were induced by different induction schemes. The results are shown in Figure 6 (A panel is albumin, B panel is urea). More albumin and urea, indicating that heparin can effectively increase the maturation of induced differentiation of hepatoid cells.
细胞诱导至25天，在诱导培养基中加入设定浓度的睾酮，放入细胞培养箱，37℃、5％CO2孵育24小时，收集上清，利用质谱技术对其中剩余的睾酮和产生的6-羟基睾酮进行定量。结果如图7所示(A幅为睾酮，B幅为6-羟基睾酮)，可以看出添加肝素的诱导方案获得的肝样细胞能够代谢更多的睾酮并产生更多的6-羟基睾酮，表明肝素能够有效地增加诱导分化肝样细胞的功能成熟。After the cells were induced for 25 days, a set concentration of testosterone was added to the induction medium, placed in a cell culture incubator, and incubated at 37°C, 5% CO2 for 24 hours, the supernatant was collected, and the remaining testosterone and the produced one were analyzed by mass spectrometry. 6-hydroxytestosterone was quantified. The results are shown in Figure 7 (A panel is testosterone, B panel is 6-hydroxytestosterone), it can be seen that the liver-like cells obtained by adding heparin induction scheme can metabolize more testosterone and produce more 6-hydroxytestosterone, It shows that heparin can effectively increase the functional maturation of induced differentiated hepatoid cells.
二、肝药物毒性评价2. Toxicity evaluation of liver drugs
利用实施例4诱导分化方案由CGMCC No.13809人胚胎干细胞CPS1-tdTomato-hESC诱导分化获得肝样细胞，在培养基中加入具有明确线粒体毒性的化合物对氨基乙酰酚(APAP)，作用浓度为2mM，放入细胞培养箱，37℃、5％CO2孵育24小时，荧光显微镜下观察，可见原来仅分布于胞浆的红色荧光开始扩散至全细胞，呈高度聚集，荧光强度明显增强，48小时后，超过80％的细胞死亡，表明APAP的肝毒性。Utilize the induction differentiation scheme of Example 4 to obtain hepatoid cells by inducing differentiation of CGMCC No.13809 human embryonic stem cell CPS1-tdTomato-hESC, and add the compound p-aminoacetylphenol (APAP) with clear mitochondrial toxicity to the medium, and the concentration is 2mM , placed in a cell incubator, incubated at 37°C, 5% CO2 for 24 hours, observed under a fluorescent microscope, it can be seen that the red fluorescence originally distributed only in the cytoplasm began to diffuse to the whole cell, and was highly aggregated, and the fluorescence intensity was significantly enhanced, after 48 hours After that, more than 80% of the cells died, indicating the hepatotoxicity of APAP.
同样，参照已有药物代谢研究体系，利用CPS1-tdtomato报告基因人胚胎干细胞诱导分化方案获得的肝细胞样细胞还能实施肝药物代谢研究，在此不再赘述。Similarly, with reference to the existing drug metabolism research system, the hepatocyte-like cells obtained by using the CPS1-tdtomato reporter gene human embryonic stem cell induction differentiation protocol can also be used for liver drug metabolism research, which will not be repeated here.
序列表sequence listing
<110> 中国人民解放军军事医学科学院野战输血研究所<110> Institute of Field Blood Transfusion, Academy of Military Medical Sciences, Chinese People's Liberation Army
<120> CPS1报告基因干细胞及其构建方法与应用<120> CPS1 reporter gene stem cell and its construction method and application
<130> CGCNB185026W<130> CGCNB185026W
<141> 2018-03-12<141> 2018-03-12
<150> 2017101565826<150> 2017101565826
<151> 2017-03-16<151> 2017-03-16
<160> 4<160> 4
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 9292<211> 9292
<212> DNA<212>DNA
<213> 携带CPS1 sgRNA编码序列的重组载体pX458-sgCPS1(人工序列)<213> Recombinant vector pX458-sgCPS1 (artificial sequence) carrying CPS1 sgRNA coding sequence
<400> 1<400> 1
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacacca gctgtgcaga aatctcgcat gttttagagc tagaaatagc aagttaaaat 300cgaaacacca gctgtgcaga aatctcgcat gttttagagc tagaaatagc aagttaaaat 300
aaggctagtc cgttatcaac ttgaaaaagt ggcaccgcgt cggtgctttt ttgttttaga 360aaggctagtc cgttatcaac ttgaaaaagt ggcaccgcgt cggtgctttt ttgttttaga 360
gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca 420gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca 420
gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac 480gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac 480
cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac 540cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac 540
gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 600gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 600
tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc 660tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc 660
agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 720agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 720
ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 780ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 780
cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 840cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 840
gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 900gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 900
ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 960ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 960
ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga 1020ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga 1020
cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga 1080cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga 1080
ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat 1140ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat 1140
tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat 1200tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat 1200
tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact 1260tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact 1260
ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata 1320ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata 1320
agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga 1380agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga 1380
agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg 1440agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg 1440
agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca 1500agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca 1500
agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc 1560agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccaccccggc 1560
tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag 1620tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag 1620
agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt 1680agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt 1680
ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg 1740ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg 1740
acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg 1800acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg 1800
acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt 1860acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt 1860
tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc 1920tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc 1920
tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca 1980tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca 1980
gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa 2040gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa 2040
atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc 2100atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc 2100
tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac 2160tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac 2160
tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg 2220tgcagctgag caaggacacc tacgacgacg acctggaca cctgctggcc cagatcggcg 2220
accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg 2280accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg 2280
acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga 2340acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga 2340
gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc 2400gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc 2400
ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg 2460ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg 2460
acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg 2520acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg 2520
acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga 2580acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga 2580
ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc 2640ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc 2640
ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc 2700ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc 2700
tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct 2760tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct 2760
ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca 2820ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca 2820
agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca 2880agggcgcttc cgccccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca 2880
acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc 2940acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc 2940
tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc 3000tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc 3000
agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc 3060agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc 3060
tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg 3120tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg 3120
aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca 3180aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca 3180
aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga 3240aggacttcct ggacaatgag gaaaacgagg aattctgga agatatcgtg ctgaccctga 3240
cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg 3300cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg 3300
acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc 3360acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc 3360
ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga 3420ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga 3420
agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct 3480agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct 3480
ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca 3540ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca 3540
ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg 3600ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg 3600
tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg 3660tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg 3660
ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga 3720ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga 3720
tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca 3780tcgaagagggg catcaaagag ctgggcagcc agatcctgaa agaacaccccc gtggaaaaca 3780
cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg 3840cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg 3840
tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc 3900tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc 3900
agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc 3960agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc 3960
ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc 4020ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc 4020
ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg 4080ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg 4080
agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa 4140agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa 4140
cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg 4200cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg 4200
acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt 4260acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt 4260
ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg 4320ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg 4320
cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc 4380cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc 4380
tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca 4440tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca 4440
agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga 4500agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga 4500
actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg 4560actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg 4560
agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc 4620agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc 4620
ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg 4680ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg 4680
gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga 4740gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga 4740
aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc 4800aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc 4800
tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc 4860tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc 4860
tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag 4920tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag 4920
ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt 4980ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt 4980
tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa 5040tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa 5040
acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga 5100acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga 5100
agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc 5160agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc 5160
actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg 5220actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg 5220
acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag 5280acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag 5280
agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct 5340agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct 5340
tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg 5400tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg 5400
acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc 5460acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc 5460
agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa 5520agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa 5520
aggaattcgg cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga 5580aggaattcgg cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga 5580
atcctggccc agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg 5640atcctggccc agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg 5640
agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg 5700agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg 5700
ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct 5760ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct 5760
ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc 5820ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc 5820
acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca 5880acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca 5880
ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg 5940ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg 5940
acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc 6000acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc 6000
tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc 6060tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc 6060
agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc 6120agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc 6120
agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg 6180agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg 6180
acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc 6240acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc 6240
acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt 6300acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt 6300
acaaggaatt ctaactagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 6360acaaggaatt ctaactagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 6360
atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 6420atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 6420
cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 6480cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 6480
ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc 6540ggggggtggg gtggggcagg acaagcaaggg ggaggattgg gaagagaata gcaggcatgc 6540
tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg 6600tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg 6600
ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc 6660ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc 6660
ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct 6720ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct 6720
tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt 6780tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt 6780
agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 6840agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 6840
agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 6900agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 6900
tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 6960tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 6960
cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga 7020cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga 7020
tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 7080tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 7080
caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg 7140caaactggaa caacactcaa ccctatctcg ggctattctt ttgattatta aggggattttg 7140
ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 7200ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 7200
aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc 7260aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc 7260
gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 7320gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 7320
ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 7380ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 7380
aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 7440aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 7440
ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 7500ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 7500
aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 7560aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 7560
atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 7620atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 7620
caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 7680caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 7680
cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 7740cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 7740
tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 7800tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 7800
tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 7860tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 7860
gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 7920gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 7920
tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 7980tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 7980
gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 8040gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 8040
aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 8100aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 8100
gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 8160gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 8160
atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 8220atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 8220
caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 8280caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 8280
ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc 8340ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc 8340
attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 8400attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 8400
agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 8460agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 8460
aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 8520aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 8520
catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 8580catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 8580
ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 8640ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 8640
tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 8700tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 8700
ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 8760ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 8760
ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 8820ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 8820
ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 8880ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 8880
gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 8940gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 8940
aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 9000aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 9000
acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 9060acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 9060
gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 9120gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 9120
gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 9180gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 9180
cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 9240cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 9240
aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt 9292aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt 9292
<210> 2<210> 2
<211> 2022<211> 2022
<212> DNA<212>DNA
<213> 左臂插入片段CPSL(人工序列)<213> left arm insert CPSL (artificial sequence)
<400> 2<400> 2
tttgtggatg attgtcaagt ttcactctcc atcactatgg aatacataac gtcatgtgta 60tttgtggatg attgtcaagt ttcactctcc atcactatgg aatacataac gtcatgtgta 60
catggtgata tgaaacgtgt ttcaaaatac ttcttagtaa ggatactttc cttgacggaa 120catggtgata tgaaacgtgt ttcaaaatac ttcttagtaa ggatactttc cttgacggaa 120
acaagtgaga gcatgaagaa tgtaatgcag cactttatat ttcatgtcaa acttatattg 180acaagtgaga gcatgaagaa tgtaatgcag cactttatat ttcatgtcaa acttatattg 180
tgtatagata agtacatcga gtataagatg accagctata tttcactaag gttgtgcata 240tgtatagata agtacatcga gtataagatg accagctata tttcactaag gttgtgcata 240
ataaatcaat ttgttgagca cttactacat acaggaacct atgttgagta ctatgatgat 300ataaatcaat ttgttgagca cttactacat acaggaacct atgttgagta ctatgatgat 300
gtcatccatt ggattaccta attggtcatt ccttgaccag gttttcatgc cttagaccat 360gtcatccatt ggattaccta attggtcatt ccttgaccag gttttcatgc cttagaccat 360
gtcatcatta gattttgtac gacattttat ttcagcagat tgtgtgaatc ttcaggaata 420gtcatcatta gattttgtac gacattttat ttcagcagat tgtgtgaatc ttcaggaata 420
ccattgcatc aacccttaaa gatctatgtg gcataagtac ataggagata attctctgct 480ccattgcatc aacccttaaa gatctatgtg gcataagtac ataggagata attctctgct 480
gttttaagat agtgcagcct acagaatggc tgtgccattc tatgcaatat gttatttgtt 540gttttaagat agtgcagcct acagaatggc tgtgccattc tatgcaatat gttatttgtt 540
agcgttcttg agaaaccaac acataaatta ttatcacttt aataaaataa tggcattgtg 600agcgttcttg agaaaccaac acataaatta ttatcacttt aataaaataa tggcattgtg 600
ttcaaacctt agcaggcctc caaggatgta atagctccgt gtttggggca ttgtaaaaca 660ttcaaacctt agcaggcctc caaggatgta atagctccgt gtttggggca ttgtaaaaca 660
ataattagca ctactaagga ggcaatagag ttaatgtggg agtgggaaca tgggggtgaa 720ataattagca ctactaagga ggcaatagag ttaatgtggg agtgggaaca tgggggtgaa 720
agggcattca atggtggagg gatcacctct ttttaaaaaa taagtatcta tcaaattacc 780agggcattca atggtggagg gatcacctct ttttaaaaaa taagtatcta tcaaattacc 780
aattcttaat gagtgaacag atagcatttt aaagagaaaa caaacatctg cattccatta 840aattcttaat gagtgaacag atagcatttt aaagagaaaa caaacatctg cattccatta 840
gacttgcact agaattcctt caagaacaca atataatccc tatataagtg taatttaaac 900gacttgcact agaattcctt caagaacaca atataatccc tatataagtg taatttaaac 900
atctacttag caatagagga gggcagatgg cagtcaactc agcatggcat tgacttgaat 960atctacttag caatagagga gggcagatgg cagtcaactc agcatggcat tgacttgaat 960
ggctaagaga gcaatttatg ttagtatttt ataaactaac tataaaatat gccttgttgt 1020ggctaagaga gcaatttatg ttagtatttt ataaactaac tataaaatat gccttgttgt 1020
ctataagttt ttgtttattt ttccagattg attagagatg gcagcattga cctagtgatt 1080ctataagttt ttgtttattt ttccagattg attagagatg gcagcattga cctagtgatt 1080
aaccttccca acaacaacac taaatttgtc catgataatt atgtgattcg gaggacagct 1140aaccttccca acaacaacac taaatttgtc catgataatt atgtgattcg gaggacagct 1140
gttgatagtg gaatccctct cctcactaat tttcaggtat agtcttttcc ttggatatag 1200gttgatagtg gaatccctct cctcactaat tttcaggtat agtcttttcc ttggatatag 1200
actggatggg agttttattt ctgtgcctcc cttaagagtg taatcagtag atgcacatct 1260actggatggg agttttattt ctgtgcctcc cttaagagtg taatcagtag atgcacatct 1260
ctcttttccc ctctttgtaa ctttcagaat agagaggaat ttttaataat ctaaaataat 1320ctcttttccc ctctttgtaa ctttcagaat agagaggaat ttttaataat ctaaaataat 1320
gatgctaaca tggtaggtca tggcttaaat gggacagttg gtgatcaagc aattgagata 1380gatgctaaca tggtaggtca tggcttaaat gggacagttg gtgatcaagc aattgagata 1380
atcaaaggcc atgaatggcc atggctctct ccttacaatt atcctttgaa taaaaagtga 1440atcaaaggcc atgaatggcc atggctctct ccttacaatt atcctttgaa taaaaagtga 1440
cagcatgaca tggaataaag atacaaactt ttatttcatt gtttctaaat gaaagccacc 1500cagcatgaca tggaataaag atacaaactt ttatttcatt gtttctaaat gaaagccacc 1500
atataaaagc aacaggttaa tgatggtcca gatgtagcac aagtgcttgt tcaattcaca 1560atataaaagc aacaggttaa tgatggtcca gatgtagcac aagtgcttgt tcaattcaca 1560
gaaaatgatt gcatgaggca tgttcagttt cacttggaca tgacccatcg aatttatcac 1620gaaaatgatt gcatgaggca tgttcagttt cacttggaca tgacccatcg aatttatcac 1620
agggagaaac agagtggaga ctcattaact ttctgcctat catatttatt ttttatgcag 1680agggagaaac agagtggaga ctcattaact ttctgcctat catatttatt ttttatgcag 1680
aatcagtttt aatccctatg ggaggagaaa taagcgtatt cacagtgaca tctgagatat 1740aatcagtttt aatccctatg ggaggagaaa taagcgtatt cacagtgaca tctgagatat 1740
aaaagaaagt ccccatggtg aggtctgaga catgggagaa agtctccatt acataaaaaa 1800aaaagaaagt ccccatggtg aggtctgaga catgggagaa agtctccatt acataaaaaa 1800
cttttatgcc ttttatccca tacccctttg aaaactgggg acagacactt gtgacttttg 1860cttttatgcc ttttatccca tacccctttg aaaactgggg acagacactt gtgacttttg 1860
tcttcattca ttaaaaattc acttttatct catggagggt gctgattcct accattatat 1920tcttcattca ttaaaaattc acttttatct catggagggt gctgattcct accattatat 1920
tttcaggtga ccaaactttt tgctgaagct gtgcagaaat ctcgcaaggt ggactccaag 1980tttcaggtga ccaaactttt tgctgaagct gtgcagaaat ctcgcaaggt ggactccaag 1980
agtcttttcc actacaggca gtacagtgct ggaaaagcag ca 2022agtcttttcc actacaggca gtacagtgct ggaaaagcag ca 2022
<210> 3<210> 3
<211> 2295<211> 2295
<212> DNA<212>DNA
<213> 右臂插入片段CPSR(人工序列)<213> right arm insert CPSR (artificial sequence)
<400> 3<400> 3
agatgcagac accccagccc cattattaaa tcaacctgag ccacatgtta tctaaaggaa 60agatgcagac accccagccc catttattaaa tcaacctgag ccacatgtta tctaaaggaa 60
ctgattcaca actttctcag agatgaatat tgataactaa acttcatttc agtttacttt 120ctgattcaca actttctcag agatgaatat tgataactaa acttcatttc agtttacttt 120
gttatgcctt aatattctgt gtcttttgca attaaattgt cagtcacttc ttcaaaacct 180gttatgcctt aatattctgt gtcttttgca attaaattgt cagtcacttc ttcaaaacct 180
tacagtcctt cctaagttac tcttcatgag atttcatcca tttactaata ctgtattttt 240tacagtcctt cctaagttac tcttcatgag atttcatcca tttactaata ctgtattttt 240
ggtggactag gcttgcctat gtgcttatgt gtagcttttt actttttatg gtgctgatta 300ggtggactag gcttgcctat gtgcttatgt gtagcttttt actttttatg gtgctgatta 300
atggtgatca aggtaggaaa agttgctgtt ctattttctg aactctttct atactttaag 360atggtgatca aggtaggaaa agttgctgtt ctattttctg aactctttct atactttaag 360
atactctatt tttaaaacac tatctgcaaa ctcaggacac tttaacaggg cagaatactc 420atactctatt tttaaaacac tatctgcaaa ctcaggacac tttaacaggg cagaatactc 420
taaaaacttg ataaaattaa atatagattt aatttatgaa ccttccatca tgatgtttgt 480taaaaacttg ataaaattaa atatagattt aatttatgaa ccttccatca tgatgtttgt 480
gtattgcttc tttttggatc ctcattctca cccatttggc taatccagga atattgttat 540gtattgcttc tttttggatc ctcattctca cccatttggc taatccagga atattgttat 540
cccttcccat tatattgaag ttgagaaatg tgacagaggc atttagagta tggacttttc 600cccttcccat tatattgaag ttgagaaatg tgacagaggc atttagagta tggacttttc 600
ttttcttttt ctttttcttt ttttcttttt gagatggagt cacactctcc aggctggagt 660ttttcttttt ctttttcttt ttttcttttt gagatggagt cacactctcc aggctggagt 660
gcagtggcac aatctcggct cactgcaatt tccgtctccc aagttcaagc gattctcctg 720gcagtggcac aatctcggct cactgcaatt tccgtctccc aagttcaagc gattctcctg 720
ctttagacta tggatttctt taaggaatac tggtttgcag ttttgttttc tggactatat 780ctttagacta tggatttctt taaggaatac tggtttgcag ttttgttttc tggactatat 780
cagcagatgg tagacagtgt ttatgtagat gtgttgttgt ttttatcatt ggattttaac 840cagcagatgg tagacagtgt ttatgtagat gtgttgttgt ttttatcatt ggattttaac 840
ttggcccgag tgaaataatc agatttttgt cattcacact ctcccccagt tttggaataa 900ttggcccgag tgaaataatc agatttttgt cattcacact ctccccccagt tttggaataa 900
cttggaagta aggttcattc ccttaagacg atggattctg ttgaactatg gggtcccaca 960cttggaagta aggttcattc ccttaagacg atggattctg ttgaactatg gggtcccaca 960
ctgcactatt aattccaccc actgtaaggg caaggacacc attccttcta catataagaa 1020ctgcactatt aattccaccc actgtaaggg caaggacacc attccttcta catataagaa 1020
aaaagtctct ccccaagggc agcctttgtt acttttaaat attttctgtt attacaagtg 1080aaaagtctct ccccaagggc agcctttgtt acttttaaat attttctgtt attacaagtg 1080
ctctaattgt gaacttttaa ataaaatact attaagaggt aatgcagttg aatctggttt 1140ctctaattgt gaacttttaa ataaaatact attaagaggt aatgcagttg aatctggttt 1140
tattttatgt tgctgtacaa aaatcagttt acttctatga taaaataggg ttttgggcca 1200tattttatgt tgctgtacaa aaatcagttt acttctatga taaaataggg ttttgggcca 1200
ggattgcatt gcttatttat tttttccatg caaacccata tagggatgag aaaattaatg 1260ggattgcatt gcttatttat tttttccatg caaacccata tagggatgag aaaattaatg 1260
ttaaaaataa gttttagctg tcacattttt cattttttct tgtccctccc ttgcttttaa 1320ttaaaaataa gttttagctg tcacattttt cattttttct tgtccctccc ttgcttttaa 1320
gtctcatcat aacattatgt ggttatatgg ctataaacca tctttgatct ggatcctttc 1380gtctcatcat aacattatgt ggttatatgg ctataaacca tctttgatct ggatcctttc 1380
taatgtataa ctacaaccac aaactgatta aagattgcca caaatatata gtaaactttc 1440taatgtataa ctacaaccac aaactgatta aagattgcca caaatatata gtaaactttc 1440
aaacaaatgt cttgcataac tcagaaaagt aacttaaccc aatcagaggc tgaccagcat 1500aaacaaatgt cttgcataac tcagaaaagt aacttaaccc aatcagaggc tgaccagcat 1500
tgtctgatgg aaatagtgcc aatgagcaca ctgatgccat atccccaatt taccaccgaa 1560tgtctgatgg aaatagtgcc aatgagcaca ctgatgccat atccccaatt taccaccgaa 1560
actatggctt tgtgaaggtc ctaagataat tatgctataa cagcttttat ttcttccttg 1620actatggctt tgtgaaggtc ctaagataat tatgctataa cagcttttat ttcttccttg 1620
ttacctcaca gtcaattgat taccactttg tatagctcta gacgaatgct taaagtaatt 1680ttacctcaca gtcaattgat taccactttg tatagctcta gacgaatgct taaagtaatt 1680
catcttctct ccccctcact tccttgttct tcctgtggtc aagactttca ttacttcttc 1740catcttctct ccccctcact tccttgttct tcctgtggtc aagactttca ttacttcttc 1740
cctgtaatct tttttttttt tttttttttt ttttgagacg gagttttgct cttgtcaccc 1800cctgtaatct ttttttttttttttttttttttttgagacg gagttttgct cttgtcaccc 1800
aggctggagt gcagtggcgc gatctcagct cactgcaacc tccatctcct gggttccagc 1860aggctggagt gcagtggcgc gatctcagct cactgcaacc tccatctcct gggttccagc 1860
gattctcctg cctcagcctc ctgagtagct gagattacag gtgcccgcca acaagcctgg 1920gattctcctg cctcagcctc ctgagtagct gagattacag gtgcccgcca acaagcctgg 1920
ctaatatttt gtatttttag tagagacggg gtttcgccat gttgggcagg ctggtcccct 1980ctaatatttt gtatttttag tagagacggg gtttcgccat gttgggcagg ctggtcccct 1980
ataatcatat aattatgtaa tccctgatca ctttgattca ttcacctgaa aatatttaat 2040ataatcatat aattatgtaa tccctgatca ctttgattca ttcacctgaa aatatttaat 2040
gagtttcttc catgtttcat gtagaatata tcacaatctc tcctactatt attctagcat 2100gagtttcttc catgtttcat gtagaatata tcacaatctc tcctactatt attctagcat 2100
gtttctgtta catatcacca gcaccacaac caccatcacc ataataatta acattatgga 2160gtttctgtta catatcacca gcaccacaac caccatcacc ataataatta acattatgga 2160
gtccatacta tttttcaggc atttgtaagt actttacatg agttaaccta gttaatccac 2220gtccataacta tttttcaggc atttgtaagt actttacatg agttaaccta gttaatccac 2220
aaaatatccc tgtgagtagg tgctgttatc tccatactat aaataagaaa acttaggcat 2280aaaatatccc tgtgagtagg tgctgttatc tccatactat aaataagaaa acttaggcat 2280
accaaagtta agtaa 2295accaaagtta agtaa 2295
<210> 4<210> 4
<211> 14920<211> 14920
<212> DNA<212>DNA
<213> 重组载体pET32-CPSLR-2A-tdTomato(人工序列)<213> Recombinant vector pET32-CPSLR-2A-tdTomato (artificial sequence)
<400> 4<400> 4
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgatta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcattt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagttttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggacac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980accggataag gcgcagcggt cggggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160cacgaggggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880ttttttcctg tttggtcact gatgcctccg tgtaaggggg attctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000ggttatgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360
gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420
cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480
gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900
tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140
cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200
aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260
ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320
tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380tccacagcaa tggcatcctg gtcatccagc gtagagttaa tgatcagccc actgacgcgt 4380
tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440
gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500
gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560
gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620
ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680
taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740
ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800
atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860
tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920
gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980gcccaacagt cccccggcca cggggcctgc caccataccc agccgaaac aagcgctcat 4980
gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040
aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100
cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160
ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gagcgataaa 5220ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gagcgataaa 5220
attattcacc tgactgacga cagttttgac acggatgtac tcaaagcgga cggggcgatc 5280attattcacc tgactgacga cagttttgac acggatgtac tcaaagcgga cggggcgatc 5280
ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa tgatcgcccc gattctggat 5340ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa tgatcgcccc gattctggat 5340
gaaatcgctg acgaatatca gggcaaactg accgttgcaa aactgaacat cgatcaaaac 5400gaaatcgctg acgaatatca gggcaaactg accgttgcaa aactgaacat cgatcaaaac 5400
cctggcactg cgccgaaata tggcatccgt ggtatcccga ctctgctgct gttcaaaaac 5460cctggcactg cgccgaaata tggcatccgt ggtatcccga ctctgctgct gttcaaaaac 5460
ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag gtcagttgaa agagttcctc 5520ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag gtcagttgaa agagttcctc 5520
gacgctaacc tggccggttc tggttctggc catatgcacc atcatcatca tcattcttct 5580gacgctaacc tggccggttc tggttctggc catatgcacc atcatcatca tcattcttct 5580
ggtctggtgc cacgcggttc tggtatgaaa gaaaccgctg ctgctaaatt cgaacgccag 5640ggtctggtgc cacgcggttc tggtatgaaa gaaaccgctg ctgctaaatt cgaacgccag 5640
cacatggaca gcccagatct gggtaccttt gtggatgatt gtcaagtttc actctccatc 5700cacatggaca gcccagatct gggtaccttt gtggatgatt gtcaagtttc actctccatc 5700
actatggaat acataacgtc atgtgtacat ggtgatatga aacgtgtttc aaaatacttc 5760actatggaat acataacgtc atgtgtacat ggtgatatga aacgtgtttc aaaatacttc 5760
ttagtaagga tactttcctt gacggaaaca agtgagagca tgaagaatgt aatgcagcac 5820ttagtaagga tactttcctt gacggaaaca agtgagagca tgaagaatgt aatgcagcac 5820
tttatatttc atgtcaaact tatattgtgt atagataagt acatcgagta taagatgacc 5880tttatatttc atgtcaaact tatattgtgt atagataagt acatcgagta taagatgacc 5880
agctatattt cactaaggtt gtgcataata aatcaatttg ttgagcactt actacataca 5940agctatattt cactaaggtt gtgcataata aatcaatttg ttgagcactt actacataca 5940
ggaacctatg ttgagtacta tgatgatgtc atccattgga ttacctaatt ggtcattcct 6000ggaacctatg ttgagtacta tgatgatgtc atccattgga ttacctaatt ggtcattcct 6000
tgaccaggtt ttcatgcctt agaccatgtc atcattagat tttgtacgac attttatttc 6060tgaccaggtt ttcatgcctt agaccatgtc atcattagat tttgtacgac attttatttc 6060
agcagattgt gtgaatcttc aggaatacca ttgcatcaac ccttaaagat ctatgtggca 6120agcagattgt gtgaatcttc aggaatacca ttgcatcaac ccttaaagat ctatgtggca 6120
taagtacata ggagataatt ctctgctgtt ttaagatagt gcagcctaca gaatggctgt 6180taagtacata ggagataatt ctctgctgtt ttaagatagt gcagcctaca gaatggctgt 6180
gccattctat gcaatatgtt atttgttagc gttcttgaga aaccaacaca taaattatta 6240gccattctat gcaatatgtt atttgttagc gttcttgaga aaccaacaca taaattatta 6240
tcactttaat aaaataatgg cattgtgttc aaaccttagc aggcctccaa ggatgtaata 6300tcactttaat aaaataatgg cattgtgttc aaaccttagc aggcctccaa ggatgtaata 6300
gctccgtgtt tggggcattg taaaacaata attagcacta ctaaggaggc aatagagtta 6360gctccgtgtt tggggcattg taaaacaata attagcacta ctaaggaggc aatagagtta 6360
atgtgggagt gggaacatgg gggtgaaagg gcattcaatg gtggagggat cacctctttt 6420atgtgggagt gggaacatgg gggtgaaagg gcattcaatg gtggagggat cacctctttt 6420
taaaaaataa gtatctatca aattaccaat tcttaatgag tgaacagata gcattttaaa 6480taaaaaataa gtatctatca aattaccaat tcttaatgag tgaacagata gcattttaaa 6480
gagaaaacaa acatctgcat tccattagac ttgcactaga attccttcaa gaacacaata 6540gagaaaacaa acatctgcat tccattagac ttgcactaga attccttcaa gaacacaata 6540
taatccctat ataagtgtaa tttaaacatc tacttagcaa tagaggaggg cagatggcag 6600taatccctat ataagtgtaa tttaaacatc tacttagcaa tagagggaggg cagatggcag 6600
tcaactcagc atggcattga cttgaatggc taagagagca atttatgtta gtattttata 6660tcaactcagc atggcattga cttgaatggc taagagagca atttatgtta gtattttata 6660
aactaactat aaaatatgcc ttgttgtcta taagtttttg tttatttttc cagattgatt 6720aactaactat aaaatatgcc ttgttgtcta taagtttttg tttatttttc cagattgatt 6720
agagatggca gcattgacct agtgattaac cttcccaaca acaacactaa atttgtccat 6780agagatggca gcattgacct agtgattaac cttcccaaca acaacactaa atttgtccat 6780
gataattatg tgattcggag gacagctgtt gatagtggaa tccctctcct cactaatttt 6840gataattatg tgattcggag gacagctgtt gatagtggaa tccctctcct cactaatttt 6840
caggtatagt cttttccttg gatatagact ggatgggagt tttatttctg tgcctccctt 6900caggtatagt cttttccttg gatatagact ggatgggagt tttatttctg tgcctccctt 6900
aagagtgtaa tcagtagatg cacatctctc ttttcccctc tttgtaactt tcagaataga 6960aagagtgtaa tcagtagatg cacatctctc ttttcccctc tttgtaactt tcagaataga 6960
gaggaatttt taataatcta aaataatgat gctaacatgg taggtcatgg cttaaatggg 7020gaggaatttt taataatcta aaataatgat gctaacatgg taggtcatgg cttaaatggg 7020
acagttggtg atcaagcaat tgagataatc aaaggccatg aatggccatg gctctctcct 7080acaagttggtg atcaagcaat tgagataatc aaaggccatg aatggccatg gctctctcct 7080
tacaattatc ctttgaataa aaagtgacag catgacatgg aataaagata caaactttta 7140tacaattatc ctttgaataa aaagtgacag catgacatgg aataaagata caaactttta 7140
tttcattgtt tctaaatgaa agccaccata taaaagcaac aggttaatga tggtccagat 7200tttcattgtt tctaaatgaa agccaccata taaaagcaac aggttaatga tggtccagat 7200
gtagcacaag tgcttgttca attcacagaa aatgattgca tgaggcatgt tcagtttcac 7260gtagcacaag tgcttgttca attcacagaa aatgattgca tgaggcatgt tcagtttcac 7260
ttggacatga cccatcgaat ttatcacagg gagaaacaga gtggagactc attaactttc 7320ttggacatga cccatcgaat ttatcacagg gagaaacaga gtggagactc attaactttc 7320
tgcctatcat atttattttt tatgcagaat cagttttaat ccctatggga ggagaaataa 7380tgcctatcat atttattttttgcagaat cagttttaat ccctatggga ggagaaataa 7380
gcgtattcac agtgacatct gagatataaa agaaagtccc catggtgagg tctgagacat 7440gcgtattcac agtgacatct gagatataaa agaaagtccc catggtgagg tctgagacat 7440
gggagaaagt ctccattaca taaaaaactt ttatgccttt tatcccatac ccctttgaaa 7500gggagaaagt ctccattaca taaaaaactt ttatgccttt tatcccatac ccctttgaaa 7500
actggggaca gacacttgtg acttttgtct tcattcatta aaaattcact tttatctcat 7560actggggaca gacacttgtg acttttgtct tcattcatta aaaattcact tttatctcat 7560
ggagggtgct gattcctacc attatatttt caggtgacca aactttttgc tgaagctgtg 7620ggagggtgct gattcctacc attatatttt caggtgacca aactttttgc tgaagctgtg 7620
cagaaatctc gcaaggtgga ctccaagagt cttttccact acaggcagta cagtgctgga 7680cagaaatctc gcaaggtgga ctccaagagt cttttccact acaggcagta cagtgctgga 7680
aaagcagcag atatcaaaat tgtcgctcct gtcaaacaaa ctcttaactt tgatttactc 7740aaagcagcag atatcaaaat tgtcgctcct gtcaaacaaa ctcttaactt tgattactc 7740
aaactggctg gggatgtaga aagcaatcca ggtccaggat ccgccaccat ggtgagcaag 7800aaactggctg gggatgtaga aagcaatcca ggtccaggat ccgccaccat ggtgagcaag 7800
ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac 7860ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac 7860
ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc 7920ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc 7920
gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc 7980gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc 7980
cagttcatgt acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag 8040cagttcatgt acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag 8040
aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt 8100aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt 8100
ctggtgaccg tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag 8160ctggtgaccg tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag 8160
atgcgcggca ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg 8220atgcgcggca ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg 8220
gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag 8280gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag 8280
gccctgaagc tgaaggacgg cggccactac ctggtggagt tcaagaccat ctacatggcc 8340gccctgaagc tgaaggacgg cggccactac ctggtggagt tcaagaccat ctacatggcc 8340
aagaagcccg tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc 8400aagaagcccg tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc 8400
cacaacgagg actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg 8460cacaacgagg actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg 8460
ttcctggggc atggcaccgg cagcaccggc agcggcagct ccggcaccgc ctcctccgag 8520ttcctggggc atggcaccgg cagcaccggc agcggcagct ccggcaccgc ctcctccgag 8520
gacaacaaca tggccgtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc 8580gacaacaaca tggccgtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc 8580
atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc 8640atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc 8640
cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg 8700cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg 8700
tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat 8760tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat 8760
tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac 8820tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac 8820
ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag 8880ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag 8880
gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg 8940gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg 8940
ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc 9000ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc 9000
caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac 9060caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac 9060
atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc 9120atggccaaga agcccgtgca actgcccggc tactactacg tggaccacaa gctggacatc 9120
acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac 9180acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac 9180
cacctgttcc tgtacggcat ggacgagctg tacaagtgaa gcggccgcga ctctagatca 9240cacctgttcc tgtacggcat ggacgagctg tacaagtgaa gcggccgcga ctctagatca 9240
taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc 9300taatcagcca taccacattt gtagagaggttt tacttgcttt aaaaaacctc ccacacctcc 9300
ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt 9360ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt 9360
ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 9420ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 9420
tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaaattcc tgcagcccaa 9480tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaaattcc tgcagcccaa 9480
ttccgatcat attcaataac ccttaatata acttcgtata atgtatgcta tacgaagtta 9540ttccgatcat attcaataac ccttaatata acttcgtata atgtatgcta tacgaagtta 9540
ttaggtctga agaggagttt acgtccagcc aagctagcct cgacattgat tattgactag 9600ttaggtctga agaggagttt acgtccagcc aagctagcct cgacattgat tattgactag 9600
ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt 9660ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt 9660
tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac 9720tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac 9720
gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg 9780gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg 9780
ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 9840ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 9840
tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat 9900tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat 9900
gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 9960gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 9960
ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc 10020ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct ccccacccccc 10020
aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg 10080aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg 10080
ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg 10140ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg 10140
tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg 10200tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg 10200
gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct 10260gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct 10260
tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc 10320tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc 10320
gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt 10380gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt 10380
ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa agccttgagg ggctccggga 10440ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa agccttgagg ggctccggga 10440
gggccctttg tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag 10500gggccctttg tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag 10500
cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct 10560cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct 10560
ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg 10620ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg 10620
ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg 10680ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtgggggg ggtgagcagg 10680
gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc gagttgctga 10740gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc gagttgctga 10740
gcacggcccg gcttcgggtg cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc 10800gcacggcccg gcttcgggtg cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc 10800
gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc gggccgggga 10860gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc gggccgggga 10860
gggctcgggg gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag 10920gggctcgggg gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag 10920
ccgcagccat tgccttttat ggtaatcgtg cgagagggcg cagggacttc ctttgtccca 10980ccgcagccat tgccttttat ggtaatcgtg cgagagggcg cagggacttc ctttgtccca 10980
aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc 11040aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc 11040
gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg 11100gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg 11100
ccgccgtccc cttctccctc tccagcctcg gggctgtccg cggggggacg gctgccttcg 11160ccgccgtccc cttctccctc tccagcctcg gggctgtccg cggggggacg gctgccttcg 11160
ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctaggctgtt 11220ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctaggctgtt 11220
gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa 11280gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa 11280
accatgggat cggccattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg 11340accatgggat cggccattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg 11340
gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg 11400gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg 11400
ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc 11460ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc 11460
ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct 11520ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct 11520
tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa 11580tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa 11580
gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg 11640gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg 11640
gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa 11700gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa 11700
gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat 11760gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat 11760
gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg 11820gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg 11820
cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc 11880cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc 11880
atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac 11940atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac 11940
cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg 12000cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg 12000
gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc 12060gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc 12060
tatcgccttc ttgacgagtt cttctgaggg gatcaattct ctagagctcg ctgatcagcc 12120tatcgccttc ttgacgagtt cttctgaggg gatcaattct ctagagctcg ctgatcagcc 12120
tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 12180tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 12180
accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 12240accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 12240
tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 12300tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 12300
gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg 12360gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg 12360
gaaagaacca gctggggctc gactagagct tgcggaaccc ttaatataac ttcgtataat 12420gaaagaacca gctggggctc gactagagct tgcggaaccc ttaatataac ttcgtataat 12420
gtatgctata cgaagttatg aattcgagct ccgtcgacag atgcagacac cccagcccca 12480gtatgctata cgaagttatg aattcgagct ccgtcgacag atgcagacac cccagcccca 12480
ttattaaatc aacctgagcc acatgttatc taaaggaact gattcacaac tttctcagag 12540ttattaaatc aacctgagcc acatgttatc taaaggaact gattcacaac tttctcagag 12540
atgaatattg ataactaaac ttcatttcag tttactttgt tatgccttaa tattctgtgt 12600atgaatattg ataactaaac ttcatttcag tttactttgt tatgccttaa tattctgtgt 12600
cttttgcaat taaattgtca gtcacttctt caaaacctta cagtccttcc taagttactc 12660cttttgcaat taaattgtca gtcacttctt caaaacctta cagtccttcc taagttactc 12660
ttcatgagat ttcatccatt tactaatact gtatttttgg tggactaggc ttgcctatgt 12720ttcatgagat ttcatccatt tactaatact gtatttttgg tggactaggc ttgcctatgt 12720
gcttatgtgt agctttttac tttttatggt gctgattaat ggtgatcaag gtaggaaaag 12780gcttatgtgt agctttttac tttttatggt gctgattaat ggtgatcaag gtaggaaaag 12780
ttgctgttct attttctgaa ctctttctat actttaagat actctatttt taaaacacta 12840ttgctgttct attttctgaa ctctttctat actttaagat actctatttt taaaacacta 12840
tctgcaaact caggacactt taacagggca gaatactcta aaaacttgat aaaattaaat 12900tctgcaaact caggacactt taacagggca gaatactcta aaaacttgat aaaattaaat 12900
atagatttaa tttatgaacc ttccatcatg atgtttgtgt attgcttctt tttggatcct 12960atagatttaa tttatgaacc ttccatcatg atgtttgtgt attgcttctt tttggatcct 12960
cattctcacc catttggcta atccaggaat attgttatcc cttcccatta tattgaagtt 13020cattctcacc catttggcta atccaggaat attgttatcc cttcccatta tattgaagtt 13020
gagaaatgtg acagaggcat ttagagtatg gacttttctt ttctttttct ttttcttttt 13080gagaaatgtg acagaggcat ttagagtatg gacttttctt ttctttttct ttttcttttt 13080
ttctttttga gatggagtca cactctccag gctggagtgc agtggcacaa tctcggctca 13140ttctttttga gatggagtca cactctccag gctggagtgc agtggcacaa tctcggctca 13140
ctgcaatttc cgtctcccaa gttcaagcga ttctcctgct ttagactatg gatttcttta 13200ctgcaatttc cgtctcccaa gttcaagcga ttctcctgct ttagactatg gatttcttta 13200
aggaatactg gtttgcagtt ttgttttctg gactatatca gcagatggta gacagtgttt 13260aggaatactg gtttgcagtt ttgttttctg gactatatca gcagatggta gacagtgttt 13260
atgtagatgt gttgttgttt ttatcattgg attttaactt ggcccgagtg aaataatcag 13320atgtagatgt gttgttgttt ttatcattgg attttaactt ggcccgagtg aaataatcag 13320
atttttgtca ttcacactct cccccagttt tggaataact tggaagtaag gttcattccc 13380atttttgtca ttcacactct cccccagttt tggaataact tggaagtaag gttcattccc 13380
ttaagacgat ggattctgtt gaactatggg gtcccacact gcactattaa ttccacccac 13440ttaagacgat ggattctgtt gaactatggg gtcccacact gcactattaa ttccacccac 13440
tgtaagggca aggacaccat tccttctaca tataagaaaa aagtctctcc ccaagggcag 13500tgtaagggca aggacaccat tccttctaca tataagaaaa aagtctctcc ccaagggcag 13500
cctttgttac ttttaaatat tttctgttat tacaagtgct ctaattgtga acttttaaat 13560cctttgttac ttttaaatat tttctgttat tacaagtgct ctaattgtga acttttaaat 13560
aaaatactat taagaggtaa tgcagttgaa tctggtttta ttttatgttg ctgtacaaaa 13620aaaatactat taagaggtaa tgcagttgaa tctggtttta ttttatgttg ctgtacaaaa 13620
atcagtttac ttctatgata aaatagggtt ttgggccagg attgcattgc ttatttattt 13680atcagtttac ttctatgata aaatagggtt ttgggccagg attgcattgc ttatttattt 13680
tttccatgca aacccatata gggatgagaa aattaatgtt aaaaataagt tttagctgtc 13740tttccatgca aacccatata gggatgagaa aattaatgtt aaaaataagt tttagctgtc 13740
acatttttca ttttttcttg tccctccctt gcttttaagt ctcatcataa cattatgtgg 13800acatttttca ttttttcttg tccctccctt gcttttaagt ctcatcataa cattatgtgg 13800
ttatatggct ataaaccatc tttgatctgg atcctttcta atgtataact acaaccacaa 13860ttatatggct ataaaccatc tttgatctgg atcctttcta atgtataact acaaccacaa 13860
actgattaaa gattgccaca aatatatagt aaactttcaa acaaatgtct tgcataactc 13920actgattaaa gattgccaca aatatatagt aaactttcaa acaaatgtct tgcataactc 13920
agaaaagtaa cttaacccaa tcagaggctg accagcattg tctgatggaa atagtgccaa 13980agaaaagtaa cttaacccaa tcagaggctg accagcattg tctgatggaa atagtgccaa 13980
tgagcacact gatgccatat ccccaattta ccaccgaaac tatggctttg tgaaggtcct 14040tgagcacact gatgccatat ccccaattta ccaccgaaac tatggctttg tgaaggtcct 14040
aagataatta tgctataaca gcttttattt cttccttgtt acctcacagt caattgatta 14100aagataatta tgctataaca gcttttattt cttccttgtt acctcacagt caattgatta 14100
ccactttgta tagctctaga cgaatgctta aagtaattca tcttctctcc ccctcacttc 14160ccactttgta tagctctaga cgaatgctta aagtaattca tcttctctcc ccctcacttc 14160
cttgttcttc ctgtggtcaa gactttcatt acttcttccc tgtaatcttt tttttttttt 14220cttgttcttc ctgtggtcaa gactttcatt acttcttccc tgtaatcttt tttttttttt 14220
tttttttttt ttgagacgga gttttgctct tgtcacccag gctggagtgc agtggcgcga 14280ttttttttttttgagacgga gttttgctct tgtcacccag gctggagtgc agtggcgcga 14280
tctcagctca ctgcaacctc catctcctgg gttccagcga ttctcctgcc tcagcctcct 14340tctcagctca ctgcaacctc catctcctgg gttccagcga ttctcctgcc tcagcctcct 14340
gagtagctga gattacaggt gcccgccaac aagcctggct aatattttgt atttttagta 14400gagtagctga gattacaggt gcccgccaac aagcctggct aatattttgt atttttagta 14400
gagacggggt ttcgccatgt tgggcaggct ggtcccctat aatcatataa ttatgtaatc 14460gagacggggt ttcgccatgt tgggcaggct ggtcccctat aatcatataa ttatgtaatc 14460
cctgatcact ttgattcatt cacctgaaaa tatttaatga gtttcttcca tgtttcatgt 14520cctgatcact ttgattcatt cacctgaaaa tattaatga gtttcttcca tgtttcatgt 14520
agaatatatc acaatctctc ctactattat tctagcatgt ttctgttaca tatcaccagc 14580agaatatatc acaatctctc ctactattat tctagcatgt ttctgttaca tatcaccagc 14580
accacaacca ccatcaccat aataattaac attatggagt ccatactatt tttcaggcat 14640accacaacca ccatcaccat aataattaac attatggagt ccatactatt tttcaggcat 14640
ttgtaagtac tttacatgag ttaacctagt taatccacaa aatatccctg tgagtaggtg 14700ttgtaagtac tttacatgag ttaacctagt taatccacaa aatatccctg tgagtaggtg 14700
ctgttatctc catactataa ataagaaaac ttaggcatac caaagttaag taaaagcttc 14760ctgttatctc catactataa ataagaaaac ttaggcatac caaagttaag taaaagcttc 14760
gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag 14820gagcaccacc accacccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag 14820
ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc 14880ttggctgctg ccaccgctga gcaataacta gcataaccccc ttggggcctc taaacgggtc 14880
ttgaggggtt ttttgctgaa aggaggaact atatccggat 14920ttgaggggtt ttttgctgaa aggaggaact atatccggat 14920
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2017101565826 | 2017-03-16 | ||
| CN201710156582 | 2017-03-16 |
| Publication Number | Publication Date |
|---|---|
| CN108660156Atrue CN108660156A (en) | 2018-10-16 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810199247.9APendingCN108660156A (en) | 2017-03-16 | 2018-03-12 | CPS1 reporter genes stem cell and its construction method and application |
| Country | Link |
|---|---|
| CN (1) | CN108660156A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110540962A (en)* | 2019-08-28 | 2019-12-06 | 北京协同创新研究院 | Method for preparing human definitive endoderm cells |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037669A (en)* | 2006-03-13 | 2007-09-19 | 上海交通大学医学院附属新华医院 | Method for inducing liver cell from human embryonic stem cells |
| US20140155468A1 (en)* | 2012-12-05 | 2014-06-05 | The Children's Hospital Of Philadelphia | Methods and compositions for regulation of metabolic disorders |
| CN105121632A (en)* | 2013-02-18 | 2015-12-02 | 大学健康网络 | Method for generating hepatocytes and cholangiocytes from pluripotent stem cells |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037669A (en)* | 2006-03-13 | 2007-09-19 | 上海交通大学医学院附属新华医院 | Method for inducing liver cell from human embryonic stem cells |
| US20140155468A1 (en)* | 2012-12-05 | 2014-06-05 | The Children's Hospital Of Philadelphia | Methods and compositions for regulation of metabolic disorders |
| CN105121632A (en)* | 2013-02-18 | 2015-12-02 | 大学健康网络 | Method for generating hepatocytes and cholangiocytes from pluripotent stem cells |
| Title |
|---|
| DAIHACHIRO TOMOTSUNE等: "Enrichment of Pluripotent Stem Cell-Derived Hepatocyte-Like Cells by Ammonia Treatment", 《PLOS ONE》* |
| PATRICK C LEE等: "Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells", 《MOLECULAR THERAPY-NUCLEIC ACIDS》* |
| SISI LAI等: "Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering", 《PLOS ONE》* |
| YI WANG等: "Generation of carbamoyl phosphate synthetase 1 reporter cell lines for the assessment of ammonia metabolism", 《JOURNAL OF CELLULAR AND MOLECULAR MEDICINE》* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110540962A (en)* | 2019-08-28 | 2019-12-06 | 北京协同创新研究院 | Method for preparing human definitive endoderm cells |
| CN110540962B (en)* | 2019-08-28 | 2022-02-25 | 北京协同创新研究院 | Method for preparing human definitive endoderm cells |
| Publication | Publication Date | Title |
|---|---|---|
| CN111893104A (en) | A structure-based method for optimal design of CRISPR proteins | |
| CN110923183A (en) | Construction method of lanosterol-producing Escherichia coli strain | |
| CN106636198A (en) | Site-directed knock-in plasmid vector for porcine mammary gland-specific high-expression lactoferrin gene and application thereof | |
| CN111041038B (en) | Synechocystis 6803 genetically engineered bacteria for efficient biosynthesis of astaxanthin and its construction method and application | |
| CN106947740A (en) | CPS1 reporter gene human liver cell systems and its construction method and application | |
| CN108660156A (en) | CPS1 reporter genes stem cell and its construction method and application | |
| CN112501139A (en) | Recombinant Newcastle disease virus strain and preparation method and application thereof | |
| CN115247153B (en) | Gene editing system for constructing HNF1A gene mutation diabetic model pig nuclear transfer donor cells and its application | |
| US20240352469A1 (en) | Enzymatically methylated dna and methods of production and therapeutic use | |
| CN115247173A (en) | Construction and application of gene editing system for porcine nuclear transfer donor cells with TMPRSS6 gene mutation in iron deficiency anemia | |
| KR20230150998A (en) | How to make Cas3 protein | |
| CN115232817A (en) | Gene editing system for constructing three-gene combined mutant miniature pig nuclear transplantation donor cells and application thereof | |
| CN115232793A (en) | Gene editing system for constructing ALS model pig nuclear transplantation donor cells with SOD1 gene mutation and application thereof | |
| CN115247164B (en) | Construction of a gene editing system for adenomatous polyposis model pigs and colorectal cancer model pigs and their applications | |
| CN115247175B (en) | Gene editing system for constructing epigenetic deregulation model pig nuclear transfer donor cells with SETDB1 gene mutation and application of gene editing system | |
| CN108949690B (en) | A method of prepare can real-time detection mescenchymal stem cell bone differentiation cell model | |
| CN115232794B (en) | Gene editing system and application thereof in construction of OCA-1B albinism model pig nuclear transfer donor cells | |
| CN115232834B (en) | Gene editing system for constructing OCA-1A albinism model pig nuclear transfer donor cells and its application | |
| CN115247191B (en) | Gene editing system and its application in constructing pig nuclear transplant donor cells with double gene mutations in nevoid basal cell carcinoma syndrome | |
| CN115161335B (en) | Gene editing system for constructing ALS model pig nuclear transfer donor cells with TARDBP gene mutation and application of gene editing system | |
| CN115232836B (en) | Construction of a gene editing system for pig nuclear transplantation donor cells in a congenital cataract model with CRYGC gene mutation and its application | |
| CN109628487A (en) | A method of growth factor of human nerve is prepared using transgene pig salivary gland | |
| CN103352042B (en) | The cDNA of coding recombinant Newcastle disease virus, the virus of being saved by this cDNA and the application in treatment malignant tumour thereof | |
| CN109402109A (en) | A kind of improved Overlap extension PCR method | |
| KR100902634B1 (en) | Nucleic Acid Delivery Complexes Comprising Recombinant HMVII Peptides |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication | Application publication date:20181016 |