技术领域technical field
本发明涉及基因工程领域，涉及猪ApoE基因敲除载体及其构建方法与应用。The invention relates to the field of genetic engineering, and relates to a porcine ApoE gene knockout vector and a construction method and application thereof.
背景技术Background technique
ApoE(载脂蛋白E)是脂代谢中重要的蛋白分子，在脂代谢过程中主要作为乳糜微粒(CM)、极低密度脂蛋白(VLDL)、中间密度脂蛋白(IDL)以及高密度脂蛋白(HDL)的重要组成部分。ApoE基因的突变极易引发不同类型的疾病包括糖尿病、动脉粥样硬化以及阿尔茨海默病(Alzheimer'sdisease，AD)。基因打靶制备的ApoE敲除小鼠在正常生长条件下9周龄时表现为主动脉弓动脉硬化斑块，当用高脂料饲喂时第7周即出现泡沫样的动脉硬化斑块。除此之外该敲除鼠在生长老年阶段会发生阿尔茨海默病类似的病变。因此用ApoE敲除小鼠作为研究动脉硬化以及阿尔茨海默病具有重要的医学研究价值。但是鉴于小鼠和人在脂代谢中的差异使用小鼠作为动脉硬化研究模型具有一定的局限性，因此使用与人代谢模式更接近的小型猪作为材料制作ApoE敲除猪，有望更好地模拟人类动脉硬化发病过程以供其机理研究。ApoE (apolipoprotein E) is an important protein molecule in lipid metabolism, mainly as chylomicron (CM), very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and high density lipoprotein in the process of lipid metabolism An important part of (HDL). Mutations in the ApoE gene can easily lead to different types of diseases including diabetes, atherosclerosis and Alzheimer's disease (Alzheimer'sdisease, AD). ApoE knockout mice prepared by gene targeting showed aortic arch atherosclerotic plaques at 9 weeks of age under normal growth conditions, and foamy atherosclerotic plaques appeared at 7 weeks when fed with high-fat diet. In addition, the knockout mice will develop Alzheimer's disease-like lesions in the growing old stage. Therefore, using ApoE knockout mice to study arteriosclerosis and Alzheimer's disease has important medical research value. However, in view of the differences in lipid metabolism between mice and humans, the use of mice as a model of arteriosclerosis has certain limitations. Therefore, using miniature pigs that are closer to human metabolic models as materials to produce ApoE knockout pigs is expected to better simulate Pathogenesis of human arteriosclerosis for its mechanism study.
基因打靶技术是利用同源重组的原理将外源基因片段插入特定位置，以期将目的基因部分或者全部敲除。同源重组是指姐妹染色单体之间或者同一染色体上含有同源序列的DNA分子之间发生序列交换的过程。随着80年代初胚胎干细胞的分离和体外培养技术的不断成熟，利用同源重组原理使得对基因进行特定修饰改造成为可能。到目前为止啮齿类动物如小鼠等已经有成熟的胚胎干细胞培养体系和基因打靶技术对基因进行需要的修饰。但是对于偶蹄类动物如猪、牛、羊等一直没有成熟的胚胎干细胞系作为基因打靶的材料，所以在这些动物上进行基因打靶具有一定的技术难度。伴随着体细胞克隆技术的不断成熟和完善，使得在这些动物中进行基因打靶成为可能。虽然体细胞的基因打靶效率很低，通常为10-6左右，但是通过正负筛选的方式仍然可以获得中靶克隆。影响打靶效率的因素有多种，其中最主要的就是同源臂的长度以及同源序列的同源性，一般来说同源臂在10kb以内和同源重组效率成正比，序列的同源性越高重组效率也会随之升高。因此在制作打靶载体过程中同源臂的长度和同源序列的同源性是首要考虑的两个问题。Gene targeting technology uses the principle of homologous recombination to insert foreign gene fragments into specific positions in order to knock out part or all of the target gene. Homologous recombination refers to the process of sequence exchange between sister chromatids or DNA molecules containing homologous sequences on the same chromosome. With the separation of embryonic stem cells and the continuous maturity of in vitro culture technology in the early 1980s, the use of homologous recombination principles made it possible to carry out specific modification of genes. So far, rodents such as mice have mature embryonic stem cell culture systems and gene targeting technology to modify genes. However, for artiodactyl animals such as pigs, cattle, sheep, etc., there have been no mature embryonic stem cell lines as gene targeting materials, so gene targeting in these animals has certain technical difficulties. With the continuous maturity and improvement of somatic cell cloning technology, gene targeting in these animals becomes possible. Although the gene targeting efficiency of somatic cells is very low, usually around 10-6 , on-target clones can still be obtained through positive and negative screening. There are many factors that affect the targeting efficiency, the most important of which are the length of the homology arm and the homology of the homologous sequence. Generally speaking, the homology arm within 10kb is directly proportional to the efficiency of homologous recombination, and the homology of the sequence The higher the recombination efficiency, the higher it will be. Therefore, the length of the homology arm and the homology of the homology sequence are the two primary considerations in the process of making the targeting vector.
发明内容Contents of the invention
本发明的目的在于提供猪ApoE基因敲除载体；本发明的另一个目的在于提供猪ApoE基因敲除载体的构建方法及其应用。The purpose of the present invention is to provide a pig ApoE gene knockout vector; another purpose of the present invention is to provide a construction method and application of the pig ApoE gene knockout vector.
为达到上述目的，本发明的技术方案是从中国农业大学自主繁育的贵州小香猪体细胞组织中提取基因组DNA，设计长短同源臂引物，通过PCR方法从基因组DNA中克隆同源臂，将同源臂分别克隆到表达载体，经过限制性内切酶酶切和测序分析确定同源臂的准确性后，采用酶切和连接的方法将长短同源臂分别连接到载体pEASY-T中，通过限制性内切酶酶切鉴定同源臂连接的正确性，为了方便去除Neo标记基因，在pApoEKO-DTA打靶载体基础上用同源重组的方法在长短臂之间插入一段受猪精原细胞特异性启动子调控的Cre基因。这样可以在杂交F2代获得去除Neo标记基因的敲除猪模型。In order to achieve the above-mentioned purpose, the technical solution of the present invention is to extract genomic DNA from the somatic tissue of Guizhou Miniature Pig independently bred by China Agricultural University, design long and short homologous arm primers, and clone the homologous arm from the genomic DNA by PCR method. The homology arms were respectively cloned into the expression vector, and after the accuracy of the homology arms was confirmed by restriction endonuclease digestion and sequencing analysis, the long and short homology arms were respectively connected into the vector pEASY-T by restriction enzyme digestion and ligation, The correctness of the homology arm connection was identified by restriction endonuclease digestion. In order to facilitate the removal of the Neo marker gene, a section of pig spermatogonia was inserted between the long and short arms by homologous recombination on the basis of the pApoEKO-DTA targeting vector. Cre gene regulated by a specific promoter. In this way, a knockout pig model that removes the Neo marker gene can be obtained in the hybrid F2 generation.
具体地，本发明提供的猪ApoE基因敲除载体，包含ApoE基因上下游同源臂的长臂和短臂，所述长臂的核苷酸序列如SEQ ID NO:1的第1～第5387位；所述短臂的核苷酸序列如SEQ ID NO:1的第9604～第11855位；在同源臂之间还具有标记基因。Specifically, the porcine ApoE gene knockout vector provided by the present invention comprises the long arm and the short arm of the upstream and downstream homologous arms of the ApoE gene, and the nucleotide sequence of the long arm is as shown in SEQ ID NO: 1 to 5387 position; the nucleotide sequence of the short arm is such as the 9604th to 11855th position of SEQ ID NO: 1; there is also a marker gene between the homology arms.
本发明的猪ApoE基因敲除载体命名为pApoEKO-DTA-M，其核苷酸序列如SEQ ID NO.1所示。The porcine ApoE gene knockout vector of the present invention is named pApoEKO-DTA-M, and its nucleotide sequence is shown in SEQ ID NO.1.
本发明的猪ApoE基因敲除载体上的标记基因包括带有猪精原细胞特异性启动子Cre以及新霉素抗性选择基因Neo。The marker gene on the porcine ApoE gene knockout carrier of the present invention includes the porcine spermatogonia-specific promoter Cre and the neomycin resistance selection gene Neo.
本发明提供了猪ApoE基因敲除载体在制备ApoE基因敲除的猪中的应用。The invention provides the application of the pig ApoE gene knockout vector in preparing ApoE gene knockout pigs.
本发明提供了猪ApoE基因敲除载体在制备动脉硬化模型动物中的应用。The invention provides the application of the pig ApoE gene knockout vector in preparing arteriosclerosis model animals.
本发明提供了一种扩增猪ApoE基因侧翼序列的引物组合，其包括扩增上下游同源臂长臂的引物对和短臂的引物对，用于扩增短臂的引物对其核苷酸序列如SEQ ID NO.2-3所示，用于扩增长臂的引物对其核苷酸序列如SEQ ID NO.4-5所示。The invention provides a primer combination for amplifying the flanking sequence of the pig ApoE gene, which includes a primer pair for amplifying the long arm of the upstream and downstream homologous arms and a primer pair for the short arm, and the primers for amplifying the short arm are aligned with their nucleosides. The acid sequence is shown in SEQ ID NO.2-3, and the nucleotide sequence of the primer used to amplify the long arm is shown in SEQ ID NO.4-5.
本发明提供了一种构建猪ApoE基因敲除载体的方法，包括如下步骤：The invention provides a method for constructing a porcine ApoE gene knockout carrier, comprising the steps of:
(1)扩增猪ApoE基因侧翼序列作为基因打靶的长臂Larm和短臂Sarm；将扩增出的长、短臂分别连入pEASY-T载体，得到长臂质粒载体和短臂质粒载体；(1) Amplify the pig ApoE gene flanking sequence as the long-arm Larm and short-arm Sarm for gene targeting; connect the amplified long and short arms into the pEASY-T vector respectively to obtain the long-arm plasmid vector and the short-arm plasmid vector;
(2)短臂质粒载体与打靶载体ploxp分别酶切、连接后得到ploxp-Sarm质粒，再与长臂质粒载体连接得到ploxp-Sarm-Larm；(2) The short-arm plasmid vector and the targeting vector ploxp were respectively digested and ligated to obtain the ploxp-Sarm plasmid, and then ligated with the long-arm plasmid vector to obtain ploxp-Sarm-Larm;
长短臂连接完成后的质粒载体用HindⅢ和Mlu Ⅰ酶切连接的方式将ploxp载体骨架上的tk负筛选基因替换成dta负筛选基因；The plasmid vector after the long-short arm connection is completed is replaced by the tk negative selection gene on the ploxp vector backbone with the dta negative selection gene by HindⅢ and MluI enzyme digestion and connection;
(3)在长、短臂之间插入猪精原细胞特异性启动子Cre，构建得到ApoE基因敲除载体。(3) A porcine spermatogonia-specific promoter Cre was inserted between the long and short arms to construct an ApoE gene knockout vector.
本发明方法中，步骤(1)中扩增短臂的引物序列如SEQ ID NO.2-3所示，扩增长臂的引物序列如SEQ ID NO.4-5所示。In the method of the present invention, the primer sequence for amplifying the short arm in step (1) is shown in SEQ ID NO.2-3, and the primer sequence for amplifying the long arm is shown in SEQ ID NO.4-5.
本发明的一个实施例中，步骤(1)的T载体为pEASY-T载体。In one embodiment of the present invention, the T vector in step (1) is pEASY-T vector.
本发明方法中，步骤(2)先将短臂与打靶载体ploxp分别用EcoRI和SalI双酶切。In the method of the present invention, in step (2), the short arm and the targeting vector ploxp are double-digested with EcoRI and SalI respectively.
其中，步骤(2)ploxp-Sarm质粒与长臂质粒载体连接前，分别用NotI单酶切，纯化回收后连接得到ploxp-Sarm-Larm载体。Wherein, step (2) prior to connecting the ploxp-Sarm plasmid with the long-arm plasmid vector, single-enzyme digestion with NotI respectively, purification and recovery, and connection to obtain the ploxp-Sarm-Larm vector.
其中，步骤(3)用同源重组的方法在长短臂之间插入一段受猪精原细胞特异性启动子调控的Cre基因，具体方法为：使用同源臂引物CR-F和CR-R从ptACE载体中扩增出同源重组的目的片段；取和同源重组目的片段混合后电转感受态细胞，培养挑取单克隆菌落鉴定正确的为打靶载体pApoEKO-DTA-M；所述同源臂引物CR-F和CR-R的核苷酸序列如SEQ ID NO.6、7所示。Wherein, step (3) uses homologous recombination to insert a section of Cre gene regulated by a porcine spermatogonia-specific promoter between the long and short arms, and the specific method is: use homologous arm primers CR-F and CR-R from The target fragment of homologous recombination was amplified in the ptACE vector; after being mixed with the target fragment of homologous recombination, the competent cells were electrotransduced, and the single clone colonies were cultured and picked to identify the correct targeting vector pApoEKO-DTA-M; the homologous arm The nucleotide sequences of primers CR-F and CR-R are shown in SEQ ID NO.6 and 7.
ptACE载体构建方法为：用PCR方法(引物序列：CRE-NHE1-F：GCTTCGTCAGAACAAACGACCCAACACCCGTGC/CRE-R：CTCTTCTTCTTGGGCATAGTAGGAGCAGGGCAGTC)从猪基因组中扩增出angiotensin-converting enzyme基因5’启动子区(组织特异性启动子序列)，同时用引物(CRE-F：GACTGCCCTGCTCCTACTATGCCCAAGAAGAAGAG/CRE-NHE1-R：TTCGAGAACTGGATCTCGGGGCAAGACAAATGTC)以ploxp上的Cre基因为模板扩增Cre基因片段。利用重叠PCR原理将组织特异性启动子序列和Cre基因片段连接，连接后用引物中添加的NheⅠ酶切位点(下划线加粗部分)插入PL452骨架质粒(购自Addgene)的Nhe Ⅰ酶切位点中获得。ptACE carrier construction method is: use PCR method (primer sequence: CRE-NHE1-F: GCTTCGTCAG AACAAACGACCCAACACCCGTGC/CRE-R: CTCTTCTTCTTGGGCATAGTAGGAGCAGGGCAGTC) amplified the 5' promoter region of angiotensin-converting enzyme gene (tissue-specific promoter sequence) from the pig genome, and used primers (CRE-F: GACTGCCCTGCTCCTACTATGCCCAAGAAGAAGAG/CRE-NHE1-R: TTCGAGAACT GGATCTCGGGGCAAGACAAATGTC) uses the Cre gene on ploxp as a template to amplify the Cre gene fragment. The tissue-specific promoter sequence and the Cre gene fragment were ligated using the principle of overlapping PCR, and the NheI restriction site added in the primer (underlined bold part) was inserted into the NheI restriction site of the PL452 backbone plasmid (purchased from Addgene) after ligation Points obtained.
采用基因抓捕方法，设计同源臂引物CR-F/CR-R(即普通引物后加上一段100bp的同源序列)用于同源重组。从ptACE载体中扩增出同源重组的目的片段包含Cre基因以及启动子区和Neo抗性筛选基因。在本发明的实施例中，ploxp-Sarm-Larm载体10ng和含有同源重组目的片段的PCR产物600ng混合后电转SW102大肠杆菌感受态细胞电转化采用的感受态细胞。电转后涂LB培养基平板培养，挑取较大的单克隆菌落提取质粒酶切鉴定结果见图1。鉴定质粒为最终的打靶载体pApoEKO-DTA-M，图谱见图2。Using the gene capture method, the homology arm primers CR-F/CR-R (that is, a 100bp homologous sequence added after the common primer) were designed for homologous recombination. The target fragment of homologous recombination amplified from the ptACE vector includes Cre gene, promoter region and Neo resistance screening gene. In an embodiment of the present invention, 10 ng of the ploxp-Sarm-Larm vector and 600 ng of the PCR product containing the target fragment of homologous recombination were mixed and then electroporated to the competent cells used for the electrotransformation of SW102 Escherichia coli competent cells. After electroporation, smear LB medium for plate culture, pick larger monoclonal colonies to extract plasmid enzyme digestion and identification results are shown in Figure 1. The plasmid was identified as the final targeting vector pApoEKO-DTA-M, and the map is shown in Figure 2.
本发明的优点：(1)插入的白喉毒素基因(在基因抓捕步骤前进行dta基因的插入)提高正负筛选的阳性克隆率，原tk基因在筛选过程中需要添加毒性因子GANC，替换成dta后不用添加任何因子即可以筛选从而提高筛选效率；(2)同时敲入特异启动子启动的Cre基因方便标记基因的去除，由于Cre基因与标记基因Neo同时位于两个loxp位点内，而且Cre基因被猪精原特异性启动子启动，因此在打靶猪杂交过程中精原细胞中特异表达的Cre基因能够有效识别loxp位点，从而将标记基因同时去除；(3)能够大片段地删除ApoE基因序列以确保其功能完全缺失，本发明同源重组完成后将缺失ApoE基因的2-4个外显子区域。Advantages of the present invention: (1) the inserted diphtheria toxin gene (the insertion of the dta gene before the gene capture step) improves the positive clone rate of positive and negative screening, and the original tk gene needs to be added with the toxicity factor GANC during the screening process and replaced with After dta, it can be screened without adding any factors to improve the screening efficiency; (2) knocking in the Cre gene activated by a specific promoter at the same time facilitates the removal of the marker gene, because the Cre gene and the marker gene Neo are located in two loxp sites at the same time, and The Cre gene is activated by the pig spermatogonia-specific promoter, so the Cre gene specifically expressed in the spermatogonia during the target pig hybridization process can effectively recognize the loxp site, thereby simultaneously removing the marker gene; (3) can delete large fragments The ApoE gene sequence is to ensure that its function is completely deleted, and the 2-4 exon regions of the ApoE gene will be deleted after the homologous recombination of the present invention is completed.
附图说明Description of drawings
图1为本发明的小型猪ApoE基因敲除载体pApoEKO-DTA-M酶切鉴定电泳图。Fig. 1 is the electrophoresis diagram of the enzyme digestion identification of the minipig ApoE gene knockout vector pApoEKO-DTA-M of the present invention.
图2为打靶载体pApoEKO-DTA-M结构示意图。Fig. 2 is a schematic diagram of the structure of the targeting vector pApoEKO-DTA-M.
图3为ApoE基因敲除后的小型猪成纤维细胞系。Fig. 3 is the minipig fibroblast cell line after ApoE gene knockout.
图4为打靶载体pApoEKO-DTA-M转染后获得的ApoE基因敲除小型猪成纤维细胞系鉴定结果。图中25为同批次克隆细胞混合物，结果表明筛选的克隆中有阳性克隆，31、37、46为该批次挑取的克隆号，结果表明37号为阳性中靶克隆，而31和46号为阴性克隆，“+”条带为建立的阳性样品，能扩增出来说明已经发生同源重组，目的片段已经插入细胞中。Fig. 4 is the identification result of the ApoE gene knockout minipig fibroblast cell line obtained after transfection of the targeting vector pApoEKO-DTA-M. 25 in the figure is the mixture of cloned cells of the same batch. The results show that there are positive clones among the screened clones. 31, 37, and 46 are the clone numbers picked in this batch. The results show that No. 37 is a positive target clone, while 31 and 46 The number is a negative clone, and the "+" band is an established positive sample, which can be amplified to indicate that homologous recombination has occurred and the target fragment has been inserted into the cell.
图5为获得的ApoE敲除小型猪基因型鉴定。图中1、2、3、4、12、14为出生小型猪耳号，阴性对照组为未敲除目的基因的小型猪基因组。Figure 5 is the genotype identification of the obtained ApoE knockout miniature pigs. 1, 2, 3, 4, 12, and 14 in the figure are the ear numbers of the born minipigs, and the negative control group is the minipig genome without knocking out the target gene.
具体实施方式detailed description
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。本发明实施例中的各类限制性内切酶以及连接酶购自NEB、使用的其它试剂均从Sigma购买，打靶载体ploxp为实验室保存，该载体的构建方法已在文献X.Yang,C.Li,X.Xu,C.Deng,Proc Natl Acad SciUSA 95,3667，Mar 31,1998中公开。Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All kinds of restriction endonucleases and ligases in the examples of the present invention were purchased from NEB, and other reagents used were purchased from Sigma. The targeting vector ploxp was preserved in the laboratory. The construction method of this vector has been described in the literature X.Yang, C Published in Li, X. Xu, C. Deng, Proc Natl Acad Sci USA 95, 3667, Mar 31, 1998.
本发明所述的小型猪为中国农业大学涿州小型猪厂饲养的贵州小香猪。实施例中所述的小型猪基因组DNA为根据常规方法从贵州小香猪肌肉组织中提取得到的基因组DNA。The miniature pigs described in the present invention are Guizhou miniature pigs raised by the Zhuozhou Miniature Pig Factory of China Agricultural University. The minipig genomic DNA described in the examples is the genomic DNA extracted from the muscle tissue of Guizhou Minipig according to conventional methods.
实施例1 小型猪ApoE基因侧翼序列长臂和短臂的获得Example 1 Obtaining the long arm and short arm of the flanking sequence of the minipig ApoE gene
分别设计小型猪ApoE基因侧翼序列长臂Larm和短臂Sarm的上、下游引物Design the upstream and downstream primers of the long arm Larm and short arm Sarm of the flanking sequence of minipig ApoE gene
以小型猪基因组DNA为模板，分别进行PCR扩增基因小型猪ApoE基因侧翼序列长臂Larm和短臂Sarm。The long arm Larm and short arm Sarm of the flanking sequences of the minipig ApoE gene were amplified by PCR using the minipig genomic DNA as a template.
扩增长臂的PCR反应为20μL反应体系：上下游引物各0.6μL，模板DNA1.0μL，5×GCBuffer 4.0μL，dNTPs0.6μL，KAPA Hifi Taq 0.4μL，灭菌ddH2O 12.8μL。The PCR reaction for amplifying the long arm is a 20 μL reaction system: 0.6 μL of upstream and downstream primers, 1.0 μL of template DNA, 4.0 μL of 5×GCBuffer, 0.6 μL of dNTPs, 0.4 μL of KAPA Hifi Taq, and 12.8 μL of sterilized ddH2 O.
PCR反应条件：95℃预变性5min；95℃变性20s，62℃退火15s，72℃延伸5min，共35个循环；72℃5min。PCR reaction conditions: pre-denaturation at 95°C for 5min; denaturation at 95°C for 20s, annealing at 62°C for 15s, extension at 72°C for 5min, a total of 35 cycles; 5min at 72°C.
扩增短臂的PCR反应为20μL反应体系：上下游引物各0.6μL，模板DNA1.0μL，5×GCBuffer 4.0μL，dNTPs0.6μL，KAPA Hifi Taq 0.4μL，灭菌ddH2O 12.8μL。The PCR reaction for amplifying the short arm is a 20 μL reaction system: 0.6 μL of upstream and downstream primers, 1.0 μL of template DNA, 4.0 μL of 5×GCBuffer, 0.6 μL of dNTPs, 0.4 μL of KAPA Hifi Taq, and 12.8 μL of sterilized ddH2 O.
PCR反应条件：95℃预变性5min；95℃变性20s，62℃退火15s，72℃延伸2min，共30个循环；72℃5min。PCR reaction conditions: pre-denaturation at 95°C for 5min; denaturation at 95°C for 20s, annealing at 62°C for 15s, extension at 72°C for 2min, a total of 30 cycles; 5min at 72°C.
分别将PCR产物进行琼脂糖凝胶电泳后，利用DNA凝胶回收试剂盒回收获得ApoE基因长臂和短臂，并将它们连接到pEASY-T载体上，连接产物分别转化大肠杆菌DH5α，分别挑取连接阳性菌落进行菌体扩培和质粒提取，获得ApoE基因同源臂pEASY-Larm和pEASY-Sarm两种质粒。After the PCR products were subjected to agarose gel electrophoresis, the long and short arms of the ApoE gene were recovered using a DNA gel recovery kit, and they were ligated to the pEASY-T vector. The ligated products were transformed into E. The connection-positive colonies were taken for cell expansion and plasmid extraction to obtain two plasmids, pEASY-Larm and pEASY-Sarm, which are homologous arms of the ApoE gene.
实施例2 打靶载体pApoEKO-DTA-M的构建Example 2 Construction of targeting vector pApoEKO-DTA-M
1、短臂质粒pEASY-Sarm与打靶载体ploxp的连接得到ploxp-Sarm1. Ligation of short arm plasmid pEASY-Sarm and targeting vector ploxp to obtain ploxp-Sarm
打靶载体ploxp和短臂质粒载体pEASY-Sarm分别用EcoRI和SalI双酶切，DNA产物纯化回收试剂盒回收ploxp载体的酶切片段，对pEASY-Sarm质粒载体酶切产物用琼脂糖凝胶试剂盒回收2.4kb短臂片段，回收后按照ploxp质粒与短臂片段1:3的比例用T4连接酶连接，转化Trans-1感受态细胞后挑取单克隆测序鉴定，进行DNA测序分析，结果表明短臂Sarm成功连接入打靶载体ploxp。The targeting vector ploxp and the short arm plasmid vector pEASY-Sarm were digested with EcoRI and SalI respectively, the DNA product purification and recovery kit was used to recover the digested fragment of the ploxp vector, and the digested product of the pEASY-Sarm plasmid vector was digested with an agarose gel kit The 2.4kb short arm fragment was recovered, and after recovery, it was ligated with T4 ligase according to the ratio of ploxp plasmid and short arm fragment at 1:3, transformed into Trans-1 competent cells, picked out monoclonal sequencing identification, and carried out DNA sequencing analysis, the results showed that the short arm fragment Arm Sarm was successfully connected into the targeting vector ploxp.
2、长臂质粒pEASY-Larm与ploxp-Sarm的连接得到ploxp-Sarm-Larm载体2. Ligation of long arm plasmid pEASY-Larm and ploxp-Sarm to obtain ploxp-Sarm-Larm vector
阳性克隆质粒和长臂质粒载体pEASY-Larm分别用NotI单酶切，DNA产物纯化回收ploxp-Sarm质粒片段，琼脂糖凝胶回收pEASY-Larm酶切下来的5.7kb长臂片段，回收产物用碱性磷酸酶处理后再次纯化回收，回收获得的片段按照质粒与长臂DNA片段摩尔比1:3的比例用T4连接酶连接，连接反应完成后转化Trans-1感受态细胞，培养24h后挑取单克隆菌落进行PCR鉴定，阳性克隆提取质粒用于酶切鉴定，酶切鉴定正确的样品送样测序。测序正确质粒即为目的ploxp-Sarm-Larm打靶载体。The positive clone plasmid and the long-arm plasmid vector pEASY-Larm were digested with NotI, the DNA product was purified and the ploxp-Sarm plasmid fragment was recovered, and the 5.7kb long-arm fragment excised by pEASY-Larm was recovered on agarose gel, and the product was recovered with alkali Purified and recovered after treatment with phosphatase, the recovered fragments were ligated with T4 ligase according to the molar ratio of plasmid and long-arm DNA fragments of 1:3, transformed into Trans-1 competent cells after the ligation reaction was completed, and picked after 24 hours of culture Monoclonal colonies were identified by PCR, positive clones extracted plasmids for enzyme digestion identification, and samples that were correctly identified by enzyme digestion were sent for sequencing. The correct sequenced plasmid is the target ploxp-Sarm-Larm targeting vector.
ploxp-Sarm-Larm打靶载体上tk基因替换:tk gene replacement on the ploxp-Sarm-Larm targeting vector:
以tk作为负筛选基因需要加入丙氧鸟苷(GANC)，但是添加的剂量需要根据实验的细胞类型进行耐受实验，因此会增加实验难度和降低筛选效率。通过直接插入白喉毒素dta基因可以提高筛选效率。将打靶载体ploxp-Sarm-Larm用HindⅢ和Mlu Ⅰ对载体进行酶切，1％琼脂糖凝胶电泳后切胶回收目的片段。用PCR方法从PGKdtabpA(Addgene plasmid#13440)中扩增dta的表达序列(引物序列：DTA-MLU1-F：GCTTCGTCAGACGCGTCTCGAGGTCGAGCCCCAGCT/DTA-HIND3-R：TTCGAGAACTAAGCTTAGGGGTTCCGCGCACATTTCC)扩增出的含dta基因的片段经过HindⅢ和Mlu Ⅰ双酶切后回收酶切片段。将ploxp-Sarm-Larm酶切胶回收产物与dta基因片段酶切产物以摩尔比1:3的比例混合用T4DNA连接酶进行连接。连接后获得打靶载体pApoEKO-DTA。Using tk as a negative selection gene requires the addition of GANC (GANC), but the added dose needs to be tested according to the cell type of the experiment, which will increase the difficulty of the experiment and reduce the screening efficiency. Screening efficiency can be increased by direct insertion of the diphtheria toxin dta gene. The targeting vector ploxp-Sarm-Larm was digested with HindⅢ and MluI, and the target fragment was recovered after 1% agarose gel electrophoresis. The expression sequence of dta was amplified from PGKdtabpA (Addgene plasmad #13440) by PCR (primer sequence: DTA-MLU1-F: GCTTCGTCAGACGCGTCTCGAGGTCGAGCCCCAGCT/DTA-HIND3-R: TTCGAGAACTAAGCTTAGGGGTTCCGCGCACATTTCC), and the amplified dta gene-containing fragment was subjected to HindⅢ and Recover the digested fragments after Mlu I double digestion. The ploxp-Sarm-Larm digestion gel recovery product was mixed with the dta gene fragment digestion product at a molar ratio of 1:3 and ligated with T4 DNA ligase. After ligation, the targeting vector pApoEKO-DTA was obtained.
3、修饰pApoEKO-DTA打靶载体得到打靶载体pApoEKO-DTA-M3. Modify the pApoEKO-DTA targeting vector to obtain the targeting vector pApoEKO-DTA-M
为了加速获得去除标记基因的打靶模型，利用同源重组的原理将pApoEKO-DTA载体的原有Neo基因前端添加猪精原细胞特异启动的Cre基因，其序列为SEQ ID NO.1的第8021～第9139位。采用基因抓捕方法，设计同源臂引物CR-F和CR-R(即普通引物后加上一段100bp的同源序列)用于同源重组。引物CR-F和CR-R的核苷酸序列分别如SEQ ID NO.6、7所示。In order to accelerate the acquisition of a targeting model that removes the marker gene, the original Neo gene of the pApoEKO-DTA vector was added to the front end of the original Neo gene of the pApoEKO-DTA vector by the principle of homologous recombination, and the Cre gene specifically activated by porcine spermatogonia was added. 9139th. The gene capture method was used to design the homology arm primers CR-F and CR-R (that is, a 100 bp homologous sequence added after the common primer) for homologous recombination. The nucleotide sequences of the primers CR-F and CR-R are shown in SEQ ID NO.6 and 7, respectively.
以ptACE载体为模板，先用引物CR-F和CR-R扩增出同源重组的目的片段(包含Cre基因以及启动子区和Neo抗性筛选基因)并用DNA产物回收试剂盒回收目的片段。吸取待打靶载体pApoEKO-DTA 10ng和上一步骤中DNA产物回收试剂盒回收到的PCR产物600ng混合后电转SW102大肠杆菌感受态细胞。电转后涂LB板培养，挑取较大的单克隆菌落提取质粒酶切鉴定，鉴定结果见图1。鉴定正确的质粒为最终的打靶载体pApoEKO-DTA-M，图谱见图2。Using the ptACE vector as a template, first use primers CR-F and CR-R to amplify the target fragment of homologous recombination (including Cre gene, promoter region and Neo resistance screening gene) and recover the target fragment with a DNA product recovery kit. Pipette 10 ng of the target vector pApoEKO-DTA and 600 ng of the PCR product recovered by the DNA product recovery kit in the previous step, mix and electroporate SW102 Escherichia coli competent cells. After electroporation, smear LB plates for culture, pick larger single clone colonies, extract plasmids, and identify them by enzyme digestion. The identification results are shown in Figure 1. The correct plasmid was identified as the final targeting vector pApoEKO-DTA-M, and the map is shown in Figure 2.
ptACE载体构建方法为：用PCR方法(引物序列：CRE-NHE1-F：GCTTCGTCAGAACAAACGACCCAACACCCGTGC/CRE-R：CTCTTCTTCTTGGGCATAGTAGGAGCAGGGCAGTC)从猪基因组中扩增出angiotensin-converting enzyme基因5’启动子区(组织特异性启动子序列)，同时用引物(CRE-F：GACTGCCCTGCTCCTACTATGCCCAAGAAGAAGAG；CRE-NHE1-R：TTCGAGAACTGGATCTCGGGGCAAGACAAATGTC)以ploxp上的Cre基因为模板扩增Cre基因片段。利用重叠PCR原理将组织特异性启动子序列和Cre基因片段连接，连接后用引物中添加的NheⅠ酶切位点(下划线加粗部分)插入PL452骨架质粒(购自Addgene)的Nhe Ⅰ酶切位点中获得。ptACE carrier construction method is: use PCR method (primer sequence: CRE-NHE1-F: GCTTCGTCAG AACAAACGACCCAACACCCGTGC/CRE-R: CTCTTCTTCTTGGGCATAGTAGGAGCAGGGCAGTC) amplified the 5' promoter region of angiotensin-converting enzyme gene (tissue-specific promoter sequence) from the pig genome, and used primers (CRE-F: GACTGCCCTGCTCCTACTATGCCCAAGAAGAAGAG; CRE-NHE1-R: TTCGAGAACT GGATCTCGGGGCAAGACAAATGTC) uses the Cre gene on ploxp as a template to amplify the Cre gene fragment. The tissue-specific promoter sequence and the Cre gene fragment were ligated using the principle of overlapping PCR, and the NheI restriction site added in the primer (underlined bold part) was inserted into the NheI restriction site of the PL452 backbone plasmid (purchased from Addgene) after ligation Points obtained.
实施例3 载体pApoEKO-DTA-M的打靶实例Example 3 Targeting example of vector pApoEKO-DTA-M
1、打靶载体转染成纤维细胞1. Targeting vector transfected into fibroblasts
利用抗性基因Amp内的单酶切位点ScaI线性化pApoEKO-DTA-M质粒，37℃酶切3-5h后乙醇沉淀回收目的片段，目的片段溶于ddH2O后调整浓度约1μg/μL备用。Linearize the pApoEKO-DTA-M plasmid using the single enzyme cutting site ScaI in the resistance gene Amp, digest at 37°C for 3-5 hours, and recover the target fragment by ethanol precipitation. Dissolve the target fragment in ddH2 O and adjust the concentration to about 1 μg/μL spare.
2、细胞系转染以验证其功能2. Cell line transfection to verify its function
转染从中国农业大学涿州小型猪基地分离的贵州小香猪成纤维细胞系，用含G418的培养基对转染细胞进行10天左右的筛选获得抗性细胞克隆点。消化选取部分细胞克隆点提取DNA进行跨长短臂PCR鉴定，用跨短臂引物(SARM-S-F：GGCTTTGGTTCCAGAGTTCACAGTC；CRE-NHE1-R:TTCGAGAACTGCTAGCGGATCTCGGGGCAAGACAAATGTC)PCR扩增鉴定中靶克隆，鉴定的阳性克隆点进一步测序验证，获得ApoE基因敲除的小型猪成纤维细胞系图3，PCR鉴定结果见图4，图中结果表明37号单克隆样本为阳性打靶细胞系，说明已经发生同源重组，目的片段已经插入细胞中。Transfect the Guizhou minipig fibroblast cell line isolated from the Zhuozhou miniature pig base of China Agricultural University, and use G418-containing medium to screen the transfected cells for about 10 days to obtain resistant cell clones. Digest and select some cell clones to extract DNA for cross-long and short-arm PCR identification, use cross-short-arm primers (SARM-S-F: GGCTTTGGTTCCAGAGTTCACAGTC; CRE-NHE1-R: TTCGAGAACTGCTAGCGGATCTCGGGGCAAGACAAATGTC) PCR amplification to identify target clones, and identify positive clones for further sequencing Verify that the ApoE gene knockout minipig fibroblast cell line was obtained in Figure 3, and the PCR identification results are shown in Figure 4. The results in the figure show that the No. 37 monoclonal sample is a positive target cell line, indicating that homologous recombination has occurred and the target fragment has been inserted in cells.
实施例4 ApoE基因敲除小型猪制备及鉴定Example 4 Preparation and Identification of ApoE Gene Knockout Minipigs
用体细胞核移植的方法将鉴定为阳性的成纤维细胞的细胞核作为供体转移至受体卵母细胞中，获得的核移植胚胎通过胚胎移植手术注入发情期母猪子宫内妊娠。总共移植1240枚胚胎，移植14头母猪，其中三头母猪成功妊娠。获得6头F0代小型猪个体，用跨短臂引物(SARM-S-F：GGCTTTGGTTCCAGAGTTCACAGTC/CRE-NHE1-R:TTCGAGAACTGCTAGCGGATCTCGGGGCAAGACAAATGTC)以6头F0代个体基因组为模板进行常规PCR扩增鉴定，能够扩增出5kb大小条带的个体为重组后的敲除猪，鉴定结果见图5，由此可见鉴定的6个样本均为阳性。The nuclei of fibroblasts identified as positive are transferred to recipient oocytes by somatic cell nuclear transfer, and the obtained nuclear transfer embryos are injected into the uterus of estrous sows for pregnancy. A total of 1240 embryos were transferred and 14 sows were transferred, of which three sows were successfully pregnant. 6 F0 generation miniature pigs were obtained, and the genomes of the 6 F0 generation individuals were used as templates for conventional PCR amplification identification using short-arm primers (SARM-S-F: GGCTTTGGTTCCAGAGTTCACAGTC/CRE-NHE1-R: TTCGAGAACTGCTAGCGGATCTCGGGGCAAGACAAATGTC), and 5kb could be amplified Individuals with large and small bands are recombined knockout pigs, and the identification results are shown in Figure 5. It can be seen that all 6 samples identified are positive.
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