技术领域technical field
本发明涉及一种孕烷X受体转基因小鼠模型，以及所述模型的构建方法及其应用，属于生物化学领域。The invention relates to a pregnane X receptor transgenic mouse model, a construction method and application of the model, and belongs to the field of biochemistry.
背景技术Background technique
孕烷X受体(Pregnane X Receptor,PXR)是属于核受体超家族的一种受体蛋白，按核受体系统命名为NR1I2，与组成型雄烷受体(Constitutive Androstane Receptor,CAR)构成机体对环境外源性化学物的主要感受器。人孕烷X受体(hPXR)基因鉴定于1998年，在各器官广泛表达，尤其在肝脏、肠组织中高表达。目前已知的PXR生物效应包括：调节代谢酶和转运蛋白基因表达水平；能量代谢调节；内源代谢物及内分泌激素表达水平调节；炎症反应调节等。基于上述重要作用，2007年美国国家研究委员会(NRC)发表的《二十一世纪毒性测试：策略及展望》中，PXR活化作用被列为体外测试毒性途径之一。在药物研发、化学品毒性检测中，需要对化学物是否影响PXR活性进行筛选。Pregnane X Receptor (PXR) is a receptor protein belonging to the nuclear receptor superfamily, named NR1I2 according to the nuclear receptor system, and constitutes the constitutive androstane receptor (CAR) The body's main receptors for exogenous chemicals in the environment. The human pregnane X receptor (hPXR) gene was identified in 1998 and is widely expressed in various organs, especially highly expressed in liver and intestinal tissues. The currently known biological effects of PXR include: regulating the expression levels of metabolic enzymes and transporter genes; regulating energy metabolism; regulating the expression levels of endogenous metabolites and endocrine hormones; regulating inflammatory responses, etc. Based on the important role mentioned above, PXR activation was listed as one of the in vitro toxicity testing pathways in the "21st Century Toxicity Testing: Strategies and Prospects" published by the National Research Council (NRC) in 2007. In drug development and chemical toxicity testing, it is necessary to screen whether chemicals affect PXR activity.
国内外已构建的hPXR转基因小鼠仅导入外显子基因序列，不能表达hPXR的剪接变异体，其基因上下游的调控序列未同时导入。因此，构建一种导入hPXR基因全长序列的转基因小鼠具有更多的人源特性和应用前景。The hPXR transgenic mice that have been constructed at home and abroad only introduce the exon gene sequence, and cannot express the splice variant of hPXR, and the upstream and downstream regulatory sequences of the gene are not simultaneously introduced. Therefore, the construction of a transgenic mouse into which the full-length hPXR gene sequence has more human characteristics and application prospects.
发明内容Contents of the invention
针对现有技术存在的问题，本发明的目的是提供一种导入hPXR基因全长序列的转基因小鼠模型，可表达hPXR主要的剪接变异体，表达水平接近生理水平，对hPXR特异性激活剂有特异性应答。In view of the problems existing in the prior art, the purpose of the present invention is to provide a transgenic mouse model that introduces the full-length sequence of hPXR gene, which can express the main splice variants of hPXR, the expression level is close to the physiological level, and has a certain effect on hPXR specific activators. specific response.
本发明的另一个目的是提供构建所述转基因小鼠模型的方法。Another object of the present invention is to provide a method for constructing the transgenic mouse model.
本发明的再一个目的是提供所述转基因小鼠模型在hPXR激活剂/减活性剂的快速筛选或体内验证中的应用。Another object of the present invention is to provide the application of the transgenic mouse model in the rapid screening or in vivo verification of hPXR activators/inactivators.
为实现上述目的，本发明采取以下技术方案：To achieve the above object, the present invention takes the following technical solutions:
首先，提供一种人孕烷X受体转基因小鼠模型，它是由人PXR转基因鼠与鼠孕烷X受体敲除小鼠PXR-KO交配繁殖得到的小鼠；其中，所述的人PXR转基因鼠是转入了含人孕烷X受体全基因序列的细菌人工染色体片段的小鼠。First, a human pregnane X receptor transgenic mouse model is provided, which is a mouse obtained by mating a human PXR transgenic mouse with a mouse pregnane X receptor knockout mouse PXR-KO; wherein, the human PXR transgenic mice are mice that have been transformed with a bacterial artificial chromosome fragment containing the full gene sequence of the human pregnane X receptor.
本发明所述的人孕烷X受体转基因小鼠模型不表达鼠源PXR，仅表达人源PXR。The human pregnane X receptor transgenic mouse model of the present invention does not express mouse PXR, but only human PXR.
本发明还提供一种构建所述人孕烷X受体转基因小鼠模型的方法，包括：构建鼠孕烷X受体敲除小鼠PXR-KO，以含人孕烷X受体全基因序列的细菌人工染色体片段构建人PXR转基因鼠，用所述鼠孕烷X受体敲除小鼠PXR-KO与所述人PXR转基因鼠种进行交配繁殖，得到所述的人孕烷X受体转基因小鼠模型。The present invention also provides a method for constructing the human pregnane X receptor transgenic mouse model, comprising: constructing the mouse pregnane X receptor knockout mouse PXR-KO to contain the full gene sequence of the human pregnane X receptor Human PXR transgenic mice were constructed from bacterial artificial chromosome fragments, and the mouse pregnane X receptor knockout mouse PXR-KO was used to mate with the human PXR transgenic mice to obtain the human pregnane X receptor transgene mouse model.
本发明优选的所述构建方法中，所述的构建鼠孕烷X受体敲除小鼠PXR-KO应用CRISPR-Cas9技术实现。In the preferred construction method of the present invention, the construction of mouse pregnane X receptor knockout mouse PXR-KO is realized by CRISPR-Cas9 technology.
本发明优选的所述构建方法中，所述的含人孕烷X受体全基因序列的细菌人工染色体片段为RP11-937I6细菌人工染色体片段，其核苷酸序列如SEQ ID NO.2所示。In the preferred construction method of the present invention, the bacterial artificial chromosome fragment containing the full gene sequence of the human pregnane X receptor is a RP11-937I6 bacterial artificial chromosome fragment, and its nucleotide sequence is shown in SEQ ID NO.2 .
本发明优选的所述构建方法中，所使用的鼠的品系优选C57BL/6J，其基因组PXR序列如SEQ ID NO.1所示。In the preferred construction method of the present invention, the mouse strain used is preferably C57BL/6J, whose genome PXR sequence is shown in SEQ ID NO.1.
本发明最优选的所述构建方法，具体包括如下步骤：The most preferred construction method of the present invention specifically includes the following steps:
1)应用CRISPR-Cas9技术构建鼠孕烷X受体敲除小鼠PXR-KO；1) Using CRISPR-Cas9 technology to construct mouse pregnane X receptor knockout mouse PXR-KO;
2)将RP11-937I6细菌人工染色体片段(其核苷酸序列如SEQ ID NO.2所示)注射到C57BL/6J小鼠受精卵的原核中，将处理后的所述受精卵移植入代孕母鼠中发育，出生后得到F0代嵌合体小鼠；2) Inject the RP11-937I6 bacterial artificial chromosome fragment (its nucleotide sequence is shown in SEQ ID NO.2) into the pronucleus of fertilized eggs of C57BL/6J mice, and transplant the fertilized eggs into surrogate mothers after treatment Developed in mice, and obtained F0 generation chimera mice after birth;
3)将2)中获得的所述F0代嵌合体小鼠与1)中得到的PXR-KO小鼠进行杂交得到F1代小鼠，从F1代小鼠中获得转入了所述RP11-937I6细菌人工染色体片段的杂合体小鼠，即为hPXR转基因鼠。3) Cross the F0 generation chimeric mice obtained in 2) with the PXR-KO mice obtained in 1) to obtain F1 generation mice, and obtain the RP11-937I6 transferred from the F1 generation mice The heterozygous mice for the bacterial artificial chromosome fragments are hPXR transgenic mice.
进一步优选的构建方法中，步骤1)、步骤2)和步骤3)分别进一步包含通过PCR扩增及对PCR扩增DNA产物进行测序来选择阳性基因型小鼠。In a further preferred construction method, step 1), step 2) and step 3) respectively further include selecting positive genotype mice by PCR amplification and sequencing the PCR amplified DNA product.
本发明还提供所述孕烷X受体转基因小鼠模型在人孕烷X受体激活剂或减活性剂的快速筛选或体内验证中的应用。The present invention also provides the application of the pregnane X receptor transgenic mouse model in rapid screening or in vivo verification of human pregnane X receptor activators or inactivators.
本发明的有益效果在于：本发明繁育出的孕烷X受体转基因小鼠表达hPXR主要的剪接变异体，表达水平接近生理水平，对hPXR特异性激活剂有特异性应答。所以，可以用于工业原料化学品、小分子化学药、农药等化学物是否为hPXR激活剂/减活性剂的快速筛选和体内验证，具有广泛的应用前景。The beneficial effect of the present invention is that: the pregnane X receptor transgenic mouse bred by the present invention expresses the main splicing variant of hPXR, the expression level is close to the physiological level, and has a specific response to the hPXR specific activator. Therefore, it can be used for rapid screening and in vivo verification of whether industrial raw materials chemicals, small molecule chemicals, pesticides and other chemicals are hPXR activators/deactivators, and has broad application prospects.
附图说明Description of drawings
图1为本发明实施例中步骤1.5所述序列在小鼠Nr1i2基因组中的位置示意图。Fig. 1 is a schematic diagram of the position of the sequence described in step 1.5 in the example of the present invention in the mouse Nr1i2 genome.
图2体现了应用本发明实施例方法构建的小鼠PXR剪接变异体丰度。Fig. 2 shows the abundance of splice variants of mouse PXR constructed by applying the method of the embodiment of the present invention.
图3体现了应用本发明实施例方法构建的小鼠PXR表达水平。Fig. 3 shows the expression level of mouse PXR constructed by applying the method of the embodiment of the present invention.
图4和图5均体现了应用本发明实施例方法构建小鼠对种属特异性激活剂的应答。Figure 4 and Figure 5 both reflect the application of the method of the embodiment of the present invention to construct the response of the mouse to the species-specific activator.
具体实施方式Detailed ways
下面通过实施例结合附图进一步说明本发明。The present invention is further illustrated below by means of embodiments in conjunction with the accompanying drawings.
本发明建立了不表达鼠源PXR，仅表达人源PXR的小鼠，其表达hPXR主要的剪接变异体，表达水平接近生理水平，对hPXR特异性激活剂有特异性应答。The present invention establishes a mouse that does not express murine PXR but only expresses human PXR, expresses the main splice variant of hPXR, the expression level is close to the physiological level, and has specific response to hPXR specific activator.
实施例Example
构建人PXR转基因小鼠及其化学物PXR激活作用鉴别应用：Construction of human PXR transgenic mice and their chemical PXR activation identification applications:
1.应用CRISPR-Cas9技术构建鼠孕烷X受体敲除小鼠PXR-KO；1. Using CRISPR-Cas9 technology to construct mouse pregnane X receptor knockout mouse PXR-KO;
1.1.构建CRISPRKo-Cas9打靶载体：根据C57BL/6J鼠基因组PXR序列(SEQ IDNO.1)设计gRNA打靶位点，并分别构建成CRISPRKo-Cas9打靶载体：1.1. Construction of CRISPRKo-Cas9 targeting vector: Design gRNA targeting sites according to the C57BL/6J mouse genome PXR sequence (SEQ ID NO.1), and construct CRISPRKo-Cas9 targeting vectors respectively:
gRNA1:5’-GACGCTCTACTTTAAGGTCA-3’(SEQ ID NO.3)gRNA1: 5'-GACGCTCTACTTTAAGGTCA-3' (SEQ ID NO.3)
gRNA2:5’-TCCCAGGGTTTCTAGTAGTC-3’(SEQ ID NO.4)gRNA2: 5'-TCCCAGGGTTTCTAGTAGTC-3' (SEQ ID NO.4)
1.2.CRISPR-Cas9基因敲除受精卵制备：通过体外转录将Cas9mRNA和gRNA质粒人工注射到小鼠受精卵中，并将受精卵回输到代孕鼠子宫；1.2. Preparation of CRISPR-Cas9 gene knockout fertilized eggs: artificially inject Cas9 mRNA and gRNA plasmids into mouse fertilized eggs by in vitro transcription, and return the fertilized eggs to the uterus of surrogate mice;
1.3.代孕并获得阳性建系鼠：代孕鼠生育小鼠后，剪取部分鼠尾通过PCR扩增并对扩增产物做DNA测序来选择阳性基因型小鼠；1.3. Surrogate and obtain positive establishment mice: After the surrogate mice give birth to mice, cut part of the mouse tail and amplify by PCR and perform DNA sequencing on the amplified product to select positive genotype mice;
1.4.繁育后代：对后代小鼠基因型的分析依旧通过PCR扩增并对扩增产物做DNA测序来确定。1.4. Breeding offspring: The genotype analysis of offspring mice is still determined by PCR amplification and DNA sequencing of the amplified products.
1.4.1.F0代获得两只阳性且都为雄性的建系鼠，分别记为F0Mouse-ID#18,F0Mouse-ID#24；1.4.1. The F0 generation obtained two positive and male establishment mice, respectively recorded as F0Mouse-ID#18 and F0Mouse-ID#24;
1.4.2.将F0建系雄鼠分别与雌性野生型C57BL/6J小鼠杂交【C57BL/6(Mouse-ID#18)×C57BL/6(WT)；C57BL/6(Mouse-ID#24)×C57BL/6(WT)】获得F1代小鼠，经鼠尾DNA PCR扩增并测序鉴定基因型各获得以下两种小鼠，分别记为：F0Mouse-ID#18-F1-Mouse-ID#2,#3,#4和F0Mouse-ID#24-F1-Mouse-ID#12,#17,#18；1.4.2. Crossbreed F0 established male mice with female wild-type C57BL/6J mice [C57BL/6(Mouse-ID#18)×C57BL/6(WT); C57BL/6(Mouse-ID#24) ×C57BL/6(WT)] F1 generation mice were obtained, and the genotypes were identified by PCR amplification and sequencing of mouse tail DNA to obtain the following two types of mice, respectively denoted as: F0Mouse-ID#18-F1-Mouse-ID# 2, #3, #4 and F0Mouse-ID#24-F1-Mouse-ID#12, #17, #18;
1.4.3.F1代小鼠单个品系组内杂交获得纯合的Nr1i2基因敲除小鼠；1.4.3. Homozygous Nr1i2 knockout mice were obtained by interbreeding within a single strain of F1 mice;
1.4.4.F1代经PCR产物DNA测序鉴定F0Mouse-ID#18-F1-Mouse-ID#2,#3,#4单链中有999碱基缺失，而F0Mouse-ID#24-F1-Mouse-ID#12,#17,#18单链中则有1038碱基缺失；1.4.4. The F1 generation was identified by DNA sequencing of PCR products, with 999 bases missing in the single strands of F0Mouse-ID#18-F1-Mouse-ID#2, #3, and #4, while F0Mouse-ID#24-F1-Mouse - There are 1038 base deletions in the single strands of ID#12, #17, and #18;
1.5.本构建步骤中设计PCR引物、DNA测序引物、gRNA靶点序列信息如下：1.5. In this construction step, design PCR primers, DNA sequencing primers, and gRNA target sequence information as follows:
PCR引物Mouse Nr1i2-F:5’-GAGAGCAAAGCATTCAGGGTACAGATT-3(SEQ ID NO.5)PCR primer Mouse Nr1i2-F:5'-GAGAGCAAAGCATTCAGGGTACAGATT-3 (SEQ ID NO.5)
PCR引物Mouse Nr1i2-R:5’-GCACTGGGAGCAAAGACAGAGCTAG-3’(SEQ ID NO.6)PCR primer Mouse Nr1i2-R:5'-GCACTGGGAGCAAAGACAGAGCTAG-3' (SEQ ID NO.6)
gRNA靶点序列1:5’-GACGCTCTACTTTAAGGTCA-3’(SEQ ID NO.3)gRNA target sequence 1: 5'-GACGCTCTACTTTAAGGTCA-3' (SEQ ID NO.3)
gRNA靶点序列2:5’-TCCCAGGGTTTCTAGTAGTC-3’(SEQ ID NO.4)gRNA target sequence 2: 5'-TCCCAGGGTTTCTAGTAGTC-3' (SEQ ID NO.4)
DNA测序引物(Forward Sequencing):5’-GGAGAAACACATAGAAACCCATCCA-3’(SEQID NO.7)DNA sequencing primer (Forward Sequencing): 5'-GGAGAAACACATAGAAACCCATCCA-3'(SEQID NO.7)
上述基因序列在小鼠Nr1i2基因组中的位置如图1所示。其中，P1代表上述PCR引物Mouse Nr1i2-F，P2代表上述PCR引物Mouse Nr1i2-R，G1代表上述gRNA靶点序列1，G2代表上述gRNA靶点序列2，D代表上述DNA测序引物。The positions of the above gene sequences in the mouse Nr1i2 genome are shown in FIG. 1 . Among them, P1 represents the above-mentioned PCR primer Mouse Nr1i2-F, P2 represents the above-mentioned PCR primer Mouse Nr1i2-R, G1 represents the above-mentioned gRNA target sequence 1, G2 represents the above-mentioned gRNA target sequence 2, and D represents the above-mentioned DNA sequencing primer.
2.将RP11-937I6细菌人工染色体片段(核苷酸序列如SEQ ID NO.2所示)注射到C57BL/6J小鼠受精卵的原核中，将处理后的所述受精卵移植入代孕母鼠中发育，出生后得到F0代嵌合体小鼠；2. Inject the RP11-937I6 bacterial artificial chromosome fragment (nucleotide sequence as shown in SEQ ID NO.2) into the pronucleus of fertilized eggs of C57BL/6J mice, and transplant the fertilized eggs into surrogate mother mice after treatment The F0 generation chimera mice were obtained after birth;
2.1.将RP11-937I6BAC菌液无内毒素大提，获得高纯度显微注射用DNA；2.1. Extract the RP11-937I6BAC bacterial liquid without endotoxin to obtain high-purity DNA for microinjection;
2.2.将步骤a无内毒素提取的RP11-937I6细菌人工染色体片段DNA人工注射到小鼠受精卵中，并将受精卵回输到代孕鼠子宫；2.2. Artificially inject the RP11-937I6 bacterial artificial chromosome fragment DNA extracted without endotoxin in step a into fertilized eggs of mice, and return the fertilized eggs to the uterus of surrogate mice;
2.3.代孕并获得阳性建系鼠：代孕鼠生育小鼠后，剪取部分鼠尾通过PCR扩增来确定BAC阳性转基因型小鼠，进一步确认可对PCR扩增DNA产物进行测序。设计3组引物，其中3对由人来源NR1I2(GenBank accession number：8856)基因组序列设计的引物和2对小鼠Rgs7(GenBank accession number：24012)基因组序列设计的引物，每组分别以小鼠内源表达Rgs7(G protein signaling 7)做内参和人来源的RP11-937I6细菌人工染色体片段中DNA片段作为转基因组，三组引物分别如下：2.3. Surrogate and obtain positive establishment mice: After the surrogate mice give birth to mice, cut part of the mouse tail and amplify by PCR to determine the BAC-positive transgenic mice, and further confirm that the PCR-amplified DNA products can be sequenced. 3 sets of primers were designed, including 3 pairs of primers designed from human NR1I2 (GenBank accession number: 8856) genome sequence and 2 pairs of primers designed from mouse Rgs7 (GenBank accession number: 24012) genome sequence. The source expresses Rgs7 (G protein signaling 7) as an internal reference and the DNA fragment in the human-derived RP11-937I6 bacterial artificial chromosome fragment is used as a transgenome. The three sets of primers are as follows:
转基因hPXR PCR引物F1:GCCAGAAGGGATTTGAGAGATGAT(SEQ ID NO.8)Transgenic hPXR PCR primer F1: GCCAGAAGGGATTTGAGAGATGAT (SEQ ID NO.8)
转基因hPXR PCR引物R1:CAGTGGAAGTAGCCTGAACAGTG(SEQ ID NO.9)Transgenic hPXR PCR primer R1: CAGTGGAAGTAGCCTGAACAGTG (SEQ ID NO.9)
内参mRgs7PCR引物F1:GTGGCTTCAGGTTTGCTGTCAGTA(SEQ ID NO.10)Internal reference mRgs7 PCR primer F1: GTGGCTTCAGGTTTGCTGTCAGTA (SEQ ID NO.10)
内参mRgs7PCR引物R1CGCATCTGGAGCAGATAAGGAAAGAT(SEQ ID NO.11)Internal reference mRgs7 PCR primer R1CGCATCTGGAGCAGATAAGGAAAGAT (SEQ ID NO.11)
转基因PCR产物大小:317bpTransgenic PCR product size: 317bp
内参PCR产物大小:515bpInternal reference PCR product size: 515bp
转基因hPXR PCR引物F2:GTATTCCAGGTGCATTCCACAT(SEQ ID NO.12)Transgenic hPXR PCR primer F2: GTATTCCAGGTGCATTCCACAT (SEQ ID NO.12)
转基因hPXR PCR引物R2:CGGCAATAATGTGGAGAAACTGAG(SEQ ID NO.13)Transgenic hPXR PCR primer R2: CGGCAATAATGTGGAGAAACTGAG (SEQ ID NO.13)
内参mRgs7PCR引物F1:GTGGCTTCAGGTTTGCTGTCAGTA(SEQ ID NO.10)Internal reference mRgs7 PCR primer F1: GTGGCTTCAGGTTTGCTGTCAGTA (SEQ ID NO.10)
内参mRgs7PCR引物R1:CGCATCTGGAGCAGATAAGGAAAGAT(SEQ ID NO.11)Internal reference mRgs7 PCR primer R1: CGCATCTGGAGCAGATAAGGAAAGAT (SEQ ID NO.11)
转基因PCR产物大小:289bpTransgenic PCR product size: 289bp
内参PCR产物大小:515bpInternal reference PCR product size: 515bp
转基因hPXR PCR引物F3:GCCTTGTTTATAGCCACTTGTGAG(SEQ ID NO.14)Transgenic hPXR PCR primer F3: GCCTTGTTTATAGCCACTTGTGAG (SEQ ID NO.14)
转基因hPXR PCR引物R3:CTGTGTTAGCCAGGATGGTCTCGAT(SEQ ID NO.15)Transgenic hPXR PCR primer R3: CTGTGTTAGCCAGGATGGTCTCGAT (SEQ ID NO.15)
内参mRgs7PCR引物F2:GCAGAAGAGGACAGATACATTCAT(SEQ ID NO.16)Internal reference mRgs7 PCR primer F2: GCAGAAGAGGACAGATACATTCAT (SEQ ID NO.16)
内参mRgs7PCR引物R2:CCTACTGAAGAATCTATCCCACAG(SEQ ID NO.17)Internal reference mRgs7 PCR primer R2: CCTACTGAAGAATCTATCCCACAG (SEQ ID NO.17)
转基因PCR产物大小:485bpTransgenic PCR product size: 485bp
内参PCR产物大小:689bpInternal reference PCR product size: 689bp
3.将2中获得所述的F0代嵌合体小鼠与PXR-KO纯合小鼠进行杂交，从F1代小鼠中获得转入了所述RP11-937I6细菌人工染色体片段的杂合体小鼠，即为所述的人源化的PXR转基因鼠；3. Cross the F0 generation chimeric mice obtained in 2 with PXR-KO homozygous mice, and obtain the heterozygous mice that have been transferred to the RP11-937I6 bacterial artificial chromosome fragment from the F1 generation mice , which is the humanized PXR transgenic mouse;
4.经传代筛选，得到纯合子子代小鼠；4. Obtain homozygous progeny mice through subculture screening;
5.6-7周龄雄性小鼠给予如下化学物：人CAR激活剂6-(4-氯苯基)咪唑并[2,1-b][1,3]噻唑-5-甲醛-O-(3,4-二氯苯基)肟(CITCO，CAS号：338404-52-7)腹腔注射20mg/kg/d连续3天，末次24h取肝；小鼠CAR激活剂1,4-双-[2-(3,5,二氯吡啶氧基)]苯(TCPOBOP，CAS号：76150-91-9)腹腔注射3mg/kg单次，72h取肝；人PXR激活剂利福平(RIF，CAS号：13292-46-1)腹腔注射10mg/kg/d连续4天，末次24h取肝；鼠PXR激活剂孕烯醇酮16α-腈(PCN，CAS号：1434-54-4)腹腔注射75mg/kg单次，24h取肝；5. Male mice aged 6-7 weeks were given the following chemicals: human CAR activator 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3 ,4-Dichlorophenyl)oxime (CITCO, CAS No.: 338404-52-7) was injected intraperitoneally at 20 mg/kg/d for 3 consecutive days, and the liver was collected at the last 24 hours; mouse CAR activator 1,4-bis-[2 -(3,5,Dichloropyridyloxy)]benzene (TCPOBOP, CAS No.: 76150-91-9) was injected intraperitoneally at a dose of 3 mg/kg, and the liver was collected after 72 hours; human PXR activator rifampicin (RIF, CAS No. : 13292-46-1) intraperitoneal injection of 10mg/kg/d for 4 consecutive days, the liver was taken at the last 24h; mouse PXR activator pregnenolone 16α-nitrile (PCN, CAS No.: 1434-54-4) intraperitoneal injection of 75mg/kg kg at a time, take the liver within 24 hours;
6.提取肝组织中总RNA；6. Extraction of total RNA in liver tissue;
7.反转录获得cDNA。7. Obtain cDNA by reverse transcription.
8.使用下述引物进行定量PCR检测：8. Use the following primers for quantitative PCR detection:
鼠ActB上游引物：GGCTGTATTCCCCTCCATCG(SEQ ID NO.18)Mouse ActB upstream primer: GGCTGTATTCCCCTCCATCG (SEQ ID NO.18)
鼠ActB下游引物：CCAGTTGGTAACAATGCCATGT(SEQ ID NO.19)Mouse ActB downstream primer: CCAGTTGGTAACAATGCCATGT (SEQ ID NO.19)
人ActB上游引物：CATGTACGTTGCTATCCAGGC(SEQ ID NO.20)Human ActB upstream primer: CATGTACGTTGCTATCCAGGC (SEQ ID NO.20)
人ActB下游引物：CTCCTTAATGTCACGCACGAT(SEQ ID NO.21)Human ActB downstream primer: CTCCTTAATGTCACGCACGAT (SEQ ID NO.21)
人PXR上游引物：AAGCCCAGTGTCAACGCAG(SEQ ID NO.22)Human PXR upstream primer: AAGCCCAGTGTCAACGCAG (SEQ ID NO.22)
人PXR下游引物：GGGTCTTCCGGGTGATCTC(SEQ ID NO.23)Human PXR downstream primer: GGGTCTTCCGGGTGATCTC (SEQ ID NO.23)
人PXR..1上游引物：CAAGCGGAAGAAAAGTGAACG(SEQ ID NO.24)Human PXR..1 upstream primer: CAAGCGGAAGAAAAGTGAACG (SEQ ID NO.24)
人PXR.1下游引物：CACAGATCTTTCCGGACCTG(SEQ ID NO.25)Human PXR.1 downstream primer: CACAGATCTTTCCGGACCTG (SEQ ID NO.25)
人PXR..2上游引物：TCAAGAATTTCCGGGTCTCTC(SEQ ID NO.26)Human PXR..2 upstream primer: TCAAGAATTTCCGGGTCTCTC (SEQ ID NO.26)
人PXR.3上游引物：TCAAGAATTTCCGGCTGCG(SEQ ID NO.27)Human PXR.3 upstream primer: TCAAGAATTTCCGGCTGCG (SEQ ID NO.27)
人PXR.2/3下游引物：CGATGGGCAAGTCCCTGAAG(SEQ ID NO.28)Human PXR.2/3 downstream primer: CGATGGGCAAGTCCCTGAAG (SEQ ID NO.28)
鼠CYP2B10上游引物：TGGAGGAACTGCGGAAAT(SEQ ID NO.29)Mouse CYP2B10 upstream primer: TGGAGGAACTGCGGAAAT (SEQ ID NO.29)
鼠CYP2B10下游引物：AACATCTGGCTGGAGAATGA(SEQ ID NO.30)Mouse CYP2B10 downstream primer: AACATCTGGCTGGAGAATGA (SEQ ID NO.30)
鼠CYP3A11上游引物：GTCAAACGCCTCTCCTTGCTG(SEQ ID NO.31)Mouse CYP3A11 upstream primer: GTCAAACGCCTCTCCTTGCTG (SEQ ID NO.31)
鼠CYP3A11下游引物：GGCTTGCCTTTCTTTGCCTTC(SEQ ID NO.32)Mouse CYP3A11 downstream primer: GGCTTGCCTTTCTTTGCCTTC (SEQ ID NO.32)
鼠PXR上游引物：GAAGGGCCATGAAACGCAATG(SEQ ID NO.33)Mouse PXR upstream primer: GAAGGGCCATGAAACGCAATG (SEQ ID NO.33)
鼠PXR下游引物：CAGCGGCATCGGACATGAT(SEQ ID NO.34)Mouse PXR downstream primer: CAGCGGCATCGGACATGAT (SEQ ID NO.34)
9.常规方法计算相对表达倍数。9. Calculate the relative expression fold by conventional methods.
结果如图2-5所示：本发明繁育的转基因小鼠表达3种主要的人PXR剪接变异体，其表达丰度与人肝组织相似；比较肝组织PXR mRNA表达水平，转基因小鼠仅表达人PXR，不表达小鼠PXR，其人PXR表达相对水平与野生型小鼠自身PXR、人肝组织自身PXR的表达水平相当；比较野生型小鼠和PXR转基因小鼠对4种种属特异性核受体激活剂(人PXR激活剂RIF，小鼠PXR激活剂PCN，人CAR激活剂CITCO，小鼠CAR激活剂TCPOBOP)应答(靶基因CYP2B10、CYP3A11诱导表达)，可见PXR转基因小鼠对人PXR激活剂RIF、小鼠CAR激活剂TCPOBOP产生预期的靶基因诱导表达。因此，本发明繁育的转基因小鼠表达人PXR主要的剪接变异体，表达水平接近生理水平，对人PXR特异性激活剂应答表现为人PXR特征，不同于小鼠。The results are shown in Figures 2-5: the transgenic mice bred in the present invention express three main human PXR splice variants, and their expression abundance is similar to that of human liver tissue; comparing the expression levels of PXR mRNA in liver tissue, the transgenic mice only express Human PXR does not express mouse PXR, and the relative expression level of human PXR is comparable to that of wild-type mouse PXR and human liver tissue itself; compare wild-type mice and PXR transgenic mice for the four species-specific nuclear Receptor activator (human PXR activator RIF, mouse PXR activator PCN, human CAR activator CITCO, mouse CAR activator TCPOBOP) response (target gene CYP2B10, CYP3A11 induced expression), it can be seen that PXR transgenic mice have no response to human PXR The activator RIF and the mouse CAR activator TCPOBOP produced the expected induced expression of target genes. Therefore, the transgenic mice bred in the present invention express the main splicing variants of human PXR, the expression level is close to the physiological level, and the response to human PXR-specific activators shows the characteristics of human PXR, which is different from mice.
本发明用到化学物有：6-(4-氯苯基)咪唑并[2,1-b][1,3]噻唑-5-甲醛-O-(3,4-二氯苯基)肟、1,4-双-[2-(3,5,二氯吡啶氧基)]苯、利福平、孕烯醇酮16α-腈。以上化学物均购自SIGMA公司，分析纯试剂，为中国检验检疫科学研究院所保存。The chemicals used in the present invention are: 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-formaldehyde-O-(3,4-dichlorophenyl)oxime , 1,4-bis-[2-(3,5,dichloropyridyloxy)]benzene, rifampicin, pregnenolone 16α-carbonitrile. The above chemicals were purchased from SIGMA Company, analytical reagents, and kept by the Chinese Academy of Inspection and Quarantine.
除非特殊定义，本发明描述所用的术语是在有关技术领域中公知的术语。标准的化学符号及缩写符号可以与其全名互换使用。Unless otherwise defined, terms used in the description of the present invention are terms commonly known in the relevant technical field. Standard chemical symbols and abbreviated symbols can be used interchangeably with their full names.
除非特殊指明，本发明所用到但未明确阐述或简单阐述的技术和方法是指本技术领域通常使用的技术和方法，可按照本领域公知的技术和方法进行。试剂盒的使用是根据制造商或供应商提供的说明书进行。Unless otherwise specified, the technologies and methods used in the present invention but not explicitly or briefly described refer to the technologies and methods commonly used in the technical field, and can be performed according to the technologies and methods known in the art. The kits were used according to the instructions provided by the manufacturer or supplier.
序列表sequence listing
<110> 中国检验检疫科学研究院<110> Chinese Academy of Inspection and Quarantine
<120> 一种孕烷X受体转基因小鼠模型、其构建方法及其应用<120> A Pregnane X Receptor Transgenic Mouse Model, Its Construction Method and Application
<130> LP0209<130> LP0209
<160> 33<160> 33
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 1340<211> 1340
<212> DNA<212>DNA
<213> C57BL/6J品系小鼠(Mus musculus strain C57BL/6J)<213> C57BL/6J strain mouse (Mus musculus strain C57BL/6J)
<400> 1<400> 1
ggtgagagca aagcattcag ggtacagatt ttctctggct tcctttgtca aggagaaaca 60ggtgagagca aagcattcag ggtacagatt ttctctggct tcctttgtca aggagaaaca 60
catagaaacc catccatggg taccacgaat aacaaggctc cgaggaagga tgggaggtat 120catagaaacc catccatggg taccacgaat aacaaggctc cgaggaagga tgggaggtat 120
ttaagcaaaa ggcctggagc ctagacttgt tctaggtaga gactctcggg ggctgtaggt 180ttaagcaaaa ggcctggagc ctagacttgt tctaggtaga gactctcggg ggctgtaggt 180
gcccgtgaaa agcaccagtc tcattttctg cagaaatatc tcagaaagtc acctcctctt 240gcccgtgaaa agcaccagtc tcattttctg cagaaatatc tcagaaagtc acctcctctt 240
tttgagtcct tgaccttaaa gtagagcgtc aatctacacc tgcctcataa aatccctggt 300tttgagtcct tgaccttaaa gtagagcgtc aatctacacc tgcctcataa aatccctggt 300
gtgtgctacc tgctgctggc agggtctgga gtcctggctc tggagctgag tgcctgatgc 360gtgtgctacc tgctgctggc agggtctgga gtcctggctc tggagctgag tgcctgatgc 360
tctcttttat cccacagaag ggccatgaaa cgcaatgtcc ggctgaggtg ccccttccgc 420tctcttttat cccacagaag ggccatgaaa cgcaatgtcc ggctgaggtg ccccttccgc 420
aagggaacct gcgagatcac ccggaagaca cgacggcagt gccaggcctg ccgtttgcgc 480aagggaacct gcgagatcac ccggaagaca cgacggcagt gccaggcctg ccgtttgcgc 480
aagtgcctgg agagtggcat gaagaaagag agtgagtggc tgcggggtag gaggctcagg 540aagtgcctgg agagtggcat gaagaaagag agtgagtggc tgcggggtag gaggctcagg 540
cttggtggac tgggcggttg ccagagagct gaggcaccag tgcaccacag cgcctaagcg 600cttggtggac tgggcggttg ccagagagct gaggcaccag tgcaccacag cgcctaagcg 600
catgcgcatt tgtgagcctg tgggactttg gtgataccag gtactagttc tcattatggg 660catgcgcatt tgtgagcctg tgggactttg gtgataccag gtactagttc tcattatggg 660
aagtgtggtg ggagatgcag gtcagggcta tgtcctctct tttttctcag ctatccctgc 720aagtgtggtg ggagatgcag gtcagggcta tgtcctctct tttttctcag ctatccctgc 720
atgattcctt ttgtccacag tcattactgt agtcctggtg gtgtatggag gttgggggag 780atgattcctt ttgtccacag tcattactgt agtcctggtg gtgtatggag gttggggggag 780
ggggatgacc ccgccggggc acatatgccg agtcctcttt gttaacacgc agtgatcatg 840ggggatgacc ccgccggggc acatatgccg agtcctcttt gttaacacgc agtgatcatg 840
tccgatgccg ctgtggagca gaggcgggcc ttgatcaaga ggaagaagag ggaaaagatt 900tccgatgccg ctgtggagca gaggcgggcc ttgatcaaga ggaagaagag ggaaaagatt 900
gaggctccac cgcctggagg gcaggggctg acggaagaac agcaggcgct gatccaggag 960gaggctccac cgcctggagg gcaggggctg acggaagaac agcaggcgct gatccaggag 960
ctgatggacg ctcagatgca aacctttgac acaactttct cccacttcaa ggatttccgg 1020ctgatggacg ctcagatgca aacctttgac acaactttct cccacttcaa ggatttccgg 1020
gtagggagct tagagctgtg accacatcca tggctatgct agctcctcaa agtccgagtc 1080gtagggagct tagagctgtg accacatcca tggctatgct agctcctcaa agtccgagtc 1080
actttcccag ggtttctagt agtcaggggc atcaggatac ccagacaact tgctagaaaa 1140actttcccag ggtttctagt agtcaggggc atcaggatac ccagacaact tgctagaaaa 1140
gcatcctgct cccaacctca aactctggga tgggaaatct tttgatgatt gcccagctga 1200gcatcctgct cccaacctca aactctggga tgggaaatct tttgatgatt gcccagctga 1200
ctgtcctcag tggataggaa ctgagttaag attaagcaat ggcaggcatg ttgcccacac 1260ctgtcctcag tggataggaa ctgagttaag attaagcaat ggcaggcatg ttgccccacac 1260
tgtgatgtga ggacagccaa gaggagaagg ggtacggact tcctgttggc tagctctgtc 1320tgtgatgtga ggacagccaa gaggagaagg ggtacggact tcctgttggc tagctctgtc 1320
tttgctccca gtgccaccca 1340tttgctccca gtgccaccca 1340
<210> 2<210> 2
<211> 20<211> 20
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 2<400> 2
gacgctctac tttaaggtca 20gacgctctac tttaaggtca 20
<210> 3<210> 3
<211> 20<211> 20
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 3<400> 3
tcccagggtt tctagtagtc 20tcccagggtt tctagtagtc 20
<210> 4<210> 4
<211> 27<211> 27
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 4<400> 4
gagagcaaag cattcagggt acagatt 27gagagcaaag cattcagggt acagatt 27
<210> 5<210> 5
<211> 25<211> 25
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 5<400> 5
gcactgggag caaagacaga gctag 25gcactgggag caaagacaga gctag 25
<210> 6<210> 6
<211> 25<211> 25
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 6<400> 6
ggagaaacac atagaaaccc atcca 25ggagaaacac atagaaaccc atcca 25
<210> 7<210> 7
<211> 24<211> 24
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 7<400> 7
gccagaaggg atttgagaga tgat 24gccagaaggg atttgagaga tgat 24
<210> 8<210> 8
<211> 23<211> 23
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 8<400> 8
cagtggaagt agcctgaaca gtg 23cagtggaagt agcctgaaca gtg 23
<210> 9<210> 9
<211> 24<211> 24
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 9<400> 9
gtggcttcag gtttgctgtc agta 24gtggcttcag gtttgctgtc agta 24
<210> 10<210> 10
<211> 26<211> 26
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 10<400> 10
cgcatctgga gcagataagg aaagat 26cgcatctgga gcagataagg aaagat 26
<210> 11<210> 11
<211> 22<211> 22
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 11<400> 11
gtattccagg tgcattccac at 22gtattccagg tgcattccac at 22
<210> 12<210> 12
<211> 24<211> 24
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 12<400> 12
cggcaataat gtggagaaac tgag 24cggcaataat gtggagaaac tgag 24
<210> 13<210> 13
<211> 24<211> 24
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 13<400> 13
gccttgttta tagccacttg tgag 24gccttgttta tagccacttg tgag 24
<210> 14<210> 14
<211> 25<211> 25
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 14<400> 14
ctgtgttagc caggatggtc tcgat 25ctgtgttagc caggatggtc tcgat 25
<210> 15<210> 15
<211> 24<211> 24
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 15<400> 15
gcagaagagg acagatacat tcat 24gcagaagagg acagatacat tcat 24
<210> 16<210> 16
<211> 24<211> 24
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 16<400> 16
cctactgaag aatctatccc acag 24cctactgaag aatctatccc aagag 24
<210> 17<210> 17
<211> 20<211> 20
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 17<400> 17
ggctgtattc ccctccatcg 20ggctgtattc ccctccatcg 20
<210> 18<210> 18
<211> 22<211> 22
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 18<400> 18
ccagttggta acaatgccat gt 22ccagttggta acaatgccat gt 22
<210> 19<210> 19
<211> 21<211> 21
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 19<400> 19
catgtacgtt gctatccagg c 21catgtacgtt gctatccagg c 21
<210> 20<210> 20
<211> 21<211> 21
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 20<400> 20
ctccttaatg tcacgcacga t 21ctccttaatg tcacgcacga t 21
<210> 21<210> 21
<211> 19<211> 19
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 21<400> 21
aagcccagtg tcaacgcag 19aagcccagtg tcaacgcag 19
<210> 22<210> 22
<211> 19<211> 19
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 22<400> 22
gggtcttccg ggtgatctc 19gggtcttccgggtgatctc 19
<210> 23<210> 23
<211> 21<211> 21
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 23<400> 23
caagcggaag aaaagtgaac g 21caagcggaag aaaagtgaac g 21
<210> 24<210> 24
<211> 20<211> 20
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 24<400> 24
cacagatctt tccggacctg 20cacagatctttccggacctg 20
<210> 25<210> 25
<211> 21<211> 21
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 25<400> 25
tcaagaattt ccgggtctct c 21tcaagaattt ccgggtctct c 21
<210> 26<210> 26
<211> 19<211> 19
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 26<400> 26
tcaagaattt ccggctgcg 19tcaagaattt ccggctgcg 19
<210> 27<210> 27
<211> 20<211> 20
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 27<400> 27
cgatgggcaa gtccctgaag 20cgatgggcaa gtccctgaag 20
<210> 28<210> 28
<211> 18<211> 18
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 28<400> 28
tggaggaact gcggaaat 18tggaggaact gcggaaat 18
<210> 29<210> 29
<211> 20<211> 20
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 29<400> 29
aacatctggc tggagaatga 20aacatctggc tggagaatga 20
<210> 30<210> 30
<211> 21<211> 21
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 30<400> 30
gtcaaacgcc tctccttgct g 21gtcaaacgcc tctccttgct g 21
<210> 31<210> 31
<211> 21<211> 21
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 31<400> 31
ggcttgcctt tctttgcctt c 21ggcttgcctt tctttgcctt c 21
<210> 32<210> 32
<211> 21<211> 21
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 32<400> 32
gaagggccat gaaacgcaat g 21gaagggccat gaaacgcaat g 21
<210> 33<210> 33
<211> 19<211> 19
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 33<400> 33
cagcggcatc ggacatgat 19cagcggcatc ggacatgat 19
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810100289.2ACN108174823A (en) | 2018-02-01 | 2018-02-01 | A kind of Pregnane X Receptor transgene mouse model, its construction method and its application |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810100289.2ACN108174823A (en) | 2018-02-01 | 2018-02-01 | A kind of Pregnane X Receptor transgene mouse model, its construction method and its application |
| Publication Number | Publication Date |
|---|---|
| CN108174823Atrue CN108174823A (en) | 2018-06-19 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810100289.2APendingCN108174823A (en) | 2018-02-01 | 2018-02-01 | A kind of Pregnane X Receptor transgene mouse model, its construction method and its application |
| Country | Link |
|---|---|
| CN (1) | CN108174823A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533925A (en)* | 2011-12-06 | 2012-07-04 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for in-vitro screening of PXR activation characteristics |
| CN104593418A (en)* | 2015-02-06 | 2015-05-06 | 中国医学科学院医学实验动物研究所 | Method for establishing humanized rat drug evaluation animal model |
| CN105950639A (en)* | 2016-05-04 | 2016-09-21 | 广州美格生物科技有限公司 | Preparation method of staphylococcus aureus CRISPR/Cas9 system and application of system in constructing mouse model |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533925A (en)* | 2011-12-06 | 2012-07-04 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for in-vitro screening of PXR activation characteristics |
| CN104593418A (en)* | 2015-02-06 | 2015-05-06 | 中国医学科学院医学实验动物研究所 | Method for establishing humanized rat drug evaluation animal model |
| CN105950639A (en)* | 2016-05-04 | 2016-09-21 | 广州美格生物科技有限公司 | Preparation method of staphylococcus aureus CRISPR/Cas9 system and application of system in constructing mouse model |
| Title |
|---|
| BREWER CT 等: "PXR variants: the impact on drug metabolism and therapeutic responses", 《ACTA PHARM SIN B》* |
| KIM H 等: "Generation of a PXR reporter human induced pluripotent stem cell line (PXR-mCherry hiPSC) using the CRISPR/Cas9 system", 《 STEM CELL RESEARCH 》* |
| STAUDINGER JL等: "The nuclear receptor PXR is a lithocholic acid sensor", 《PNAS》* |
| XIAOCHAO MA等: "The Pregnane X Receptor Gene-Humanized Mouse: A Model for Investigating", 《DRUG METABOLISM AND DISPOSITION》* |
| 刘艳 等: "人类孕烷受体遗传变异对遗传药理学和药物代谢的影响", 《中国临床药理学与治疗学》* |
| 王超: "《基因修饰小鼠制备常用技术》", 1 December 2013, 中国农业大学出版社* |
| 魏群: "《生物工程技术实验指导》", 30 September 2002* |
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Transposon-induced epigenetic silencing in the X chromosome as a novel form of dmrt1 expression regulation during sex determination in the fighting fish | |
| Varrault et al. | Zac1 regulates an imprinted gene network critically involved in the control of embryonic growth | |
| Li et al. | The splicing regulator PTBP2 controls a program of embryonic splicing required for neuronal maturation | |
| CN105555132B (en) | Mrap2 knockouts | |
| Zekri et al. | Control of fetal growth and neonatal survival by the RasGAP-associated endoribonuclease G3BP | |
| Baines et al. | Disruption of the Sec24d gene results in early embryonic lethality in the mouse | |
| Coleman et al. | Rapid knockout and reporter mouse line generation and breeding colony establishment using EUCOMM conditional-ready embryonic stem cells: a case study | |
| Thorvaldsen et al. | Developmental profile of H19 differentially methylated domain (DMD) deletion alleles reveals multiple roles of the DMD in regulating allelic expression and DNA methylation at the imprinted H19/Igf2 locus | |
| EP2218786A1 (en) | Mammalian artificial chromosome vector comprising human cytochrome p450 gene (cluster), and non-human mammalian animal retaining the same | |
| Emmer et al. | Murine Surf4 is essential for early embryonic development | |
| Oliver et al. | Disruption of Visc-2, a brain-expressed conserved long noncoding RNA, does not elicit an overt anatomical or behavioral phenotype | |
| Hims et al. | A humanized IKBKAP transgenic mouse models a tissue-specific human splicing defect | |
| CN110438160B (en) | Construction method and application of a Cd2ap gene knockout animal | |
| Liu et al. | Functional SNPs of INCENP affect semen quality by alternative splicing mode and binding affinity with the target Bta-miR-378 in Chinese Holstein bulls | |
| WO2005003359A1 (en) | Method and system of constructing transgenic organism | |
| Schmouth et al. | Modelling human regulatory variation in mouse: finding the function in genome-wide association studies and whole-genome sequencing | |
| Zhang et al. | Novel mutations in the Myo5a gene cause a dilute coat color phenotype in mice | |
| CN118910047B (en) | ADAMTS1 gene distal enhancer related to sheep tail type and application thereof in sheep fat tail early screening | |
| Xu et al. | Ectopic expression of retrotransposon-derived PEG11/RTL1 contributes to the callipyge muscular hypertrophy | |
| CN115044619A (en) | A method for constructing a systemic inducible Ppp3ca knockout mouse model | |
| Kumar et al. | Unexpected embryonic stem (ES) cell mutations represent a concern in gene targeting: lessons from “fierce” mice | |
| US7582741B2 (en) | Conditional disruption of dicer1 in cell lines and non-human mammals | |
| Dawlaty et al. | Gene targeting methods for studying nuclear transport factors in mice | |
| US6984771B2 (en) | Mice heterozygous for WFS1 gene as mouse models for depression | |
| CN114045290B (en) | Construction method and application of keratin gene modified mouse animal model |
| 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 | ||
| RJ01 | Rejection of invention patent application after publication | Application publication date:20180619 | |
| RJ01 | Rejection of invention patent application after publication |