人胎肝来源PACT等重要功能基因的基因打靶研究
发布时间：2018-11-28 12:47
【摘要】：肝脏是人体内多种物质代谢的主要器官,具有重要的生理功能。4-6月孕龄的人胎肝还是造血、免疫系统干祖细胞的主要来源,其中可能存在大量具有重要功能的新基因。本室通过胎肝大规模cDNA测序已经建立了22周孕龄人胎肝基因表达图谱,同时正在进行从胎肝中大规模发掘重要功能新基因的探索,包括PACT、HPO、CKIP-1、NPDC-1、LMBP等。 基因打靶技术是一种通过同源重组按预期方式对靶基因进行定点修饰而改变生物活体遗传信息,进而观察相应基因功能的实验手段。应用此技术,我们共构建成功了上述5种基因的打靶载体,并将打靶载体导入小鼠胚胎干细胞,筛选获得了PACT、HPO、CKIP-13种基因的中靶细胞。各自的基因型均已经过PCR和Southern印迹法鉴定。通过囊胚显微注射技术,我们获得了PACT、HPO、CKIP-13种基因的嵌合体小鼠。其中HPO、CKIP-1这2种基因的嵌合体小鼠不能将突变等位基因遗传至子代。而PACT嵌合体小鼠中的突变等位基因可以遗传至子代,获得了可存活、可繁殖、表型正常的PACT杂合体小鼠。将PACT杂合体小鼠自交,在子代中未发现PACT纯合体小鼠。根据野生型与杂合型小鼠数目约1∶2的比例,推测其可能在胚胎期纯合致死。 我们对PACT杂合体小鼠自交子代做了大量的胚胎期追踪分析,发现PACT纯合体基因敲除小鼠在胚胎期6.5至7.5天死亡,胚胎发育停滞,原肠形成失败。这揭示了PACT在高等动物早期胚胎发育过程中不可或缺的重要性。通过RT-PCR显示,7.5天的PACT纯合体基因敲除胚胎中,p53的靶基因p21、Mdm2的mRNA水平显著升高。因为PACT在体外实验中可以强烈的抑制p53的转录活性,所以我们推测PACT的缺失可能导致p53转录活性的失控,其下游基因过度表达而导致胚胎期致死效应。目前,更进一步的表型分析和死亡机制研究正在进行中。
[Abstract]:The liver is the main organ of the metabolism of various substances in human body and has important physiological function. The fetal liver of 4-6 months gestational age is still hematopoietic, the main source of stem progenitor cells of immune system, and there may be a large number of new genes with important function. The gene expression map of human fetal liver at 22 weeks of gestation was established by large-scale cDNA sequencing of fetal liver in our laboratory. At the same time, the discovery of new genes of important function from fetal liver, including PACT,HPO,CKIP-1,NPDC-1,LMBP and so on, was carried out on a large scale. Gene targeting is an experimental method to modify the genetic information of living organisms by homologous recombination and modify the target gene in the expected way, and then observe the function of the corresponding gene. Using this technique, we successfully constructed the targeting vectors of the five genes mentioned above, and introduced the target vectors into mouse embryonic stem cells to screen the middle target cells of the PACT,HPO,CKIP-13 gene. Their genotypes have been identified by PCR and Southern blotting. By microinjection of blastocyst, chimeric mice with PACT,HPO,CKIP-13 gene were obtained. The chimeric mice of the two HPO,CKIP-1 genes could not inherit the mutant alleles to their offspring. The mutant alleles in PACT chimeric mice could be inherited to their offspring, and PACT heterozygotes with viable, reproductive and normal phenotypes were obtained. No PACT homozygous mice were found in the offspring of PACT heterozygote mice. According to the proportion of wild and heterozygous mice about 1:2, it is inferred that the mice may die in homozygote at embryonic stage. We have done a lot of embryo tracing analysis on PACT heterozygote mice. We found that PACT homozygous gene knockout mice died from 6.5 to 7.5 days of embryo stage, embryo development stagnated and protointestinal formation failed. This reveals the importance of PACT in the early embryonic development of higher animals. RT-PCR showed that the mRNA level of p53 target gene p21 Mdm2 was significantly increased in 7.5 day PACT homozygous knockout embryos. Because PACT can strongly inhibit the transcriptional activity of p53 in vitro, we speculate that the deletion of PACT may lead to the loss of p53 transcriptional activity, and the overexpression of the downstream gene may lead to the lethal effect of embryo. Further studies on phenotypic analysis and death mechanisms are currently under way.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：Q789
本文编号：2362861
[Abstract]:The liver is the main organ of the metabolism of various substances in human body and has important physiological function. The fetal liver of 4-6 months gestational age is still hematopoietic, the main source of stem progenitor cells of immune system, and there may be a large number of new genes with important function. The gene expression map of human fetal liver at 22 weeks of gestation was established by large-scale cDNA sequencing of fetal liver in our laboratory. At the same time, the discovery of new genes of important function from fetal liver, including PACT,HPO,CKIP-1,NPDC-1,LMBP and so on, was carried out on a large scale. Gene targeting is an experimental method to modify the genetic information of living organisms by homologous recombination and modify the target gene in the expected way, and then observe the function of the corresponding gene. Using this technique, we successfully constructed the targeting vectors of the five genes mentioned above, and introduced the target vectors into mouse embryonic stem cells to screen the middle target cells of the PACT,HPO,CKIP-13 gene. Their genotypes have been identified by PCR and Southern blotting. By microinjection of blastocyst, chimeric mice with PACT,HPO,CKIP-13 gene were obtained. The chimeric mice of the two HPO,CKIP-1 genes could not inherit the mutant alleles to their offspring. The mutant alleles in PACT chimeric mice could be inherited to their offspring, and PACT heterozygotes with viable, reproductive and normal phenotypes were obtained. No PACT homozygous mice were found in the offspring of PACT heterozygote mice. According to the proportion of wild and heterozygous mice about 1:2, it is inferred that the mice may die in homozygote at embryonic stage. We have done a lot of embryo tracing analysis on PACT heterozygote mice. We found that PACT homozygous gene knockout mice died from 6.5 to 7.5 days of embryo stage, embryo development stagnated and protointestinal formation failed. This reveals the importance of PACT in the early embryonic development of higher animals. RT-PCR showed that the mRNA level of p53 target gene p21 Mdm2 was significantly increased in 7.5 day PACT homozygous knockout embryos. Because PACT can strongly inhibit the transcriptional activity of p53 in vitro, we speculate that the deletion of PACT may lead to the loss of p53 transcriptional activity, and the overexpression of the downstream gene may lead to the lethal effect of embryo. Further studies on phenotypic analysis and death mechanisms are currently under way.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：Q789
【引证文献】
相关博士学位论文 前1条
1 尹秀山;CKIP-1基因的敲除小鼠模型建立及其在骨骼、免疫系统中的生理功能研究[D];中国人民解放军军事医学科学院;2009年
，本文编号：2362861
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