睾丸特异性表达新基因T279及TSC21在小鼠睾丸精子发生过程中的功能研究

发布时间：2018-05-03 06:15

本文选题：T279 + TSC21　；参考：《郑州大学》2017年博士论文

【摘要】：第一部分:睾丸特异性新基因T279及TSC21基因敲除小鼠模型的建立目的构建基因打靶载体,通过基因打靶技术,建立睾丸特异性新基因T279及TSC21基因敲除小鼠模型,在活体实验动物水平研究这两个睾丸特异性新基因在小鼠睾丸精子发生过程中的功能。方法订购含T279基因及TSC21基因的129 BAC克隆,PCR扩增Retrieve同源臂及Loxp-neo-Loxp片段,利用PL253质粒进行目片段的连接和同源重组,构建打靶载体并电转ES细胞,利用Southern blot方法鉴定同源重组阳性的ES细胞克隆。鉴定后的ES细胞克隆通过显微注射的方法注入囊胚的胚泡中,再将囊胚植入假孕母鼠的子宫中发育,产生嵌合体小鼠,通过回交、互交,从出生的子代小鼠中通过genotyping技术可鉴定、筛选出纯合子的T279及TSC21基因敲除小鼠,再经繁育即可建立T279及TSC21基因敲除小鼠品系。结果经过限制性内切酶酶切鉴定及DNA测序鉴定,T279及TSC21基因敲除打靶载体构建成功。Southern blot结果显示成功筛选到打靶序列同源重组阳性的ES细胞克隆。鉴定后的ES细胞克隆注入囊胚胚泡后植入假孕母鼠的子宫中发育,顺利得到嵌合体小鼠,通过回交、互交,从出生的子代鼠中鉴定并筛选出了纯合子的T279及杂合子的TSC21基因敲除小鼠。结论睾丸特异性新基因T279及TSC21基因敲除小鼠模型构建成功,繁育顺利。第二部分:睾丸特异性新基因T279及TSC21在小鼠睾丸精子发生过程中的功能研究目的繁育并回交扩增睾丸特异性新基因T279及TSC21基因敲除小鼠品系,通过表型分析,初步探索睾丸特异性新基因T279及TSC21在雄性小鼠睾丸精子发生过程中的作用,为男性不育症的诊断和治疗探索新的途径。方法通过RT-PCR和Real-Time PCR等实验技术,分析睾丸特异性新基因T279及TSC21在小鼠体内的表达情况,繁育并回交扩增睾丸特异性新基因T279及TSC21基因敲除小鼠品系,通过交配实验、子代分析、附睾精子质量分析、睾丸组织切片HE染色、生精功能评估等技术,观察睾丸特异性新基因T279及TSC21基因敲除缺失对雄性小鼠睾丸精子发生过程和生育能力造成的影响。结果T279基因完全敲除缺失很可能会导致雄性小鼠精子畸形率的升高,但是T279基因敲除雄性小鼠的生育能力并未受到明显的影响。TSC21基因部分敲除对雄性小鼠的精子发生过程及生育能力没有造成明显的影响,目前尚没有TSC21基因敲除纯合子后代出生。结论睾丸特异性新基因T279及TCS21很可能在雄性小鼠睾丸精子发生过程中起重要的作用。T279基因完全敲除缺失很可能会导致雄性小鼠精子发生过程异常,进而引起雄性小鼠精子畸形率的升高,主要表现为精子头部畸形。TSC21基因完全缺失很可能导致子代胚胎发育过程中因发育异常引起胚胎期死亡。
[Abstract]:The first part: the establishment of a new testicular specific gene T279 and TSC21 knockout mouse model to construct a gene targeting vector. Through gene targeting technique, a new testis specific gene T279 and TSC21 knockout mouse model was established, and the two new testicular specific genes in the mouse testis spermatogenesis were studied at the level of living experimental animals. Function in the process. Method order 129 BAC clones containing T279 gene and TSC21 gene, PCR amplification of Retrieve homologous arm and Loxp-neo-Loxp fragment, use PL253 plasmids to connect and reorganize the target fragments, construct targeting carrier and electrically transfer ES cells, use Southern blot method to identify homologous recombination positive ES cell clones. Identified ES The cell clones inject into the blastocyst of the blastocyst by microinjection, then the blastocyst is implanted into the uterus of the pregnant mouse, and the chimerism mice are produced. Through the backcross and intercross, the T279 and TSC21 knockout mice of the homozygote can be identified from the offspring of the birth offspring by genotyping technique, and then the T279 and TS can be established by breeding. C21 gene knockout mouse strains. Results through restriction endonuclease digestion identification and DNA sequencing identification, T279 and TSC21 gene knockout target vector construction successful.Southern blot results showed that the target sequence homologous recombinant ES cell clones were successfully screened. The identified ES cell clones were implanted into the blastocyst of the embryo and implanted into the offspring of pregnant mice. In the uterus, the chimerism mice were successfully obtained, and the T279 and heterozygote TSC21 gene knockout mice were identified and screened from the offspring of the offspring from the offspring of the offspring. Conclusion the new testes specific gene T279 and TSC21 gene knockout mice were constructed successfully, and the second part: the new testis specific gene T279 and TS The function of C21 during the spermatogenesis of testicular spermatogenesis in mice. Aim to breed and backcross the new testicular specific gene T279 and TSC21 knockout mice. Through phenotypic analysis, the role of testis specific new gene T279 and TSC21 in the spermatogenesis of male mice was preliminarily explored to diagnose and treat male infertility. To explore new ways. Methods RT-PCR and Real-Time PCR were used to analyze the expression of new testicular specific gene T279 and TSC21 in mice, propagation and backcross amplification of new testis specific gene T279 and TSC21 knockout mice, through mating experiment, progeny analysis, epididymal sperm quality analysis, testicular tissue section The effects of T279 and TSC21 knockout deletion on testicular spermatogenesis and fertility of male mice were observed by HE staining and spermatogenic function evaluation. Results the complete knockout deletion of T279 gene might lead to the increase of sperm malformation rate in male mice, but the T279 gene knockout male mice. The ability has not been significantly affected by.TSC21 gene knockout. There is no significant effect on the spermatogenesis and fertility of male mice. There is no TSC21 gene knockout homozygote birth. Conclusion the new testicular specific gene T279 and TCS21 may play an important role in the process of testicular spermatogenesis in male mice. The complete knockout deletion of.T279 gene may lead to abnormal spermatogenesis in male mice and the increase of sperm malformation rate in male mice. The main manifestation is that the complete deletion of the.TSC21 gene of the sperm head deformity may lead to the embryonic death in the development of the offspring embryo due to abnormal development.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R698.2

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