Transgenic Mice Expressing hnRNP B0 bound to Single-Stranded Telomere DNA

异质核核糖核蛋白（hnRNP） B0是一种核RNA结合蛋白hnRNP A2/B1的睾丸特异性剪接异构体。体外分析表明B0特异性结合单链端粒DNA，与端粒的维持和功能有关。为了阐明B0在体内的作用，我们利用发育工程技术对B0进行了进一步的研究。1)在翻译延伸因子启动子（pEF-BOS）表达载体中引入人hnRNP B0的编码序列。2)将pEF-BOS-B0构建物微注射到BDF1小鼠受精卵中，按照前面描述的方法生成转基因小鼠。结果获得7只携带B0转基因的独立创始人小鼠（B0- tg小鼠）。其中4只在体内稳定表达B0转基因，3只出现出生率下降。3)对携带B0基因单拷贝的257号小鼠进行系统分析，发现B0- tg小鼠的出生率下降，更多的不是发育异常引起的，而是雄性小鼠生育能力差引起的。在B0纯合子雄性小鼠中，B0蛋白表达水平升高，减数分裂后，精子发生停止于单倍体圆形精细胞阶段，圆形精细胞凋亡。结果表明，在染色体同源重组发生的减数分裂前期厚成体期就已经出现了分子水平的异常。然而，端粒长度并没有像最初预期的那样明显改变，个体寿命的变化目前也不清楚。除睾丸外，其他脏器未见形态学改变。综上所述，利用发育工程技术的研究明确了B0蛋白在体内仅在睾丸中起作用，并抑制精子发生的成熟。目前，在分子水平上进行了进一步的分析，包括端粒在减数分裂中的功能。B0-Tg小鼠作为雄性不育的模型动物似乎提供了重要的信息。少

Heterogeneous nuclear ribonucleoprotein (hnRNP) B0 is a testis-specific splicing isoform of hnRNP A2/B1 which is a kind of nuclear RNA binding proteins. The in vitro analysis demonstrated that B0 specifically binds to single-stranded telomere DNA and has a relation to the maintenance and function of telomere. For purpose of clarifying in vivo role of B0, we further studied about B0 using a developmental engineering.1) The coding sequence of human hnRNP B0 was introduced in the pEF-BOS (translation elongation factor promoter) expression vector.2) pEF-BOS-B0 construct was microinjected in fertilized eggs obtained from BDF1 mice and transgenic mice were generated as previously described. As the result, seven independent founder mice carrying B0 transgene (B0-TG mice) were obtained. Four stably expressed B0 transgene in whole body, and three of them showed decreased birthrate.3) Systematical analysis of #257 founder which carrying a single copy of B0 revealed that the decreased birth rate … More of B0-TG mice was not caused by the developmental anomalies but was caused by the poor fertility of male mice. In B0-homozygote male mice, which expressed increased level of B0 protein, the spermatogenesis stops in the stage of the haploidic round spermatid after the meiosis, and the round spermatids fall into apoptosis. It was clarified that the abnormality of molecular level is already seen at the Pachyten stage of the meiosis prophase in which the homologous recombination of the chromosome occur. However, the telomere length did not apparently change as it anticipates at the beginning, and change in the individual lifespan are not clear at present, either. There was no morphological change in the organs except for the testis.In summary, it was clarified by the study using developmental engineering that B0 protein has in vivo role only in the testis and suppresses the maturation of spermatogenesis. At present, the further analysis of molecular level is advanced, including and it the telomere function in meiosis. The B0-Tg mice seem to give important information as a model animal of male infertility. Less