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Attempt to control sex ratio in the livestock by the transgene of anti-SRY ribozyme or antisence SRY

尝试通过抗SRY核酶或反义SRY转基因来控制家畜性别比

基本信息

批准号：
13460127
负责人：
SONOKI Shigenori
金额：
$ 9.54万
依托单位：
Azabu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13460127/
关键词：
anti SRY ribozyme antisence SRY Livestock sex ratio transgenic mice SRY ribozyme antisence アンチセンスRNA pCAAGS polIIIプロモーター性決定性比制御

项目摘要

The purpose of this study is to conduct fundamental research and development to increase the ratio of females in livestock by means of genetic engineering ; i.e. antisense mRNA or anti-SRY ribozyme is expressed as a transgene in a transcript of SRY, the sex determining gene in mammals. We created transgenic mice (C3B6F1) in which antisense SRY was expressed using pCAGGS, an expression vector that has a strong promoter/enhancer complex, and analyzed the phenotypes of their reproductive organs. We have created 12 transgenic mice so far, and the phenotype has matched the genotype in all mice. In the first offspring (F1) of these mice (founder), there were some mice whose phenotype was female, was female, but for which PCR analysis returned positive to Zfy, the Y-chromosome marker. We carried out genetic analysis of these mice by designing a new Zfy-detecting primer and adding Ubel, a gene that shows sexual dimorphism in the base sequence. However, no transsexual mouse in which the phenotype contradicted the genotype was confirmed. With regard to anti-mouse SRY ribozymes, we designed several types, such as hammerhead ribozymes and a tRNA-ribozyme complex. We examined the cutting effect of these ribozymes on SRY mRNA in vitro, and the effect was confirmed. In addition, we constructed pCAGGS and po1III promoter-based expression vectors so that these ribozymes could be expressed in vivo. An experiment in which anti-SRY ribozyme po1III vector constructs were introduced to cells containing SRY genes as transgenes suggests that the ribozymes inhibit SRY mRNA expression in vivo. We also examined whether ultra-low temperature freezing is an effective method of storing spermatozoa and early embryos to produce transgenic livestock, and explored more effective in vitro production systems for early embryos. Both returned good results.

本研究的目的是通过基因工程的方法进行基础研究和开发，即在哺乳动物性别决定基因SRY的转录本中表达反义mRNA或抗SRY核酶作为转基因，以提高家畜的雌性比例。我们创建了转基因小鼠（C3 B6 F1），其中反义SRY表达使用pCAGGS，一种表达载体，具有强大的启动子/增强子复合物，并分析了它们的生殖器官的表型。到目前为止，我们已经创造了12只转基因小鼠，并且表型与所有小鼠的基因型相匹配。在这些小鼠（创始者）的第一个后代（F1）中，有一些小鼠的表型是雌性，但PCR分析对Y染色体标记Zfy呈阳性。我们通过设计新的Zfy检测引物并添加Ubel（一种在碱基序列中显示两性异形的基因）对这些小鼠进行了遗传分析。然而，没有一个变性小鼠的表型矛盾的基因型得到证实。在抗小鼠SRY核酶方面，我们设计了几种类型的核酶，如锤头状核酶和tRNA-核酶复合物。我们在体外检测了这些核酶对SRY mRNA的切割效果，并证实了这种效果。此外，我们构建了pCAGGS和po 1 III启动子为基础的表达载体，使这些核酶可以在体内表达。将抗SRY核酶po 1 III载体构建体引入含有SRY基因作为转基因的细胞的实验表明，核酶在体内抑制SRY mRNA表达。我们还研究了超低温冷冻是否是生产转基因家畜的精子和早期胚胎的有效方法，并探索了更有效的早期胚胎体外生产系统。两者都取得了良好的效果。