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

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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.