CRISPR-Cas9介导的输卵管特异性表达转基因鸡研究

用生物反应器生产医学临床应用的药用蛋白而言，转基因鸡较转基因家畜更有优势，因为鸡具有世代间隔短、后代繁殖快、产蛋量高等优点，在生物制药领域有着美好的应用前景。目前转基因鸡存在的问题是转基因效率低下。 虽然卵清蛋白启动子能有效诱导外源基因在鸡输卵管特异性表达，但未能实现高水平表达的目的。以CRISPR-Cas9介导的基因组编辑技术有望解决这一难题。本研究拟将靶向作用于鸡内源性卵清蛋白基因位点的sgRNA和Cas9 慢病毒载体与含有靶位点两侧翼同源臂的2A-EGFP慢病毒供体载体共同注射2.5日龄（Stage14 HH）鸡胚背主动脉，人工孵化至出壳。 通过精液PCR进行基因型分型，以此筛选G0转基因公鸡。通过侧交实验和人工孵化获得G1、G2转基因公鸡和输卵管特异性表达EGFP的转基因母鸡。

As animal bioreactors to produce recombinant pharmaceutical protein for treatment of human diseases, transgenic chickens have some advantages over transgenic livestock, such as short generation time, large egg production, and the easy scale-up of transgenic flocks. Therefore, transgenic chicken have an extensive future in bio-pharmaceuticals. However, low transgenic efficiency in chicken is always a problem. Although chicken ovalbumin promoter appears to be effective in inducing tissue-specific expression of delivered genes in transgenic chickens, but not sufficient for higher level production of foreign protein in the chicken oviduct. CRISPR/Cas9-mediated genome editing may be a promising approach to deal with the trouble. Here, we try to generate knock-in transgenic hens with oviduct-specific expression of EGFP by means of CRISPR-Cas9 system. A lentiviral vector, which contains Cas9 and the sgRNA targeted for chicken endogenous ovalbumin, and a lentiviral donor vector containing a reporter gene(EGFP) flanked homogenous recombination (HR) arms are to be co-injected to the dorsal aorta of 2.5–day old（Stage 14 HH）embryos, which will be artificially incubated to hatch. G0 transgenic roosters are screened with PCR for genotyping in semen. G1 and G2 transgenic roosters and hens with oviduct-specific expression of EGFP are to be generated by cross-testing and artificial egg incubation.