DNA甲基化对鸡输卵管特异性表达输异基因的调控机制研究

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。研究DNA 甲基化调控输异基因在输卵管特异性表达的机制，有望提高转基因效率。本研究拟PCR扩增鸡卵清蛋白启动子和EGFP基因；构建重组慢病毒表达载体。采用体外经过去甲基化/非去甲基化处理的重组慢病毒载体分别转染293FT细胞， 浓缩的重组慢病毒感染体外培养的鸡PGCs,分别将这两种经过遗传修饰的PGCs注射至2.5日龄胚胎的背主动脉中，以PCR扩增精液样品中EGFP来筛选孵出的G0转基因公鸡。通过侧交实验和孵化获得G1、G2转基因后代。分析输卵管和其它器官中CpG甲基化状态和EGFP表达量之间的对应关系，揭示CpG甲基化调控输异基因输卵管特异性表达的机制。

As animal bioreactors to produce recombinant pharmaceutical protein used in treatment of human diseases, transgenic chickens have some adavatages over transgenic livestock, such as short generation time, egg production,and the easy scale-up of transgenic flocks..Therefor, transgenic chicken have a extansive future in bio-pharmaceuticals.However, low transgenic efficiency in chicken is a problem. Studies on modulatiaon mechanism in oviduct-specific expression of a transgene in chicken may lead to overcome this problem. By means of PCR, chicken ovalbumin promotor and enhanced green fluorescent protein(EGFP) are to be amplified, and recombinant lentiviral expression vectors are to be constructed. Demethylated / undemethylated recombinant lentiviral expression plasmids are used to transfect 293FT cells respectively to produce concentrated lentiviral particles, which are utilized to infect in vitro-cultured chicken PGCs. These two kinds of genetic modified PGCs are injected into the dorsal aorta of 2.5-d old embryos ,which are incubated until hatching.Transgenci roosters(GO) are screed for EGFP with PCR.To generate G1 and G2 transgenic chickens,the transgenic chickens are mated with wild-type hens.With CT-conversion and bisulfite sequencing of genomic DNA, RT-PCR detection of DNA methyltransferase(DNTMs), and chromatin immunoprecipitation analysis of methyl CpG binding protein( MBDs), CpG methylation status in tissues of oviduct and other organs are investigated , and the relationship between the CpG methylation and EGFP expression level in these tissues are analysed ,which will contribute to learn modulation mechanism of DNA methylation in oviduct expression of a transgene in chickens.