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

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.

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。

（1）克隆了6 个不同长度（3.6kb，2.6kb,1.9kb,1.7kb,1.6kb,1kb）的卵清蛋白启动子，并构建了相应的荧光素酶表达载体。.（2）经荧光素酶检测，1.6kb 启动子活性最高，3.6kb，1.9kb 启动子次之。.（3）构建了上述三个不同长度启动子调控的EGFP表达载体，慢病毒包装后感染293FT 细胞，同样显示1.6kb启动子活性最强。.（4）优化了慢病毒包装方法，使浓缩后的慢病毒滴度达到了1×109 TU/ml以上。.（5）利用大鼠肝脏细胞（BRL）制备条件培养基、STO 细胞作为饲养层，对分离自6日龄鸡胚性腺的原始生殖细胞（PGC）进行了初步鉴定和传代培养，并利用浓缩后的慢病毒对原代PGC 进行了体外感染实验。.（6）从青年产蛋鸡的输卵管中分离和培养输卵管上皮细胞，并对其特性进行了初步鉴定。这为应用输卵管上皮细胞体外研究卵清蛋白表观遗传机制和转录调控机制创造了条件。.（7）经过艰苦训练，熟练掌握了2.5日龄鸡胚背主动脉注射技术；将慢病毒注射至2.5日龄鸡胚，在发育至7.5天的鸡胚心脏和性腺中观察到了EGFP表达。这为利用遗传修饰的PGC 注射2.5日龄鸡胚背主动脉制备转基因鸡奠定了基础。.（8）在本实验室建立了改良的换壳培养（surrogate egg shell culture）技术平台。将慢病毒或转座子（pCAGcopGFP）+转座子酶（piggyBacTransposase）注射受精蛋胚盘下腔，换壳培养，从发育至3天的胚胎和7.5天胚胎性腺中均观察到了EGFP表达。这为遗传材料直接注射胚盘下腔制备转基因鸡奠定了基础。.（9）借助甲基化特异性PCR，对已构建的3个慢病毒载体中卵清蛋白启动子（长度不等）甲基化位点进行了检测。.（10）慢病毒感染输卵管上皮细胞，培养液中添加去甲基化试剂，实时定量PCR检测EGFP表达，未达到预期目的。