转座子介导克隆及分析鸡胚早期心脏发育相关基因

Human health is threatened by congenital heart disease seriously, but the understanding of molecular mechanisms of early heart development is still relatively rough and a large number of unknown genes are waiting for exploiting. As a heart development model, chick embryos have unique advantages such as in vitro incubation, translucent, easy to observe, short development time, low research cost and etc. but due to the limitations of traditional techniques, the research progress of functional genomics in chick embryo heart is slow. Transposon-mediated gene trap technology is the latest generation of high-throughput exploiting platform of functional genomics, and the transposons, such as PB, whose stable integration of DNA and sustained expression in chick embryo have been verified. Funded by Youth Foundation and Postdoctoral Science Foundation of Jiangsu Province, the applicant have carried out electroporation optimization of chick embryo in vivo and preliminary studies of properties of transposons in chick embryo transfer. Current project is aim to construct gene trap vectors based on the recent optimized PB transposon and verified the trapping function at cellular level, then optimize the methods of electroporation of chick embryo heart cells, and trap genes related with early heart development in chick embryos. The project is expected to initially establish the study platform of functional genomics of heart development in chick embryo based on PB transposon-mediated gene trap technique, obtain genes related with cardiac development with independent intellectual property, and promote the research progress of molecular regulation mechanism of heart development.

先天性心脏病严重威胁着人类健康，但目前对早期心脏发育分子机制的认识还比较粗略，大量未知基因有待挖掘。鸡胚作为心脏发育研究模型具有体外孵化，半透明，易观察，发育时间短，研究成本低等独特优势，但由于传统技术限制，鸡胚心脏发育功能基因组学的研究进展缓慢。转座子介导的基因捕获技术是最新一代功能基因高通量的挖掘研究平台，PB等转座子介导DNA在鸡胚内稳定整合和持续表达已经得到验证。申请人在江苏省青年基金和博士后基金资助下开展了活体鸡胚电穿孔优化和转座子在鸡胚中转座特性等前期研究，本项目拟利用最近优化的PB转座子技术，构建基因捕获载体并在细胞水平进行验证，然后通过优化鸡胚心脏细胞活体电穿孔技术，尝试进行鸡胚早期心脏发育功能基因捕获研究。项目研究预期将初步建立以PB转座子介导基因捕获技术为基础的鸡胚心脏发育功能基因组学研究技术，获得具有自主知识产权的心脏发育相关基因，促进心脏发育分子调控机制研究进展。

心脏病严重威胁着人类健康，但目前对早期心脏发育分子机制的认识还比较粗略，大量未知基因有待挖掘。鸡胚作为心脏发育研究模型具有体外孵化，半透明，易观察，发育时间短，研究成本低等独特优势，但由于传统技术限制，鸡胚心脏发育功能基因组学的研究进展缓慢。转座子介导的基因捕获技术是最新一代功能基因高通量的挖掘研究平台，PB等转座子介导DNA在鸡胚内稳定整合和持续表达已经得到验证。本项目利用转座子技术，构建并优化一系列基因捕获载体并在鸡细胞系和斑马鱼进行验证，然后通过优化的鸡胚显微注射技术，尝试进行鸡胚早期心脏发育功能基因捕获研究。获得以下研究结果。.1.采用鸡蛋开窗法将 Sleeping Beauty (SB)单质粒转座载体 pT2-SV40-EGFP-SB11 注射至孵化 36 h 鸡胚盘。结果表明，EGFP 在鸡胚胎中获得较高的表达率(40%~89%)，本实验初步证明了经改良的鸡蛋开窗法可用于鸡胚转基因研究，并初步建立了 SB 转座子介导外源基因在鸡胚中转染方法。2.为了比较SB(Sleeping Beauty)、PB(PiggyBac)和Tol2 3种转座子介导的基因捕获效率，构建了基于PB、SB和TOL2三种转座子的启动子捕获载体pT3-PST-Pro-Trap。将此载体分别和三种转座酶mRNA注射斑马鱼一细胞期胚胎，结果表明： PB、SB和TOL2三组胚胎均能表达EGFP，说明在转座子介导下，启动子捕获载体可插入内源基因启动子下游，从而驱动 EGFP被表达。3.为了提高基因捕获效率，进一步优化基因捕获载体。构建了PB转座子介导的启动子和poly(A)捕获载体pPB-RG-trap。将载体注射斑马鱼胚胎，有较强绿荧光表达，表明该载体基因捕获效率较高，可用于鸡胚胎早期心脏发育功能基因捕获研究。4.将pPB-RG-trap注射HH10期鸡胚心管，检测结果表明，注射组ED4和ED8期鸡胚发育滞后于正常对照组，在心脏、肠、胃及皮肤出现绿色荧光，且心脏发育出现异常。再通过3’RACE克隆30个基因，经序列测定和比对拟递交至GenBank数据库。.本项目研究初步建立了以PB转座子介导基因捕获技术为基础的鸡胚心脏发育功能基因组学研究技术，获得具有自主知识产权的心脏发育相关基因，促进心脏发育分子调控机制研究。