Molecular cloning and characterization of differentiation-inducing factors into the cardiac myocyte lineage using an expression cloning approach

由于胚胎干细胞（ES）能够自我更新并根据适当的信号分化为特化细胞，因此胚胎干细胞被认为是治疗心脏病的功能性心肌细胞的有希望的来源。然而，迄今为止，胚胎干细胞向心肌细胞的体外分化仍然效率低下，其分子机制尚未完全阐明。因此，鉴定分化诱导因子进入心肌细胞谱系已成为了解心肌细胞分化和心脏发育的分子机制的关键，并促进胚胎干细胞在心脏病中的治疗应用。在本研究中，我们试图利用表达克隆方法识别分化诱导因子进入心肌细胞谱系。我们用DMSO诱导小鼠胚胎癌细胞系P19CL细胞向心肌细胞分化。我们利用从分化细胞中分离的mRNA构建cDNA文库。然后我们生成了表达cDNA文库的重组逆转录病毒。在心脏特异性α-MHC启动子转录控制下表达EGFP的ES细胞克隆被表达cDNA文库的重组逆转录病毒感染。从分化细胞中提取Gemonic DNA到心肌细胞中，并进行PCR分离整合的DNA到基因组DNA中。我们获得了G蛋白β多肽2样1(Gnb2I1, Rack1)，氧化酶组装1样1(Oxa1l)， glutaredoxin 5同源物（Glrx5）和极光激酶A相互作用蛋白1（Aurkaip1）作为分化诱导因子进入心肌细胞谱系的候选基因。我们正在研究它们在胚胎干细胞向心肌细胞分化中的作用。我们的研究将为心肌细胞分化的分子机制提供新的见解，并为胚胎干细胞定向分化为心肌细胞提供新的方法。

Since embryonic stem (ES) cells are capable of self-renew and differentiation into specialized cells in response to appropriate signals, ES cells are proposed as a promising source of functional cardiac myocytes for cardiac diseases. However, to date the in vitro differentiation of ES cells into cardiac myocytes remains inefficient and its molecular mechanisms are not fully elucidated. Therefore, the identification of differentiation-inducing factors into the cardiac myocyte lineage has become critical for understanding the molecular mechanisms of differentiation into cardiac myocyte and heart development, and facilitates therapeutic applications of ES cells in cardiac diseases. In the present study, we sought to identify differentiation-inducing factors into the cardiac myocyte lineage using an expression cloning approach. We induced differentiation of the mouse embryonic carcinoma cell line, P19CL cells, into cardac myocytes using DMSO. We made cDNA libraries using mRNA isolated from differentiating cells. We then generated recombinant retroviruses expressing the cDNA libraries. ES cell clones that express EGFP under the transcriptional control of a cardiac-specific α-MHC promoter were infected with recombinant retroviruses expressing the cDNA libraries. Gemonic DNA was extracted from differentiated cells into cardiac myocytes and performed PCR to isolate the integrated cDNAs into the genomic DNA. We obtained G protein beta polypeptide 2 like 1 (Gnb2I1, Rack1), oxidase assembly 1-like (Oxa1l), glutaredoxin 5 homolog (Glrx5), and aurora kinase A interacting protein 1(Aurkaip1) as candidate genes for differentiation-inducing factors into the cardiac myocyte lineage. We are studying their functions in differentiation of ES cells into cardiac myocyte. Our study will provide novel insights into the molecular mechanisms of cardiac myocyte differentiation and the development of novel approaches for the directed differentiation of ES cells into cardiac myocytes.