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

由于胚胎干细胞能够在适当的信号作用下自我更新和分化为特化细胞，因此胚胎干细胞被认为是一种很有前途的心脏疾病功能心肌细胞来源。然而，到目前为止，ES细胞体外分化为心肌细胞的效率仍然很低，其分子机制还不完全清楚。因此，识别分化为心肌细胞系的诱导因子，对于理解分化为心肌细胞和心脏发育的分子机制，以及促进ES细胞在心脏疾病中的治疗应用具有重要意义。在目前的研究中，我们试图使用表达克隆的方法识别心肌细胞谱系中的分化诱导因子。我们用DMSO诱导小鼠胚胎癌细胞系P19CL细胞分化为Cardac肌细胞。我们利用从分化细胞中分离的mRNA构建了cDNA文库。然后，我们构建了表达这些文库的重组逆转录病毒。在心脏特异性α-MHC启动子的转录控制下表达绿色荧光蛋白的ES细胞克隆被表达该文库的重组逆转录病毒感染。从分化的细胞中提取Gemonic DNA，转化为心肌细胞，进行聚合酶链式反应，分离整合到基因组DNA中的cDNA。我们获得了G蛋白β多肽2样蛋白1(Gnb2I1，Rack1)、氧化酶组件1样蛋白1(Oxa11)、谷氧还蛋白5同系物(Glrx5)和极光蛋白A相互作用蛋白1(Aurkaip1)作为心肌细胞系诱导分化因子的候选基因。我们正在研究它们在ES细胞分化为心肌细胞中的作用。我们的研究将对心肌细胞分化的分子机制提供新的见解，并为ES细胞定向分化为心肌细胞提供新的途径。

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.