NCAM L1小基因模型的建立及其剪接模式研究

Establishment of NCAM L1 minigene model and its splicing patterns in different cell lines

目的:建立可用于前体mRNA可变剪接分析的NCAM L1小基因模型。方法:以人基因组DNA为模板,通过PCR扩增获得NCAM L1小基因片段,并将其克隆至真核表达载体中,构建小基因的质粒。在此基础上,用NCAM L1小基因模型转染HeLa、COS-1、PFSK及R28细胞,并用RT-PCR进行被剪接的小基因产物的半定量检测。结果:NCAM L1小基因在4种细胞中的剪接模式不同,在PFSK以及Hela细胞中,存在2种剪接亚型,而在COS-1以及R28细胞中只有一种剪接亚型存在。结论:所构建的NCAM L1小基因可用于细胞水平的基因剪接分析。

Objective:To establish a minigene model of neural cell adhesion molecule L1(NCAM L1)gene and to study its aplicing patterns in different cell lines.Methods:Using human genetic cDNA as template,the NCAM L1 minigene fragment was amplified and inserted into eukaryotic expression vector.The minigene was transfected into 4 cell lines and the splicing patterns of NCAM L1 minigene in these cell lines were studied.Results:The splicing patterns of NCAM L1 minigene were different in individual cell lines.In PFSK and Hela cell lines,two splicied isoforms were generated but in COS-1 and R28 cell lines,only one isoform existed.Conclusion:NCAM L1 minigene model can be used in alternative splicing analysis.