甲状腺髓样癌家系RET基因点突变质粒及稳定细胞系构建

Plasmid and stable cell linereconstruction of RET point mutation in medullary thyroid carcinoma pedigree

目的构建中国大陆甲状腺髓样癌RET基因常见突变位点的质粒并验证其表达。方法分析中国大陆甲状腺髓样癌家系RET基因,进行RET基因外显子高通量"二代"测序分析。体外构建RET基因相应位点突变并研究在NIH3T3细胞中的表达水平。结果通过对野生型RET质粒进行点突变PCR、克隆测序后,成功获得相应位点点突变慢病毒质粒。经过慢病毒感染NIH3T3细胞,Western Blot验证目的基因为稳定表达。结论成功构建了载有RET基因突变的稳定细胞系,为研究RET不同位点突变提供了良好平台。

OBJECTIVE The plasmid construction and validation of RET point mutation in vitro.METHODS The RET gene exon in the mainland familial medullary thyroid carcinoma family was analized with muon capture ＂two generation＂ sequencing of.In vitro,the RET relative point mutationswere reconstructed in NIH3T3 cells and the expression levels were studied.RESULTS The corresponding lentiviral plasmids of RET point mutations were successfully obtained after point mutating the wild RET plasmids,it was verified that the target genes were expressed in NIH3T3 cells stably by Western Blot.CONCLUSIONS Stable cell lines carrying RET point mutations were reconstructedsuccessfully,which provide a good platform for studying various point mutations.