滤纸挑选克隆方法在筛选稳定表达细胞株中的应用

Application of filter paper method in screening of stable expression cell lines

目的:利用滤纸挑选克隆方法简单、有效地筛选稳定表达VEGF-C shRNA的乳腺癌MCF-7细胞株。方法:用已构建的表达VEGF-C shRNA的重组质粒载体pSIREN-VEGF-C转染乳腺癌MCF-7细胞,利用嘌呤酶素筛选阳性克隆;以滤纸法挑选阳性克隆并扩大培养成稳定表达的细胞株;荧光定量PCR及蛋白质印迹法检测稳定表达的细胞株中VEGF-C的表达情况。结果:用滤纸法挑选并培养出的稳定表达细胞株中,VEGF-C的mRNA及蛋白表达均明显下调,抑制率分别为95%及100%,P<0.05。结论:利用滤纸挑选克隆方法成功筛选稳定表达VEGF-C shRNA的乳腺癌MCF-7细胞株,并将其命名为MCF-7/V-C。

OBJECTIVE： To screen the MCF-7 cell lines with stable expression shRNA targeting VEGF-C gene by the filter paper method.METHODS： The recombinant vector（pSIREN-VE-GF-C） was transfected into human breast cancer cell MCF-7 by liposome.The positive transfected cell clones were selected with puromycin and screened by the filter paper method.After culture expansion,the stable cell lines were attained.The expression of VEGF-C in the stable cell lines was detected by the real-time quantitative PCR and Western blot assay,respectively.RESULTS： The expressions of VEGF-C mRNA and protein were decreased markedly in the stable cell lines compared with the control group and the inhibitive rates were 95% and 100% respectively（P〈0.05）.CONCLUSION： The MCF-7 cell lines with stable expression shRNA targeting VEGF-C gene screening by the filter paper method have been constructed successfully and named MCF-7/V-C.