Hong Fan1, Jun Xu, Shouwei Wu, Zhujiang Zhao, Jianqiong Zhang, Wei Xie. 1. State Education Ministry Laboratory of Developmental Genes and Human Disease, Jiangsu Provincial Key Laboratory of Gene Diagnosis and Therapy, Genetics Research Center, Southeast University Medical School, Nanjing, Jiangsu, 210009 P. R. China.
Abstract
OBJECTIVE: To construct the specific stable expression and high efficiency small interfering RNA(siRNA) expression vector that can block DNMT1 gene function. METHODS: Using vector-based RNA interference technique, the authors constructed a vector to transcribe functional short interfering RNA (RNAi). After transfection by lipofectmine (TM) reagent, the treated cells were selected by G418. The expression levels of RNA and protein of DNMT1 were analyzed by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting. The status of methylation of E-cadherin was analyzed by methylation-specific PCR(MSP). RESULTS: The expression level of endogenous DNMT1 mRNA in transfected SMMC-7721 cell lines with DNMT1 RNAi construct was 43% less than that in control cell 7721-pSU cell lines. The protein level in the former was about 10% less than that in the latter. The efficiency of the siRNA of DNMT1 was found to be higher than 90%. Demethylation of promoter of E-cadherin was obtained due to the inhibition of DNMT1. CONCLUSION: DNMT1 siRNA stable expressing vector was obtained by gene-recombined technology. There was no complete sameness between the levels of protein and RNA in gene silenced cell lines. The efficiency of the siRNA should be confirmed by Western-blotting.
OBJECTIVE: To construct the specific stable expression and high efficiency small interfering RNA(siRNA) expression vector that can block DNMT1 gene function. METHODS: Using vector-based RNA interference technique, the authors constructed a vector to transcribe functional short interfering RNA (RNAi). After transfection by lipofectmine (TM) reagent, the treated cells were selected by G418. The expression levels of RNA and protein of DNMT1 were analyzed by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting. The status of methylation of E-cadherin was analyzed by methylation-specific PCR(MSP). RESULTS: The expression level of endogenous DNMT1 mRNA in transfected SMMC-7721 cell lines with DNMT1 RNAi construct was 43% less than that in control cell 7721-pSU cell lines. The protein level in the former was about 10% less than that in the latter. The efficiency of the siRNA of DNMT1 was found to be higher than 90%. Demethylation of promoter of E-cadherin was obtained due to the inhibition of DNMT1. CONCLUSION:DNMT1 siRNA stable expressing vector was obtained by gene-recombined technology. There was no complete sameness between the levels of protein and RNA in gene silenced cell lines. The efficiency of the siRNA should be confirmed by Western-blotting.