Abstract:

Objective To construct a small interfering RNA（siRNA） eukaryotic expression vector specific for methylene tetrahydrofolate reductase（MTHFR） gene and to observe its silencing effect on MTHFR gene. Methods The expression vectors of PsiRNA-MTHFR were constructed by gene recombination and then were nucleofected into the primary cultured MEPM cell. At 48 h and 5 d after nucleofection, the expression of MTHFR in the levels of mRNA and protein was detected by real-time quantitative polymerase chain reaction（Real-Time PCR） and Western blot. Results The eukaryotic expression vector of PsiRNA-MTHFR, which significantly down-regulated mRNA and protein of MTHFR at 48 h and 5 d after nucleofection, were successfully constructed. Conclusion Eukaryotic expression vector of siRNA specific for MTHFR is successfully contructed, which lays the basis for its application in the mechanism research of MTHFR gene regulating embryo palate shelves fusion.

Key words: methylene tetrahydrofolate reducetase gene, RNA interference, eukaryotic expression