Suppression Of FGF5 Expression In Fetal Fibroblasts Of Mice By RNA Interference

In this experiment,the RNAi has been used to knock down the expression of fibroblast growth factor 5(FGF5) in fetal fibroblast cells derived from eGFP transgenic mice.Firstly,three shRNA targeting three different regions(316～335bp,499～518bp and 766～785bp) of FGF5 mRNA were designed,then synthesized and annealed to double strands.Secondly,the shRNA templates were ligated into psilencer 3.0 which had H1 promoter to drive shRNA synthesis.Then the H1 and shRNA was cut simultaneously.Thirdly,the H1 and shRNA was inserted into pCDsR which has a red fluorenscent protein expression cassette,and then named as pCDsR-shRNA316(V_Ⅰ),pCDsR-shRNA499(V_Ⅱ),pCDsR-shRNA766(V_Ⅲ)。Meanwhile,the fibroblast cells were cultured.After that the shRNA vectors were respectively transfected into the eGFP cells,and the total RNA were extracted,then reverse transcribed to cDNA.Subsequently the cDNA were detected by the method of SYBR GREENⅠReal Time PCR.The result is that the expression of FGF5 has been knocked down for 6.7 times with the transfeced into the vector V_Ⅰ;The expression of FGF5 has been knocked down for 12.5 times with the transfeced into the vector V_Ⅱ;there is not obvious gene expression silence with the transfeced into the vector V_Ⅲ.The conclusion is that in FGF5 mRNA of mice,the region of 766～785bp is not effective RNAi region;the region of 316～335bp,499～518bp are effectinve RNAi region,and the region of 499～518bp is the most effective one.The result is vital for subsequent study on the silence of other growth factor genes,and also providing data to establish RNAi transgenic mice model.