Studies On ShRNA Interference Of Buffalo Fetal Skin Fibroblasts Hlc Gene Expression

In this study,according to buffalo Hlc sequence,we designed and synthesized nucleotide fragments which can interference Hlc express,and we constructed plasmid which can express the nucleotide.The empty plasmid transfection of buffalo fetal skin fibroblasts' system was optimized,by liposome mediated method.The optimized conditions are as the follow:plasmid concentration is 2?g/ml;liposome concentration is 5?g/ml;the complexes incubate for 30 min;and the optimal transfection efficiency is 30.87%.In order to determine the interference effect of plasmid,three interference plasmids and one empty plasmid were transferred into buffalo fetal fibroblasts respectively,and qPCR was used to detected buffalo Hlc gene expression in the cell after transfecti for 24 h and 48 h.The results showed that the transfected cells with three kinds of interference plasmids can be reduced in different degree of buffalo Hlc gene expression,and the interfere effect after transfection for 48h was better than 24h,indicating that we have successfully constructed three kinds of effective interference plasmids which can interfere with cell Hlc gene express.Since then,in order to study the e:ffect of knock down the Hlc gene's express on cell growth,we observed the transfected cells' morphology in 24 h and 48 h after transfection.Results,showed that after knocking down Hlc gene's expression,cells' growth was restrained,even lead to cell death,suggesting that the buffalo's Hlc gene's correct expression play an important role in cell growth and division.Subsequently,to select the stable transfected cells,we designed two experiments.First of all,the cell sensitivity of buffalo fetal skin fibroblasts was tested with blasticidin.Results showed that the lowest concentration of blasticdin in 10 days to kill all the cells is 2.6 ?g/ml.So the concentration of blasticdin to isolated positive transgenic cells was set as 2.6 ?g/ml.Second,under the condition of the optimal transfection,we transfected three kinds of interfere plasmids into cells,and using drug screening to obtain stable transfected cells.In this experiment,only the transfected cells with empty plasmid get cloned spots,while the transfected cells with three kinds of interference plasmids have died in the drug-screening process.We guess that the interference of the cells themselves endogenous genes affected the cell growth and division.In conclusion,we studied the functions of the buffalo Hlc.By QPCR method proved that we have successfully constructed the vectors,which could effectively knockdown cells' Hlc expression.Vectors were transfection into cells by liposome mediated method,and knock down Hlc gene's expression,which affects the normal cells division and growth,even leading to cell death.To sum up,Hlc plays an irreplaceable role in buffalo fetal skin fibroblasts.