Construction And Functional Analysis Of Skin-specific Expression Vector And Interference Vectors Of Tibetan Sheep Lhx2 Gene

The objective of this study was to construct Lhx2 gene(one of the LIM homeodomain family members)skin-specific expression vector and mi RNA interference vectors of Tibetan sheep,in order to explore possible regulation mechanism of Lhx2 gene in the growth and development of follicle cells at cellular level and molecular level.Healthy adult Tibetan sheep were used to clone Lhx2 gene from the skin tissues and construct a p CDs Red2-KL vector and four interference vectors,followed by transfection into HHFIRSC in vitro by lipofectamin 3000.Detect the m RNA and protein levels of Lhx2 gene and its downsteam target gene Notch1,Hes1 and ?-catenin genes by Real-time PCR and Western blotting.The main findings are as follows:(1)A 1221 bp CDS of Lhx2 gene of Tibetan sheep was cloned,which encodes406 amino acids.Compared with the predicted CDS sequence of sheep,there was only 1 silent mutation in the Lhx2 gene of Tibetan sheep,but no difference in the amino acid sequence.(2)Based on the p CDs Red2-KI eukaryotic vector,a skin-specific eukaryotic expression vector p CDs Red2-KL containing Lhx2 gene was constructed with KAP6.1promoter.Four interference vectors were constructed with pc DNA6.2-GW/Em GFP-mi R linear vector.These vectors were transfected into HHFIRSC in vitro by lipofectamin,respectively,with transfection efficiency of approximately80%.(3)The recombinant plasmid p CDs Red2-KL was transfected into HHFIRSC.Real-time PCR and Western blot analysis demonstrated that the Tibetan sheep Lhx2 gene was overexpressed(P<0.01),and the m RNA of Notch1 and Hes1 genes,downstream target genes of Lhx2,were significantly upregulated.Meanwhile,Notch1 and ?-catenin proteins were also significantly upregulated.(4)The four interference vectors were transfected into HHFIRSC.Real-time PCR and Western blot analysis demonstrated that no interference vector can inhibitm RNA expression of Lhx2 gene,but the Lhx2 protein levels were significantly down regulated by pc DNA6.2-GW/Em GFP-mi R?~? vectors,suggesting that the three vectors might inhibit the translation of Lhx2 gene to silence its expression.The silence effects of pc DNA6.2-GW/Em GFP-mi R?~? vectors on Lhx2 gene were 52.78%,19.44% and 36.11%,respectively.(5)With the decrease of Lhx2 protein expression,the Notch1 protein levels also decreased significantly in interference experimental groups,consisting with the results of overexpression studies.These results suggest that Lhx2 gene plays a positive regulation on Notch1 gene expression,and could regulate the function of skin follicle through Notch signaling pathway.