Generation Of Transgenic Mice And Transgenic Sheep Of Skin-specific Expressed ?-catenin Gene

?-catenin is a multifunctional molecule that functions as a crucial effecter in both cell-to-cell adhesion and Wnt signaling.Studies have shown that ?-catenin plays an important role in hair follicle morphogenesis and hair follicle stem cell differentiation.Abnormal expression of ?-catenin causes the hair follicle formation blocked during embryogenesis and the hair loss after birth.To gain a better understanding of its role in the development of hair follicles,we cloned the cDNA sequence of the?-catenin gene from the skin of sheep,performed a variety of bioinformatics analyses and produced the transgenic model of mouse and sheep.According to the homology alignment of various species,we firstly cloned the full-length sequence of ovine ?-catenin and analyzed the structure of ?-catenin protein.The expression of?-catenin mRNA was detected in the heart,liver,spleen,lung,kidney,skin,muscle,and adipose tissue.Expression levels were maximal in the lung and minimal in the muscle,and the difference in expression in these tissues was significant(P<0.01).Western blot analysis revealed the presence of the ?-catenin protein in all tissues examined;expression was highest in spleen and lowest in the skin and adipose tissues,and the difference in expression in these tissues was significant(P<0.01).We cloned the skin-specific expressed promoter K14 and constructed the skin-specific expression fusion vector of K14-?-catenin-IRES-EGFP.Ten transgenic mice(F0 generation)were obtained by prokaryotic injection.Quantitative RT-PCR showed that five of the positive mice skin-specifically expressed the ?-catenin gene,and the gene highest expressed in skin of 59#mouse.Vivo imaging system detects the green fluorescent protein expression in skin tissue of 59#mouse.We obtained the linear fragment of K 14-promoter and ?-catenin by endonuclease digestion from K14-?-catenin-EGFP vector and produced transgenic sheep by prokaryotic injection.193 embryos were observed by superovulation treatment with 19 donors.158 embryos were transplanted into 64 receptors.38 recipients pregnant and a total of 51 live offspring were obtained.Nine transgenic lambs were obtained by identification of PCR.Wool quality detection showed that the wool length of B025 is the longest in all the lambs.Compared with the 6-monthage performance,the wool diameter of B025 at 12-month age is even thinner.So B025 can be used as excellent provenance for further passage test.