Generation Of Transgenic Mice And Transgenic Sheep Of Skin-specific Expressed Wnt10b Gene

Wnt10b is a member of Wnt family,which has been reported to play various roles in the immune system,mammary gland,adipose,bone,and skin.Wnt10b is known as a strong candidate for the "First Epithelial Signal" operating in hair follicle morphogenesis and self-renewal of hair follicles.The recent studies demonstrated that Wnt10b promoted the differentiation of immature epithelial cells toward hair shaft and IRS lineages potentiates hair shaft growth via canonical pathway,suggested it may play an important role in hair follicle development by promoting differentiation of epithelial cells.Our conclusions are summarizes as follow:1)We cloned KAP6.1 promoter,extended the sequence from-1,042 to-1,523bp.Transient transfection results demonstrated that the presence of the 5'-flanking region from-1,042 to-1,523bp increased the transcriptional activity of the promoter.Within this region,we identified putative binding sites for several transcription factors,such as NF-kappa-B/p65,AP-1,and C/EBP-a.Based on the analysis of prediction results,we performed EMSA to detect binding activity.The EMSA result demonstrated that the NF-kappa-B protein bound specifically to KAP6.1 promoter in sFFCs.2)According to the homology alignment of varies species,we cloned the full-length of sheep Wnt10b gene.The expression of WntlOb mRNA was detected in the heart,liver,spleen,lung,kidney,skin,muscle,and the adipose tissue.Expression levels were maximal in the lung and the adipose.We identified WntlOb-AS,a novel splice variant lacking of exon 4,can lead to a frame shift mutation putative encoding a shorter protein in Merino sheep skin.Using confocal laser scanning microscope,we analyzed dynamic subcellular localization process of fusion protein WntlOb-EGFP/Wnt10-AS-EGFP in sFFCs.The results showed that the fusion protein Wnt10b-EGFP firstly appeared in the cytoplasm,then moved across the plasma membrane,and finally presented in extracellular matrix,the process was similar to the localization patterns occurred in human and mouse.However,the Wnt10b-AS-EGFP was not observed in any of the process.The Wnt10b-AS was more efficient in regulating the Wnt signaling pathway than Wnt10b.We predicted that Wnt10b-AS might exist at transcription level,having similar functions with Wnt10b but via different molecular mechanism.3)We constructed skin-specific expression vector of KAP6.1-Wnt10b.Five transgenic mice(FO generation)were obtained by prokaryotic injection.Quantitative RT-PCR showed that the WntlOb,frizzled,and ?-catenin gene were highly expressed in skin tissue of 66#mice.We observed the morphology and density of hair follicle in skin of transgenic mice.The result of frozen sections in 66#mice showed that the time point of hair follicle undergo from anagen to telogen was delayed in transgenic positive mice,compaired with negative individuals.The density of hair follicle in 29#and 66#mice was higher in transgenic positive mice within littermates of the same sex.4)We obtained the fragment of KAP6.1 promoter and Wnt10b gene by digestion KAP6.1-Wnt10b expression vector and then produced transgenic sheep by prokaryotic injection.177 embryos were obtained by superovulation treatment with 18 donors.166 embryos were transplanted into receptors and a total of 21 offspring were obtained.The wool quality of two transgenic sheep W002 and W003 were better than negative individuals.WntlOb and ?-catenin might be candidate for improving wool quality.