Production Of Transgenic Cashmere Goat Of Over Expression Neuropilin-1 And It’s Signaling Pathway In Secondary Hair Follicle

Neuropilin-1(Neuropilin-1.NRP-1) is a transmembrane glycoprotein. NRP-1, as a new type of cell surface receptor, promotes the formation of blood vessels by mediating the VEGF pathway. Experimental results show that NRP-1 is mainly through the VEGFR-2 pathway to increase the expression of VEGF. The formation of blood vessels was inhibited in the transgenic mouse model of knockout NRP-1. According to the transcriptome sequencing data of primary hair follicle and secondary follicle in the laboratory, we found that the expression of NPR-1 in primary and was 3.16 times than that in secondary hair follicles. Based on the above phenomena and theory, the technical route of NRP-1 transgenic cashmere goat model production was to established in this study, so that to observe the function of NRP-1 in secondary follicle growth and development. Meanwhile mass spectrometry analysis, immune coprecipitation, Western bolt, fluorescence quantitative PCR, mammalian two hybrid system etc. were used to further explore the NRP-1 related signal pathway and its function in secondary follicle growth.1. Establishment of NRP-1 transgenic cashmere goat model.NRP-1 transgenic expression vector was constructed and transfected into Alpas cashmere goat fetal fibroblasts, and NRP-1 over expressed transgenic Alpas cashmere goat embryos were produced by nuclear transplantation method. Results showed that the overexpression of NRP-1 transgenic vector was well constructed, and 33 NRP-1 positive transgenic monoclonal cell lines were obtained. After nuclear transplantation,154 transgenic clone embryos were obtained and transfered to 55 recepients. Although live transgenic kids were not obtained, the results will be benefit to establish of cashmere goat transgenic model.2.NRP-1 related signal pathway in secondary follicleDuring establishment of NRP-1 transgenic cashmere goat modle,, NRP-1 related signal pathway in secondary follicle was detected as well.NRP-1 interacting proteins were precipitated with NRP-1 precipitation in secondary dermal papilla cells, and the proteins were separated by SDS-PAGE electrophoresis. The protein bands were observed after Coomassie brilliant blue staining, and the identification of protein structure was analysed by protein mass spectrometry detection method. Through the analysis of literature and database, Prdx2 and CAT were identified as the proteins that might interacted with NRP-1. The interaction between NRP-1 and Prdx2 was confirmed by mammalian two hybrid system. The expression NRP-1 and Prdx2 RNA and protein was positively correlated by real-time fluorescent quantitative PCR and Western blotting method.In conclusion, the proceduce of NRP-1 over expression transgenic goat model production was established on the premise of that NRP-1 effects on VEGF pathway. The role of NRP-1 in the VEGF pathway was explored preliminarily, and the proteins interacting with NRP-1 were screened and identified. The results provide theoretical and experimental basis to understand the mechanism of secondary follicle growth related signaling pathway.