Construction Of Edar Knockout Cashmere Goat Fetus Fibroblast Cell Line

Gene targeting technology emergenced in the1980s is a kind of biological technolog, that can careful modify genetic information. In this study Edar gene knockout goat fetus fibroblast cell line was built with gene targeting technology. The cycles of the hair follicle is a complex physiological process, that is closely linked to many genes. Goat hair follicles are divided into primary follicles and secondary follicles, the primary follicles develop into wool and secondary follicles develop into cashmere. The Edar gene encode a signal receptor molecule in the process of primary follicle development. The Edar gene belongs to the member of tumor necrosis factor superfamily, is only related to the development of ectoderm such as sweat glands, hair, feathers, and tooth. In this study the goat fetal fibroblasts of Edar gene knockout was built by Cre-LoxP recombination system, so that to understand the effects of Edar gene on goat hair follicle development.Keratinl4promoter was cloned correctly, and the eukaryotic expression vectors that started a red fluorescent protein gene by K14and CMV promoter were constructed respectively, and then transfected them into goat fetal fibroblasts cells, epidermal cells and primary dermal papilla cells, respectively. Six kinds of positive cells transfected by pK14-DsRed vector and pCMV-DsRed vector were obtained. Compared the difference expression of red fluorescent protein gene in that six kinds of positive cells by real-time PCR method. The results were found that the efficiency of startup the exogenous gene by the K14promoter in goat fetal fibroblasts cells was lower than that in epidermal cells and primary dermal papilla cells, and the expression of exogenous gene was tissue-specific.Correctly cloned cashmere goat’s part of the Edar gene cDNA sequence, and obtained nearly4Kbp genome sequence of both sides of the12th exon on Edar gene. The suitable fragment as homologous arm of knockout vector was choosed, and the length of upstream homologous arm was3058bp, downstream homologous arm was3765bp.Two knockout vectors of pKnock-Edar and pK14-Cre were constructed successfully. With linear pKnock-Edar plasmid transfection of goat fetal fibroblasts, resistant cells were obtained through drug screening, and the resistance cells were cultured for enlargement by monoclonal cell cultivation methods. A knockout positive cell line was identified by polymerase chain reaction, and was carried on the pK14-Cre plasmid transfection. Finally a cell line that can be used for the preparation of the Edar gene knockout cashmere goat was obtained.