Study Of Shanbei White Cashmere Goat FGF5 Gene And Establishment Of Transgenic Goats

Cashmere goats pledge skin contains two distinct types of hair follicle: the primary hair follicles(PHF) bear guard hairs which are characteristically medulated; the secondary hair follicles(SHF) are more numerous than primary follicles and produce nonmedulated fine cashmere fibres. Cashmere is a superior type of textile material with the reputation of “soft golden”. Commerial value of cashemere fibres is mainly determined by its diameter, staple length and cashmere yield. One of improtant quality standards of cashmere traits is staple length. Fibroblast growth factor 5(FGF5) has been identified as an important genetic regulator of hair fibre length by far; studies shown that FGF5 and FGF5-short(FGF5s) which is generated by m RNA alternative splicing are both involved in the regulation of hair cycle. Therefore, FGF5 of Shanbei White cashmere goat was used as our subject. Firstly, the tissue distribution and temporal expression in skin expression of FGF5 and FGF5 s were invastigated by real-time q PCR. Then, by overexpressed or co-overexpressed FGF5 and FGF5 s in dermal papilla cells of PHF/SHF to investigate whether the cashmere goat FGF5 could block the activity of anagen dermal papilla cells and the relationship between FGF5 and FGF5 s. In the end, based on Piggy Bac transposon, FGF5 s stable transfection of Caprine fetal fibroblasts was constructed. And transgenic cashmere goats were produced by using somatic cell nuclear transfer technology.The main results are as follow:1. Two transcripts of cashmere goat FGF5 were cloned for the first time: the length of the CDS in FGF5 is 813 bp, encoding a protein of 270 amino acid residues. The c DNA of FGF5 s contained a CDS of 378 bp encoding a protein of 125 amino acid residues. Alignment of the FGF5 CDS with that of FGF5 s showed that the complete exon 2 has been deleted in FGF5 s by alternative splicing. According to the tissue distribution anlysis we found that high expression levels of FGF5 m RNA were detected in brain, rumen and skin; while FGF5 s m RNA was high expressed in subcutaneous fat, heart and skin. In addition, the temporal expression analysis further showed that the expression of two transcripts varied throughout the cashmere goat hair growth cycles. FGF5 m RNA expression levels were significantly higher than FGF5 s at anagen(P<0.05) and telogen(P<0.05), yet no significant m RNA expression differences were found between FGF5 and FGF5 s at catagen(P>0.05). The highest expression level of FGF5 was observed at telogen; however, that of FGF5 s was found at catagen. Moreover, expression levels of FGF5 and FGF5 s both drop to the lowest point at the anagen.2. Recombinant adenovirus carrying cashmere goat FGF5 and FGF5 s complete CDS fragments were successfully constructed, and further packaged and amplified in HEK-293 A cell lines. Then the optimum MOI of Ad-FGF5 and Ad-FGF5 s infected the dermal papilla cells of cashmere goat PHF/SHF is 300.3. The dermal papilla cells of cashmere goat PHF/SHF were isolated from cashmere goat pledge skin. The expression patterns of two markers(?-SMA and CD133) were analysed by using immunocytochemistry in the two types of cultured dermal papilla cells.4. Real-time q PCR results indicated that overexpression of FGF5 in dermal papilla cells of SHF downregulated the m RNA expression of Noggin(P<0.05), IGF-1(P<0.01) and Versican(P<0.01) significantly, and the decrease of Noggin m RNA was the most; the expression of BMP4 m RNA was upregulated about 2.62 times. No significant m RNA expression alterations of these four factors were observed in dermal papilla cells of SHF only overexpressed FGF5s(P>0.05). However, the effects of FGF5 overexpression on noggin, IGF-1, versican and BMP4 in dermal papilla cells of SHF were partly reversed after FGF5 s overexpression. Similar results were also observed in dermal papilla cells of PHF.5. A stable tansgenic fibroblast line which hair-follicle-specific expressed cashmere goat FGF5 s based on Piggy Bac transposon was estabilshed. And 4 transgene cashmere goats were obtained by using somatic cell nuclear transfer technology.In summary, the effects of anagen relative factors affected by two transcripts of Shanbei white cashmere goat FGF5 gene in anagen dermal papilla cells were studied for the first time; and Shanbei white cashmere goat FGF5 s acted as an inhibitor of FGF5. Four FGF5 s transgene cashmere goat was obtained by somatic nuclear transfer technology, provided basis for cultivating new cashmere goats with high quanlity cashmere products.