Transgenic Cashmere Goat Of Hair Follicle Cells Specific Expression Thymosin Beta 4

Transgenic animal technology, based on cytogenetics, molecular genetics, embryonic development and recombinant DNA technology, is one of most rapid development new morden biotechnology in the 21st Century. The somatic cells cloning technology has been a hot research areas since the "Dolly" was born in the early 1997s, and has got a broad application in the transgenic livestock production. This technique was adopted in our research. One expression vector which specifically expressed thymosin beta 4 (Tβ4) in hair follicle cells was constructed initially and successfully. The pKAP6.1 expressed specifically in hair follicles, was used as the promoter of Tβ4, and neomycin resistance gene (neor) and a red fluorescence protein gene DsRed2 were used as selectable markers. The expression vector was transfected in fetal fibroblasts of cashmere goat in vitro. The positive clones with red fluorescence were screened by G418. The transgenic cloning embryos of cashmere goats were obtained by somatic cell nuclear transfer technique firstly. And through the embryo transplantation, transgenic cashmere goats were produced. Our research lays a foundation for acquiring the transgenic cashmere goats and the further study on effects of Tβ4 on regulating hair follicle development and growth cycle.1. The construction of hair follicle cells specific expression vector for Tβ4 The DNA sequence of the promoter of keratin associated proteins 6.1 gene and T04 cDNA sequence were obtained from ovine total DNA and hircine total RNA by PCR and RT-PCR respectively, then linked with T-vector pMD19T to get an intermediate construction p19TKT. At the next step, the foundation of pCDsRed2 made a good winning by way that the CMV promoter sequence was linked on the upstream of DsRed2 reporter gene between Kpn I and Sma I sites. Finally pCDsR-KT, one of the hair follicle cells specific expression vector, was accomplished by linkage of pCDsRed2 linearized with EcoR I+Hind III and KT fragment digested from p19TKT.2. In vitro culture of cashmere goat fetal fibroblast cells and gene transfectionThe caprine fetal fibroblast cells (CFFCs) were successfully isolated by attachment of tissue pieces from a fetus of cashmere goat. The CFFCs were transfected with the mixture of linearized pCDsR-KT and LipofectamineTM 2000, the positive clones with red fluorescence were screened by G418. The transgenic cells' sex diagnosis and exogenous gene's integration were made by ploymerase chain reaction. Morphology and growth character were observed and recorded, the growth curves of the CFFCs and transgenic cells were drawn and the chromosomal analysis of CFFCs and transgenic cells were performed.The transgenic cells with red fluorescence were screened by G418. The exogenous gene has been integrated into genomes of the transgenic cells. Comparative analysis suggested that the transgene and in vitro screening did not cause abnormally growing. Both CFFCs and transgenic cells contained normal chromosome number consisting of 60 chromosomes (2n=60). The rates of the chromosomal normality were 76.7%(23/30) and 83.3%(25/30) accordingly. The transgenic cells should be competent of transgenic cloning. 3. Production of transgenic cashmere goat embryos by sometic cell nuclear transferThe dissection method was used to recover cumulus oocyte complexies (COCs) from cashmere goat ovaries. Oocytes were matured in vitro, and then the first polar body and nucleus of matured oocytes were aspirated through micromanipulation method, then the transgenic donor cells were transferred to the anucleated oocyte. The donor cell-enucleated oocyte complexies were fused by electronic methods. The activated complexies were cultured in vitro, and 4-8 cells stage transgenic embryos were transplanted into the oviduct of recipient goat. Under the condition of this research, the rates of oocytes maturation were 77.5%, survival rates after micro-manipulation were 93.6%, the rates of fusion were 82.4%, cleavage rates were 93.5%, the rates of 8-cell stage embryos were 24.5%, the blastocysts rates were 14.3%. Identification of the genomic DNA of transgenic 8-cell stage embryos and transgenic blastocysts by ploymerase chain reaction proved that the exogenous gene have been integrated into genomes. Red fluorescent protein was detected in transgenic 8-cell stage embryos and transgenic blastocysts.Oocytes were matured in vitro and activated by different protocols in order to offer an effectiveness method for the activation of somatic cell nuclear transfer of goat. The matured oocytes were treated with IA23187+6-DMAP, Ionomycin+6-DMAP,7% ethanol+6-DMAP, IA23187+CHX, Ionomycin+CHX and 7% ethanol (?)CHX respectively. The blastocyst rates were higher significantly in IA23187+6-DMAP and Ionomycin+6-DMAP. IA23187 or ionomycine combined with6-DMAP after maturation is the optimal condition for goat oocytes activation.4. Production of transgenic cashmere goat and PCR identificationTransgenic embryos which produced by sometic cell nuclear transfer were transplanted into the oviduct of recipient goats. After the pregnant goat management and statistics, batchs of 19 transgenic cashmere goats were obtained. Identification of the blood genomic DNA of transgenic goats by PCR proved that the total of 19 cashmere goats were transgenic goats, the Tβ4 gene have been integrated into genomes of 15 transgenic goats. A part sequence of pCDsR-KT have been integrated into genomes of other 4 transgenic goats, the neomycin resistance gene (neor) have been detected from 3 of them, the total of 4 cashmere goats'genomes contain CMV promoter sequence.