Production Of Transgenic Cashmere Goat Embryos For Insulin Like Growth Factor-Ⅰ Gene By Somatic Cell Nuclear Transfer

Transgenic animal technology has got a broad application and a great progress in many fields such as biomedicine,agriculture and even in basic biology since it was established in the early 1980s.And the application of this technology is ascending in the field of animal husbandry.The use of gene transfer exhibits a broad prospect in unconventional animal production lies in three aspects:the improvement of product quality and quantity,disease resistance,and the production of valuable proteins in the mammary gland.However,only after the somatic cells cloning technology was established,the study on the transgenic animals could really exhibit more extensive application prospects in large animals.At present,transgenic animals are obtained utilizing somatic cells transferred with exogenous genes through nuclear transplantation technique,which is the main technical route of making transgenic animals.This technique is adopted in our research.Two expression vectors which specifically expressed insulin like growth factorâ… (IGF1) in follicle cells were constructed initially and successfully.The pKAP6.1 and pUHS,which express specifically in hair follicles,were used as the promoters of IGF1.And,the neomycin resistance gene(neo^r) and a red fluorescence protein gene DsRed2 were used as selectable marker.The two expression vectors were 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.Our research lays a foundation for finally acquiring the transgenic cashmere goats and for the further study of effects of IGF1 on regulating hair follicle development and growth cycle.1 The construction of hair follicle cells specific expression vectors for IGF1The DNA sequence of the promoter of keratin associated proteins 6.1 gene and IGF1 cDNA sequence were obtained from ovine total DNA and total RNA by PCR and RT-PCR respectively,then linked with T-vector pMD19T to get an intermediate construction p19TKI.At the next step,the foundation of pCDsRed2 makes a good winning by way that the CMV promoter sequence is linked on the upstream of DsRed2 reporter gene between Kpnâ… and Smaâ… sites.Then,one of the hair follicle cells specific expression vectors,pCDsR-KI was accomplished by linkage of pCDsRed2 which was linearized with EcoRâ… and KI fragment digested from p19TKI.The DNA suquence of the another promoter of murine ultra high sulfur keratin proteins(UHS) was amplified by PCR from murine genomic DNA.Then PCR products and IGF cDNA fragments,as well as the cloning vector pMD19T were linked to be an intermediate vector p19TUI.IGF1 cDNA fragment which was digested by SalI/SphI(then formed a blunt end in the Sphâ… site by Klenow fragment treatment) from p19TKI,and pCDsRed2 linearized by Sacâ… /EcoRâ… ,then formed a blunt end in the EcoRâ… site by Klenow Fragment to form pCDsR-UI,another hair follicle cells specific expression vector for IGF1.The evidence from restriction analysis and partial DNA sequencing showed that the two expression vectors were both consistent with anticipation.2 Isolation and in vitro culture of cashmere goat fetal fibroblast cellsThe hircine fetal fibroblast cells(HFFCs) were successfully isolated by attachment of tissue pieces from a fetus of cashmere goat.The isolated cells were purified and proliferated in DMEM/F12+ 10%FBS media at 37℃in a humidified 5% CO2 incubator.Then the cells were cryopreserved within primary passage in DMEM/F12+20%FBS media which contains 10%of DMSO.Morphology and growth character were observed and recorded,the growth curves of the cells within 1^st,2^nd and 6^th passages were drawn and the chromosomal analysis of the cells within 2^nd and 6^th passages were performed.The result showed the growth of the cells were prosperous and the similar trendline of cell growth were observed among 1st,2nd and 6th passaage although the speed was slow down with the passage number increased.Both the 2nd and 6th passage cells contained normal chromosome number consisting of 60 chromosomes(2n=60).The rates of the chromosomal normality were 82.0%and 77.6%accordingly.3 Gene transfection into cashmere goat fetal fibroblast cellThe 2^nd passage of HFFCs were transfected with the mixture of linearized pCDsR-KI and Lipofectamine^TM 2000,and the mixture of linearized pCDsR-UI and Lipofectamine^TM respectively.The ratio between the liposome and DNA,as well as the transfecting duration time were optimized in our experiments.After screening the two group cells with 800μg/ml of genetycin for 12 to 15 days at 37℃in a humidified 5%CO₂ condition,23 from KI and 11 from UI cell colonies with red fluorescent were obtained.Comparative analysis suggested that the transgene and in vitro screening did not caused abnormally growing and exceptional karyotype. Identification of the genomic DNA of two group cells by ploymerase chain reaction proved that the exogenous gene have been integrated into genomes of the two groups of cells respectively.4 The transgenic fetal fibroblast cells nuclear transfer of cashmere goatA total of 738 cumulus oocyte complexies(COCs) were collected from 136 cashmere goat ovaries during the non-breeding season,and 1915 COCs were collected from 384 ovaries during the breeding season comparatively.The maturation rate of COCs from the non-breeding season was 73.4%,which was a little bit higher than COCs from the beeding season that was 65.5%.In vitro maturation of COCs were divided into three groups in 18h,20h and 24h.The rate of maturation for oocytes from 20h(80.6%) and 24h(82.2%) were both significantly higher than 18h (67.1%),P＜0.05.Parthenogenetic ooctyes were used as models to investigate the effect of two different activation methods with the parameters of 7%ethanol for 7 minutes plus 2mM 6-DMAP for 4h,and 5μM IA23187 for 5rain plus 2mM 6-DMAP for 4hr,the cleavage rates after 48h were 86.4%and 88.7%accordingly with no significant difference,P＞0.05.Three different fusion condition(130V/mm,190V/mm and 200V/mm) were investigated,and the result were 32.8%,62.4%å’Œ42.9%. Cleavage rates after 48h IVD in SOFaa containing BSA following nuclear transfer were KI group(79.3%) and UI group(62.5%).The cleaved embryos were transferred into SOFaa containing 4%FBS for 7 to 9 days.There was a difference in blastocyst developmental result between the KI and UI group.A total 31(15.3%) blastocysts were obtained from KI group whereas there is no balstocyst but only two(3.1%) morula stage embryos from UI group.Seventeen out of the 31 blastocysts expressing red fluorescence but there is no expression in UI group.The identification of integration was performed in 2 out of the 17 red fluorescent embryos by polymesrase chain reaction showed were all positive.OPS vitrification method was used for cryopreservation of some of the transgenic embryos.The survival rate after thawing was 80%.