【作者】 郭旭东；

【导师】 旭日干；

【作者基本信息】 内蒙古大学 ， 动物学， 2008， 博士

【摘要】 转基因动物技术自上世纪八十年代确立以来,在医药、农业以及基础生物学领域都有十分广泛的研究和应用,也取得了令人瞩目的成绩。在畜牧业生产中,这项技术的应用方兴未艾。在改良动物遗传性状以提高生产性能、加强畜禽抗病育种以及通过乳腺生物反应器生产药用蛋白等非常规畜牧生产方面,转基因技术正显示出广阔的应用前景。但是,只有当体细胞核移植技术真正确立以后,转基因家畜的研究和制备效率才得以大幅提高。利用转染了外源基因的体细胞通过核移植技术获得转基因克隆动物是目前转基因动物制备的主要技术路线。本研究即采用了这种转基因方法,以毛囊特异表达的启动子pKAP6-1、pUHS分别引导胰岛素样生长因子（IGF1）基因,以新霉素抗性基因和红色荧光蛋白基因作为双选择标记,首次构建成功2个毛囊细胞中特异性表达IGF1的表达载体;利用体外转染技术将此2个表达载体分别导入绒山羊胎儿成纤维细胞,通过G418筛选出稳定表达红色荧光蛋白的2种克隆细胞;以体细胞核移植技术制备绒山羊转基因克隆胚胎。本研究为转基因绒山羊的培育,以及探讨IGF1在绒山羊毛囊发育和生长周期中的调控作用提供了研究基础。1.IGF1毛囊细胞特异性表达载体的构建通过PCR和RT-PCR方法从绵羊总DNA和总RNA中分别扩增出角蛋白关联蛋白6-1基因（KAP6-1）启动子区序列和绵羊胰岛素样生长因子（IGF1）编码区cDNA序列,以pMD19T为克隆骨架构建成中间克隆载体p19TKI。将CMV启动子序列连接在pDsRed2-1表达框架的红色荧光蛋白报告基因（Red2）上游KpnⅠ+SmaⅠ位点构成pCDsRed2;pCDsRed2经EcoRⅠ酶切,与p19TKI的KI片段连接构成IGF1毛囊细胞特异表达的第一种载体pCDsR-KI。以PCR方法从小鼠基因组DNA扩增出超高硫角蛋白基因（UHS）启动子区序列,与pMD19T、IGF1 cDNA（SalI+SphI酶切片段）构建成中间克隆载体p19TUI;pCDsRed2经SacⅠ+EcoRⅠ酶切、Klenow Fragment补平EcoRⅠ端,与p19TUI的UI片段（经过SacⅠ+SphⅠ酶切、Klenow Fragment补平SphⅠ端）连接构成毛囊细胞特异表达的第二种载体pCDsR-UI。限制性内切酶分析和部分序列测定结果表明,本研究所构建的两个表达载体pCDsR-KI和pCDsR-UI,其结构与序列均与预期设计完全符合。2.绒山羊胎儿成纤维细胞的分离与体外培养绒山羊胎儿成纤维细胞以组织块贴附方法进行分离,在DMEM/F12+10%FBS培养液,37℃、5%CO₂、饱和湿度条件下进行体外培养。原代成纤维细胞以DMEM/F12+20%FBS+DMSO培养液冷冻保存。在显微镜下观察和记录了绒山羊胎儿成纤维细胞的形态和生长特性,绘制出第1、2和6代胎儿成纤维细胞的生长曲线,通过制作第2、第6代细胞中期染色体标本,分析核型特征。经以上分析证明,我们分离和培养的绒山羊胎儿成纤维细胞在体外培养环境下,其生长力旺盛,1、2、6代细胞生长曲线的各个时期特征基本保持一致,随代次增加,生长速度渐缓;第2、6代细胞的染色体数目正常（2n=60）,染色体正常比率分别为82.0%和77.6%。3.绒山羊胎儿成纤维细胞的外源基因转染脂质体Lipofectamine^TM 2000与线性化表达载体pCDsR-KI混合（转基因A组）、脂质体Lipofectamine^TM Reagent与线性化表达载体pCDsR-UI混合（转基因B组）后,分别转染至第2代绒山羊胎儿成纤维细胞。并对转染条件进行了优化,探明了脂质体和DNA的最佳组合条件及转染时间。以800μg/ml G418作为抗性筛选的浓度,在DMEM/F12+10%FBS培养液、37℃、5%CO₂、饱和湿度条件下连续培养12～15天,分别获得了两组表达红色荧光蛋白的转基因克隆细胞,其中A转染组共有23个阳性克隆、B转染组有11个阳性克隆。经过比较转基因细胞和对照组细胞的生长曲线和染色体核型分析证实,转基因和体外筛选过程并没有导致细胞生长和染色体核型的异常。通过对两组转基因细胞基因组DNA特异性区域的PCR扩增,证实外源基因IGF1及其表达控件已稳定整合在转基因细胞基因组中。4.绒山羊转基因胎儿成纤维细胞的核移植在非繁殖季节,从136个绒山羊卵巢采集到卵母细胞复合体738枚;在繁殖季节,从384个卵巢共采集卵母细胞复合体1915枚。经体外成熟培养,非繁殖季节的卵母细胞成熟率为73.4%（542/738）,略高于繁殖季节的成熟率65.5%（1254/1915）;体外成熟培养时间分别设定为18、20和24hr进行分组培养,其中20hr组成熟率80.6%（79/98）、24hr组成熟率82.2%（83/101）均显著高于18hr组成熟率67.1%（51/76）,P＜0.05。以7%乙醇7 min+2mM 6-DMAP 4 hr和5μMIA23187 5min+2mM 6-DMAP 4hr两种激活方法处理成熟卵母细胞进行孤雌激活,48hr后统计卵裂率为86.4%和88.7%,二者之间差异不显著（P＞0.05）。选择IA23187激活方法用于重构胚的激活。分别设定三种不同强度的融合条件（130V/mm、190V/mm和200V/mm）进行电融合率,融合率分别为32.8%、62.4%和42.9%。分别以转染有pCDsR-KI的A组成纤维细胞和转有pCDsR-UI的B组细胞作为核供体,以去核成熟卵母细胞作为胞质受体制备转基因克隆胚胎。以SOFaa+BSA培养液培养48hr,统计卵裂率,结果A组为79.3%（161/203）,B组为62.5%（40/64）。经过SOFaa+4%FBS体外培养7～9天,A组转基因得到囊胚数为31枚,囊胚发育率为15.3%（31/203）;B组转基因未能获得囊胚,得到桑椹胚2枚,桑椹胚发育率为3.1%（2/64）。所得到的31枚囊胚中,表达红色荧光蛋白的囊胚数17枚,而B组桑椹胚不表达红色荧光。从红色荧光囊胚中挑选2枚以PCR方法检测外源基因整合情况,结果全部为阳性。以OPS玻璃化冷冻方法保存了部分转基因克隆囊胚。更多还原

【Abstract】 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 factorⅠ（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 KpnⅠand SmaⅠsites.Then,one of the hair follicle cells specific expression vectors,pCDsR-KI was accomplished by linkage of pCDsRed2 which was linearized with EcoRⅠand 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 SphⅠsite by Klenow fragment treatment） from p19TKI,and pCDsRed2 linearized by SacⅠ/EcoRⅠ,then formed a blunt end in the EcoRⅠsite 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%和42.9%. 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%.更多还原