口蹄疫病毒VP2基因重组表达载体的构建及其稳定表达细胞系的建立

本试验旨在构建Asia 1型口蹄疫病毒(FMDV)的VP2基因重组表达载体,并建立稳定表达VP2基因的BHK-21细胞系。利用RT-PCR方法扩增Asia 1型FMDV的cDNA,获得VP2基因完整的编码区,构建可表达VP2的带有绿色荧光蛋白标记基因的pIRES2-EGFP-VP2重组表达载体。经鉴定正确后,利用LipofectamineTM2000将重组质粒转染293T细胞,通过Western Blot技术检测VP2蛋白的瞬时表达;然后再转染BHK-21细胞,通过G418筛选,形成单克隆细胞系,经荧光筛选到表达EGFP的抗性单克隆细胞,扩繁培养克隆细胞,再通过West-ern Blot技术检测其VP2的表达,最终筛选到稳定表达FMDVVP2基因的BHK-21细胞系。结果表明,VP2基因重组表达载体pIRES2-EGFP-VP2构建正确,能够在293T细胞内瞬时表达;转染BHK-21细胞,经G418筛选到表达VP2基因的BHK-21细胞克隆,经长达60d的传代,获得了稳定表达VP2基因和EGFP的BHK-21细胞系。上述结果表明,我们建立了稳定表达VP2基因的BHK-21细胞系,为进一步探讨VP2基因在FMD...     

转EGFP基因山羊克隆胚的发育

利用脂质体包裹含EGFP基因的质粒,并将之导入山羊乳腺上皮细胞,经G418筛选获得阳性细胞,以阳性细胞作为核供体,利用核移植技术构建转基因克隆胚。结果表明：用转基因山羊乳腺上皮细胞作为核供体,电融合法更适合构建转基因克隆胚。转基因山羊克隆胚体外培养最佳方案是用SOFaa培养液,在培养72 h后加入10%的正常山羊血清（Normal goat serum,NGS）。荧光显微镜下观察到转基因克隆胚中EGFP的表达,大部分克隆胚发育到8-16细胞以后的时期,绿色荧光蛋白才开始逐渐表达,随着发育时间的延长,绿色荧光蛋白的表达也逐渐增强。说明外源基因在胚胎早期发育阶段可以表达,EGFP可以作为报告基因来实现对外源基因整合及表达的监测。     

稳定表达猪圆环病毒2型衣壳蛋白P815细胞系的建立

应用脂质体介导的基因转染方法,将表达猪圆环病毒2型(porcine circovirus,PCV2)衣壳蛋白的重组质粒pcDNA3.1-orf2转染P815细胞,筛选G418抗性单克隆细胞,并经PCR、RT-PCR和间接免疫荧光鉴定,随后对目的蛋白表达稳定性进行了检测。结果表明PCV2orf2基因已经整合进P815细胞基因组DNA中,ORF2编码的衣壳蛋白在获得的阳性P815细胞中稳定表达,建立了稳定表达PCV2衣壳蛋白的P815细胞系。该细胞系的建立为PCV2新型疫苗的研究提供了必要的实验材料,同时也可为其他病毒疫苗的研究提供有价值的参考。     

TALEN介导猪瘟病毒结构蛋白基因E0敲入山羊乳腺上皮细胞β-乳球蛋白基因座

验证整合于山羊β-乳球蛋白基因座的猪瘟病毒E0(CSFV E0)基因是否能够利用内源性启动子调控该基因表达,为制备转CSFV E0基因山羊做好准备。利用脂质体转染的方法将实验室已构好的针对山羊β-乳球蛋白基因第一外显子的TALENs表达载体和包含CSFV E0基因的打靶载体共转染山羊乳腺上皮细胞,用药物(G418)进行筛选获得单克隆,并对获得的克隆进行5′端和3′端鉴定,鉴定都为阳性的克隆进行激素诱导24h后,收集培养液和细胞。通过RT-PCR和Western blot技术检测证明,转基因阳性细胞能够表达并分泌出CSFV E0蛋白。本试验为以转CSFV E0基因细胞为供核体进行核移植制作转基因动物奠定了基础。     

稳定表达乙型脑炎病毒NS1蛋白细胞系的建立

为建立稳定表达乙型脑炎病毒(JEV) NS1蛋白的真核细胞系,本研究将编码JEV NS1蛋白的人工合成基因克隆到真核表达载体pCAGG-TK-neo中,构建了重组质粒pCAGG-opti-NS1.重组质粒经脂质体转染RK-13细胞,以含G418的选择性培养基选择培养,经细胞克隆纯化,以间接免疫荧光试验(IEA)筛选表达目的基因的细胞.结果表明,转染的RK-13细胞经G418加压及IFA筛选后,获得表达JEV NS1蛋白的阳性RK-13细胞系.经RT-PCR、western blot和IFA鉴定,该细胞系在传代至第15代后仍然可以稳定表达NS1蛋白.本实验获得了能够稳定表达JEV NS1蛋白的细胞系,为进一步开展JEV相关的研究奠定了基础.     

大肠杆菌植酸酶appA2基因联合人MxA基因真核表达载体的构建及表达研究

本研究旨在探讨通过构建同时表达大肠杆菌植酸酶appA2及人抗黏液病毒基因A(Myxovirus resistance gene,MxA)的真核表达载体,比较双基因表达载体和相应单基因表达载体中外源基因的表达效率;从pcDNA-ap-pA2载体上扩增CMV-appA2-BGHpA片段,通过MluI酶切位点连接到pcDNA-MxA载体中,构建重组载体pcDNA-appA-MxA(下称AMP),分别将pcDNA3.1(+)、pcDNA-MxA、pcDNA-appA2及AMP质粒转染猪PK15细胞,经G418筛选后,取细胞通过实时荧光定量PCR测定细胞内appA2及MxA的表达,同时部分细胞通过Westernblot测定细胞内MxA蛋白表达量,采用改进的钼酸铵显色法测定细胞内植酸酶活性;酶切及测序结果表明成功构建了AMP载体;QRT-PCR结果表明,转AMP细胞组MxA表达水平是转pcDNA-MxA组的1.118倍,转AMP细胞组appA2表达水平是转pcDNA-appA2组的1.134倍,上述各组间差异显著(P0.05)。灰度分析结果表明,AMP细胞组细胞内MxA表达量是转染pcDNA-MxA细胞组的1.07倍;植酸酶活性测定试验结果表明,转AMP、pcDNA-appA2细胞组及对照组细胞内植酸酶酶活和对照组平均酶活分别为0.294、0.235及0.082FTU·μL-1,2个试验组间差异不显著,试验组和对照组间差异极显著(P0.01);试验结果表明本研究构建双基因表达载体中外源基因的表达效率比相应单基因表达载体表达效率高,本研究为以后多基因共表达及制备多基因转基因动物奠定了基础。     

绿色荧光蛋白表达载体的改造和表达

为获得增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)的真核表达载体pEFP-N1-MSC,并观察其在猪成纤维细胞中的表达情况,用Nhe Ⅰ和Age Ⅰ消除pEGFP-N1质粒上的多克隆位点(MSC),然后补平连接,获得增强型绿色荧光蛋白编码基因的真核表达载体pEGFP-N1-MSC质粒,将pEGFP-N1-MSC质粒转化DH5α感受态细胞,于Kanr LB平板上筛选阳性克隆.重组子经Nhe Ⅰ和Age Ⅰ双酶切鉴定,将该载体转染猪成纤维细胞,24 h后观察EGFP表达情况,48 h后添加G418进行筛选.结果表明:改造的pEGFP-N1-MSC真核表达载体,成功转染猪成纤维细胞,在倒置荧光显微镜下呈现绿色光,获得可产生绿色荧光的pEGFP-N1-MSC载体,为构建猪双正选择同源重组载体奠定了基础.     

PRV SA株gB基因和EGFP基因在BHK细胞中的融合表达研究

为了研究gB蛋白在细胞内的作用位点,试验以猪伪狂犬病病毒(Pseudorabies virus,PRV)SA株为模板,设计特异性引物,扩增gB基因保守片段;以pEGFP-N1质粒为模板扩增EGFP基因;以gB基因与EGFP基因胶回收产物为模板,用gB基因上游引物及EGFP基因下游引物通过重叠延伸PCR(SOE PCR)技术获得gB/EGFP融合基因;构建pcDNA3.1/gB/EGFP重组表达质粒,转染乳仓鼠肾细胞(BHK细胞),采用Western-blot技术及荧光显微镜检测荧光蛋白在细胞内的表达情况。结果表明:试验成功扩增到300 bp的gB基因、760 bp的EGFP基因及1 060 bp的gB/EGFP融合基因;成功构建出pcDNA3.1/gB/EGFP重组表达质粒并转染BHK细胞;Western-blot技术及荧光显微镜检测显示,融合蛋白在BHK细胞中成功表达。说明通过荧光蛋白研究目的蛋白的作用位点是可行的。     

抗猪繁殖与呼吸综合征病毒转基因细胞系的建立

采用脂质体法将具有抑制猪繁殖与呼吸综合征病毒(PRRSV)复制的shRNA质粒pEGFP-N1-shRNA导入PK-15细胞中,经G418药物筛选后,分离扩增绿色荧光蛋白阳性细胞,获得抗PRRSV转基因PK细胞系。通过对转染方法和条件的优化,确立最佳的转染及筛选步骤;对得到的阳性转基因细胞进行冷冻-解冻,并作PCR检测。结果表明,G418最佳筛选浓度为600μg/mL,脂质体与质粒的最佳转染比例为7∶2,最佳转染时间为24 h。本研究成功建立抗PRRSV转基因细胞系,为进一步的功能验证及体细胞核移植奠定了基础。     

牛输卵管上皮细胞转人胶原蛋白cDNA基因及转基因克隆胚胎

以2岁奶牛输卵管为材料,用胰酶消化法分离得到了牛输卵管上皮细胞。输卵管上皮细胞在DMEM／F12培养基中生长良好。用一代牛输卵管上皮细胞为靶细胞,对其进行了电穿孔转染,转染对象是分子大小为31085bp的以β-casein启动子为基础的含有人胶原蛋白cDNA基因和EGFP、Neor双标记基因的质粒。电穿孔试验发现,牛输卵管上皮细胞在低渗缓冲液中电转染可以获得阳性转染子,其中以90mOsm／kg为最佳。电压以800V为好,电压高和低都不利于电穿孔的成功。成功转染的细胞用800mg/L G418进行筛选,在10d后获得了较大的阳性细胞克隆簇。对获得的阳性细胞克隆簇进行扩大后,得到了较纯的阳性细胞克隆系,流式细胞仪检测其纯度为81．6％。在荧光显微镜下选择阳性细胞作为核移植供体细胞进行了转基因克隆试验,结果显示以转基因和非转基因细胞为核供体获得的重构胚融合率差异显著（51．9％比63．2％）,获得的桑椹胚／囊胚率差异不显著（20．3％比25．5％）。对获得的具有绿色荧光的胚胎进行DNA分析,结果显示这些胚胎成功转入了外源基因,并且转入的外源基因结构完整。     

Cloning of pig parotid secretory protein gene upstream promoter and the establishment of a transgenic mouse model expressing bacterial phytase for agricultural phosphorus pollution control

This study examined the feasibility of using the promoter of the pig parotid secretory protein (PSP) gene for expression of the phytase transgene in mouse models. The pig parotid secretory protein gene is specifically expressed at high levels in the salivary glands. The 10-kb upstream promoter region of the gene necessary for tissue-specific expression has been identified. We have constructed phytase transgenes composed of the appA phytase gene from Escherichia coli driven by the upstream promoter region of the pig PSP gene with a 3' tail of either bovine growth hormone or the pig PSP gene polyadenylation signal. Transgenic mouse models with the construct showed that the upstream region of the pig PSP gene is sufficient for directing the expression of phytase transgenes in the saliva. Expression of salivary phytase reduced fecal phytate by 8.5 and 12.5% in 2 transgenic mouse lines, respectively. These results suggest that the expression of phytase in salivary glands of monogastric animals offers a promising biological approach to relieve the requirement for dietary phosphate supplements and to reduce phosphorus pollution from animal agriculture.     

猪前脂肪细胞为核供体生产表达绿色荧光蛋白的转基因克隆胚胎

分离培养了成年猪前脂肪原代细胞，并对前脂肪细胞向成熟脂肪细胞进行诱导分化，油红O染色法鉴定了脂肪细胞。利用脂质体介导的方法将质粒pEGFP—N1转染前脂肪细胞，经过G418筛选获得了阳性细胞株。然后分别以前脂肪细胞、转绿色荧光蛋白（GFP）前脂肪细胞及胎儿成纤维细胞为核供体进行体细胞核移植，结果表明：前脂肪细胞（8．8％）和胎儿成纤维细胞（8．3％）的囊胚发育率无显差异著（P〉0．05）；与前脂肪细胞相比，转基因前脂肪细胞的囊胚发育率降低，但差异不显著（3．7％ vs．8．8％，P〉0．05）。前脂肪细胞为核供体生产转基因克隆胚胎，能够获得转基因囊胚。虽然囊胚发育率不高，但为生产脂肪细胞转基因克隆猪奠定了基础。     

Development potential of transgenic somatic cell nuclear transfer embryos according to various factors of donor cell

The present study was conducted to establish an efficient production system for bovine transgenic somatic cell nuclear transfer (SCNT) embryos, the effect of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos were examined with their expression rates of a marker gene. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6, as a carrier. In Experiment 1, three types of bovine cells were transfected at passages 2 to 4, and then trypsinized and GFP-expressing cells were randomly selected and used for SCNT. Developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In all cell types used, GFP expression rates of SCNT embryos gradually decreased with the progression of embryo development. In Experiment 2, the effect of passage number of cumulus cells in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed, but significantly higher GFP expression was shown in blastocysts reconstructed with cumulus cells at early passage. In Experiment 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 microm) or small cell (<30 microm)] at passages 2 to 4 were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells as well as fetal cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.     

Effect of Transgene Introduction and Recloning on Efficiency of Porcine Transgenic Cloned Embryo Production In Vitro

Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNβ-Z or LNβ-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro . Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNβ-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 ± 6.4%; 12.0 ± 5.7%) or EGFP (57.5 ± 6.3%; 10.1 ± 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 ± 8.2%; 12.3 ± 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency.     

Selection of donor nuclei in somatic cell-mediated gene transfer using a co-transfection method

In this study, we introduced a co-transfection method for the selection of donor nuclei in somatic cell-mediated nuclear transfer. Two vectors were constructed in our experiment. One was pMSCV-GFP carrying the neomycin-resistant gene (Neo(r)) and the green fluorescent protein (GFP) reporter gene; the other was pBC1-GFP carrying the mammary gland-specific promoter and target gene GFP. Ovine adult fibroblasts were co-transfected with pMSCV-GFP and pBC1-GFP. The data from this work demonstrated that the GFP genes in both vectors could successfully co-integrate into the genomes of ovine adult fibroblasts in three of the four transgenic cell clones assayed. Furthermore, PCR analysis of transgenic embryos proved that the GFP genes in both vectors could co-integrate into the genomes of the reconstructed embryos. Subsequently, analysis of the developmental rate of the reconstructed embryos after nuclear transfer indicated that the blastocyst rate from the co-transfected donor cells was similar (approximate 8 percent) to that from individual pMSCV-GFP transfected donor cells. The influence of co-transfection resulting in modification of donor nuclei on development of reconstructed embryos was also investigated. The results of flow cytometric analysis indicated that the co-transfected ovine fibroblasts had similar quiescent characteristics in terms of cell cycle (G0+G1 percent: 73.20 +/- 4.04) to the individual pMSCV-GFP transfected fibroblasts (G0+G1 percent: 70.77 +/- 1.19) after they were treated with serum starvation for five days. Our results suggest that the co-transfection method can be used for selection of donor cell clones in somatic cell-mediated gene transfer experiments. It can be potentially extended to applications related to expression of functional protein in mammary glands and other transgenic research relevant to nuclear transfer.     

Production of transgenic bovine cloned embryos using piggybac transposition

Transgenic research on cattle embryos has been developed to date using viral or plasmid DNA delivery systems. In this study, a different gene delivery system, piggybac transposition, was employed to investigate if it can be applied for producing transgenic cattle embryos. Green or red fluorescent proteins (GFP or RFP) were transfected into donor fibroblasts, and then transfected donor cells were reprogrammed in enucleated oocytes through SCNT and developed into pre-implantation stage embryos. GFP was expressed in donor cells and in cloned embryos without any mosaicism. Induction of RFP expression was regulated by doxycycline treatment in donor fibroblasts and pre-implantational stage embryos. In conclusion, this study demonstrated that piggybac transposition could be a mean to deliver genes into bovine somatic cells or embryos for transgenic research.     

牛朊蛋白基因真核表达载体的构建及其在牛骨髓间充质干细胞中的稳定表达

试验构建牛朊蛋白(prion protein,PRNP)基因的真核表达载体,为进一步研究牛朊蛋白的生理功能和从细胞水平研究抗疯牛病转基因克隆牛奠定基础。采用重叠延伸PCR(splicing overlap extension PCR,SOE-PCR)法扩增获得牛PRNP基因序列,并克隆到带有DsRED2报告基因的真核表达载体pDsRED2-N1中,将双酶切、PCR、测序鉴定的阳性质粒经脂质体转染牛骨髓间充质干细胞(BMSC);通过荧光显微镜观察转染细胞,并用800 μg/mL G418对转染的细胞进行药物筛选。琼脂糖凝胶电泳显示基因合成的片段大小和构建的载体大小与预期相符;重组表达载体转染BMSC后有红色荧光出现;通过药物筛选出了稳定转染的细胞单克隆。通过SOE-PCR成功扩增了牛PRNP基因序列,并构建成真核表达载体,得到稳定表达目的蛋白的BMSC细胞。     

拟蜘蛛牵丝蛋白基因细毛羊毛囊特异表达载体的构建及其转基因细胞株的筛选

试验旨在获得具有毛囊表达特性的转蜘蛛牵丝蛋白基因细胞株。根据GenBank上发表的棒络新妇属蜘蛛的cDNA片段合成拟蜘蛛牵丝蛋白基因单体S,加倍后连入pEGFP-N1框架载体以构建真核表达载体pK-2S,转染新疆美利奴细毛羊皮肤成纤维细胞后筛选单克隆,并通过PCR在DNA、RNA水平检测阳性克隆。基因合成后测序结果显示序列正确,酶切得到正确目的条带,筛选出阳性克隆并且可以在基因组及cDNA上扩增出目的片段。本研究成功将蜘蛛牵丝蛋白基因转入新疆美利奴细毛羊细胞,并在RNA水平表达,为培育毛囊特异表达蜘蛛牵丝蛋白基因并具有更高机械性能羊毛的新型细毛羊品种奠定基础。     

Effect of transfection and passage number of ear fibroblasts on in vitro development of bovine transgenic nuclear transfer embryos

The objective of this study was to determine if the transfection of human prourokinase (ProU) gene and passage number of transfected ear fibroblasts affected in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human ProU was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker and human ProU gene into a pcDNA3 plasmid and transfected into bovine ear fibroblasts using a lipid mediated method. Abattoir derived oocytes were enucleated at 18-20 hr post maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the couplets were cultured in modified synthetic oviductal fluid (mSOF) medium for 168 hr. In Experiment 1, significantly lower rate in blastocysts formation (10.3%) was observed in transfected donor cells at early passage than that in nontransfected counterparts (22.1%, P<0.05). In Experiment 2, development to blastocysts and GFP expression in blastocysts were not significantly different between early (3-7) and late (8-12) passage donor cells (10.3 vs. 11.3% and 54.5 vs. 41.7%, respectively). This study indicates that in vitro development of bovine transgenic NT embryos is negatively influenced by transfection of human ProU gene into donor fibroblasts. However, passage number of transfected ear fibroblasts does not affect in vitro development of bovine transgenic NT embryos.