【作者】 沈伟；

【导师】 陈宏； 邓继先；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2004， 博士

【摘要】 在以往的转基因动物研究中，由于基因表达调控元件的人工拼接和外源基因在动物基因组中随机整合所带来的“位置效应”，致使转基因动物外源基因的表达水平不高并且差异较大。在这种情况下，以基因同源重组为理论基础的基因打靶技术能全面克服此问题，将外源基因定位整合到家畜体细胞的乳蛋白基因座制备乳腺生物反应器，能够充分利用内源乳蛋白基因固有的高效表达调控机制。它借助体细胞核移植技术，在一定程度上能避开乳用家畜胚胎干细胞建系这一难题。由此可见，这是当前动物乳腺生物反应器生产的理想模式。本研究就是利用基因打靶技术将人组织型纤溶酶原激活剂突变体（ht-PAm)基因定位整合到山羊胎儿成纤维细胞的β-casein 基因座，然后将中靶细胞进行核移植，以期获得能稳定、高效表达 ht-PAm 的山羊乳腺生物反应器。 克隆关中奶山羊β-casein 基因 5?端的 6.3 kb 片段，进行 DNA 序列分析并鉴定，从而获得了包含较完整乳蛋白基因调控元件的山羊β-casein 基因 5?调控序列。将其与改构的ht-PAm 连接，从而使该基因的 ATG 与β-casein 基因的 ATG 相重合。分别克隆了关中奶山羊β-casein 基因第 7 外显子和第 8、9 外显子，将两侧带有 loxP 位点的 neo 基因插入到内含子 7 中，tk 基因插入到外显子 9 外侧。从而构建了含有 neo 和 tk 正负筛选标记基因的β-casein 基因打靶载体 pGBC4htPAm，该载体可以用 NotI 位点进行切割，用于细胞的转染整合。利用细胞转染试验验证了 neo 基因、tk 基因的有效性，并利用 Cre 重组酶体外验证了Cre/loxP 系统的有效性。 采集胚龄 35 d 的关中奶山羊胚胎，利用胰蛋白酶消化法得到关中奶山羊胎儿成纤维细胞。SRY-PCR 法进行胚胎性别鉴定，3 只为雄性，1 只为雌性。对胎儿成纤维细胞进行 40代连续培养，染色体核型分析跟踪证明了该细胞用于基因打靶的可行性。同时实验发现，LIF 和 EGF 等细胞因子对胎儿成纤维细胞生长有较明显的促进作用。 将线性化pGBC4htPAm通过电击转染或脂质体转染整合到山羊胎儿成纤维细胞基因组中，利用 G418 和 GANC 进行抗性细胞克隆的药物筛选，共获得抗性细胞克隆 1204 个，其中生长状态较好的 656 个，3?同源臂重组区域 PCR 检测获得阳性细胞克隆 48 个，对其中的 7 个细胞克隆进行 3?同源重组区域基因序列测定，有 3 个克隆转基因整合位点重组后的基因序列正确，其中 2 个 5?端同源臂 PCR 产物的酶切分析验证为正确同源重组。比较分析认为，电击法比脂质体法更有利于山羊胎儿成纤维细胞的基因打靶。<WP=6>II ht-PAm 在山羊β-casein 基因座定位整合与中靶体细胞核移植的研究 采集屠宰山羊卵巢获取卵母细胞并体外成熟培养，成熟率为 71.2％。经 0.5% FBS 饥饿1～3 d 的转基因体细胞做核供体，经去除第一极体和原核的 MII 期卵母细胞做核受体进行核移植。核移植胚电融合后用 Ionomycin 和 6-DMAP 进行激活处理。然后转移到 CR1aa培养液中与单层卵丘细胞共培养。重构胚的融合率 55.2%，其中用作核供体的 3 个细胞系的融合率存在差异, 体内卵的融合率（71％）与体外卵的融合率（48％）差异极显著。基因打靶体细胞核移植后共得到克隆胚 600 枚，其中部分体外发育到桑椹胚或囊胚。手术法将发生卵裂且形态正常的转基因克隆胚（2-细胞期或以上）移植到同期发情的山羊输卵管中，16 只受体山羊中有 6 只一直没有返情，其中 3 只可能妊娠。 利用克隆的关中奶山羊β-casein 基因 5?调控序列、外显子 7 到外显子 9 以及 3?侧翼序列、改构的 ht-PAm 基因构建了 ht-PAm 基因的乳腺表达载体。利用脂质体转染法使 ht-PAm基因在泌乳期家兔的乳腺上皮细胞中进行了瞬间表达，纤溶实验结果表明乳腺细胞分泌的t-PA 突变体具有较强生物活性。利用不同来源的脂质体或 DMSO 作介质，利用睾丸介导制备转 ht-PAm 基因家兔，共得到乳腺生物反应器转基因兔模型 89 只（F1代仔兔共 184 只），阳性率为 48.4％，其中新利用的 DMSO 法的转基因阳性率为 56.3％。 构建了通用型山羊β-casein 基因座基因打靶载体 pGBC-GFP-neo，载体包含了正负筛选标记基因 neo 和 tk，以及无启动子的 GFP 基因。打靶载体线性化后用脂质体包裹转染山羊乳腺上皮细胞，利用 G418 和 GANC 进行抗性细胞克隆的药物筛选，共得到抗性细胞克隆 51 个，对抗性克隆利用激素诱导β-casein 基因启动子指导 GFP 表达，得到 GFP 表达阳性细胞克隆 17 个，对其中 4 个生长状态良好的细胞克隆 PCR 检测验证后用于核移植，克隆胚发育率为 59.5%, 部分发育到桑葚胚或囊胚。 山羊体细胞基因打靶过程中，靶受体细胞常出现过快衰老现象。对山羊打靶衰老细胞进行染色体端区的长度分析发现，衰老细胞的端区长度比原代细胞端区长度缩短了2.56 kb。以小鼠胎儿成纤维细胞为材料研究发现，打靶细胞染色体端粒的长度以每代 47 bp 碱基缩短。在打靶衰老细胞中，或细胞随着增龄，p16INK4a 5?-调控区 DNA 甲基化程度逐渐降低；利用 RT-PCR 与 Northern blot 证明，衰老细胞与年轻细胞中更多还原

【Abstract】 The production of recombinant protein is one of the major successes of biotechnology; animalcells are required to synthesize proteins with the appropriate post-translational modifications.Transgenic animal mammary gland bioreactors are being used for this purpose. Producingmammary gland bioreactor showed great advantage over many years, but the level of transgenicexpression was low in transgenic animals and the diversity was greater because of the positioneffect of transgene and the artificial recombination of the gene elements. Gene targeting based onthe principle of gene homologous recombination had been studied and applied, because thetransgene could be integrated precisely in the chromosome. Gene targeting is a more powerfulmethod to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cellsprovides an wonderful means of cell-mediated transgensis. Here we describe efficient andreproducible gene targeting in goat fetal fibroblasts to place the human tissue-type plasminogenactivator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goatby nuclear transfer. To construct the gene targeting vector pGBC4htPAm, the Guanzhong milk goat beta-caseingenomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kbfragment including goat beta-casein gene 5?flanking sequence, and the right arm was 2.4 kbfragment including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned inthe goat beta-casein gene exon 2. The bacterial neomycin (neo) gene as positive selection markergene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negativeselection marker gene, was just outside the right arm. The validity of the positive-negativeselection vector was tested , and targeting homologous recombination were elevated to 3.7-foldwith the tk gene. The DNA fragment between two loxP sequences was deleted effectively usingCre recombinase in vitro. Goat fetal fibroblasts were isolated from day 35 fetuses of Guanzhong milk goats andcultured to subconfluence before transfection, about fibroblasts were electoporated with linearpGBC4htPAm. Transfected cells were cultured in 96-wellplate for 24 h without selection, thenadded the drug G418(600 μg/ml) and GANC(2 μmol/l). With LipefectaminTM-2000, cells weretransfected with linear pGBC4htPAm. These transfected cells were cultured for 24 h withoutselection, then added the drug G418(800 μg/ml). After 12 days of selection, well separated cloneswere isolated and expanded in 24-wellplate, then added the drug G418(300 μg/ml) and GANC(2μmol/l). 1204 clones were selected, and only 656 clones could grow and be tested by PCRscreening for targeting. The results demonstrated that 48 targeting cell clones with homologousrecombination events were obtained using 3?-PCR, and 3 cell clones were verified by DNA<WP=8>IV ht-PAm 在山羊β-casein 基因座定位整合与中靶体细胞核移植的研究sequence analysis on the homologous recombination region, 2 ones were verified by 5?-PCR. Goat cumulus-oocyte complexes (COCs) recovered by surface-follicles cutting frombreeding seasons were matured in vitro, and 71.2% COCs were matured. The 3 targeted cellclones were nuclear transfer as donor cell after 1~3 days culturing with 0.5% FCS, and thereceptor cells were MII oocytes without nuclear and first polar bodies. These reconstructedembryos were fused for 40 μs under 1.2 kv/cm voltage, and activated using 5 μmol/l Ionomycinfor 5 min，2 mmol/l 6-DMAP for 4 h. 55.2% reconstructed embryos were fused and co-culturedwith cumulus cells in CR1aa. The results showed that the percentages of fused embryos weredifferent from different oocyte types (matured in vivo or in vitro) and different transgenic somaticcell lines. 600 reconstructed embryos had been obtained, and some could develop to morula orblastocyst in vitro. These developed cloned embryos were transferred to 16 recipients, and 6recipients did not returned更多还原