【作者】 王凤阳；

【导师】 殷震；

【作者基本信息】 中国人民解放军军需大学 ， 预防兽医学， 2001， 博士

【摘要】 转基因动物技术作为生命科学领域中的一项重要的研究手段，广泛应用于医药、农业及生物学领域。自1980年Gordon等首次将显微注射技术用于小鼠受精卵的基因导入以来，人们对于转基因技术的研究与探索从未停止，因此，在显微注射技术不断发展和完善的同时，还相继建立了胚胎干细胞介导的基因转移，重组逆转录病毒介导的基因转移和精子载体法等转基因技术。但这些转基因技术普遍存在着操作复杂、投入较高、外源基因在受体细胞基因组上的整合率较低等不足之处。为了克服这些不足之处，本研究在多年跟踪国际转基因动物研究前沿的基础上，从选择高效的基因转移的介导方式和理想的受体细胞两个方面同时着手，进行了基因转移新技术的研究。 在众多的真核细胞基因转移技术中，动物病毒载体系统以其简单的基因组结构、比较清楚的分子生物学背景、易于改造和操作、转染率高等特点而最为引人注目，迄今已有数种病毒被改造为基因转移载体。其中，逆转录病毒载体-包装细胞系统被认为是在具有分裂功能的细胞中进行基因转移和表达最为成功的真核细胞基因转移技术。因此本研究选择重组逆转录病毒作为基因转移的介导方式。将3.7kb的LacZ基因片段分别插入到骨架为Moloney小鼠白血病病毒（MoMLV）、含有标记基因Neo~R的逆转录病毒载体pLXSN、pLNCX的5’ETR和CMV Promoter的下游，构建了含有LacZ的重组逆转录病毒载体pLNCL、pLLSN。运用脂质体、磷酸钙两种介质，以包装效率高、安全性好的包装细胞系PA317对pLNCX、pLXSN、pLLSN和pLNCL进行包装，得到了PA-1、PA-2、PA-3和PA-4四株产毒细胞。透射电镜下观察经差速离心法浓缩后的病毒悬液，观察到了具有逆转录病毒形态特征（球形，有囊膜，直径平均约为100nm）的重组病毒粒子。 精原干细胞属于未分化细胞，具有自增殖能力，可产生不断更新的干细胞及可定向分化的精原细胞，每次分裂形成的两个细胞，一个通过初级精母细胞、次级精母细胞、精子细胞等过程发育为精子，一个仍为干细胞，保持原始未分化状态和继续分裂增殖的能力。如果重组逆转录病毒介导的外源基因转染入精原干细胞，外源基因就会整合入精原干细胞基因组。由精原干细胞发育成熟的精子，就能够通过整合的方式携带外源基因，外源基因也会随干细胞增殖而复制并在干细胞中得到长期保留。这就克服了以受精卵作为目的基因载体时需要不断采卵和反复注射带来的巨额成本和繁重的工作强度等不足之处，从而提高转基因动物的生产效率。 由于以精原干细胞作为外源基因转染的靶细胞以建立转基因动物的研究具有极其重要的理论意义和实际应用前景。1997年，Kim JH等和本室赖良学等分别进行了脂质体包裹外源基因，曲细精管内转染精原干细胞的研究，并且各自得到了附睾精子和子代鼠整合检测的阳性PCR结果。Kim JH等还在睾丸组织切片中检测到了外源基因的表达。 中文摘要一 基于本研究以小鼠曲细精管内精原干细胞作为9懈塌因转染的靶细胞以建立转基因小鼠的目时为了 烙的重斑议臼色录病毒体内感染精原干细胞的效果，进行了含有ixc、NeoR基因的重组逆转录病毒体夕陋澡NIH3Th细胞的研究。将含有aH基因的浓缩病毒渤叨材摘滦NllB℃细胞，经多聚甲醛固定、Xwi染色，观察臣了蓝染细胞说明sxc基因获得表达。同时，将来自四株产毒细胞的浓缩病毒悬液分另u体夕h孜染NIH3Th细胞以测喉空逾跺嫡度，通过比较，含有标记基因Nr、由产毒细胞系PAZ所产生的浓缩重组逆转录病毒的感染滴度最高，过臣了3X106cllvilll。 在＄e了含有标记基因 Ne、感搁傲颤丝了 3 X 106 dilllTll的重组逆转录病毒的前提下经过注射条件和参数的多次优化，将pM台盼蓝、Poo按一定比例配制域注白比狡，单似收拄时ldsl6日龄公鼠寒丸曲细精管以体内感染精原干细胞。注射后4（hi，公圃倚溉进行精液品质（精子活率、精子顶体完整率、精于畸形率三项指标）的匀觎o并利用根据M炉基因序列设计的三对引物对精子基因组DNA进干了IWI；1和套式ICIt检狈。实验结果表明，注射液对公鼠精液品质无不良影响，不影响公鼠繁殖力。10 ff｝BfftZ；fR7so（品中，有8份为PCR和套式PCR附仕，说明Nd基因已经坐尧淮公鼠精子基因组中，从而首次成功地实现了M炉基因在公鼠生殖系细胞的转移。注射后45d，与母鼠交配，共获得子代鼠48只。采取鼠尾，提取基因组，利用根据Nej基因序歹uM的一对引物对子代鼠基因组DNA进行PCR检测，阳性鼠9只。经 Southern blotting检狈，阳性鼠 4只，其中 3只母鼠，l只公鼠。将这 4 ／q FO代小鼠与4F＄：FH因小鼠交配，共获得F；代小鼠15只。应用PCR方法（引物同FO代小鼠进行检测中使用的5！物）对这些F；代小励锁0，结果F；代1号母鼠听生5只子鼠中有 4只为阳胜。获得的转基因小鼠阳性率达到了 83％（4／48）。从而首次建立了重组逆转录病毒介导，以公鼠曲细精管内精原干细胞为9陇塌因转染的靶细胞的基因转移技术。并利用该技术成功的?更多还原

【Abstract】 Cunent gene transfer methods in transgenic animals include embiyo microzrijection, embiyonic stem cell mediated and retrovims mediated They have been videIy applied to construct transgeriic large animal as well as mice. However, when these methods were used to construct transgenic animals, there are some shortcomings, for example, time consuming and high cost In order to overcome these shortcomings we bad developed a new method to construct transgenic mice. Two important considerations had been taken into account, i.e. the gene transfer efficiency and the target cell. Recombinant retrovirus is selected as gene transfer method and spermtogonial stem cells were used as target cells. Recombinant retroviral vectors (pLLSN and pLNCL) cartying LazZ were constnLcted. The two vectors, together with pLNCX and pLXSN canying NeoR were transfected into PA3 17 for packaging by means of Lipofectin and calcium phosphate precipitation respectively. After G41 8 selection at a concentration of O.3g/L for 2 weeks, G41 8-resistant PA317 colonies were obtained and amplified, and four G41 8-resistant PA3 17 cell lines (PA-I, PA-2, PA-3, PA-4) were obtainecL The supernatant of the cell culture was harvested, concentrated and observed under transmission electron microscope, the mature vinis particles possessed the typically morphological feature of retrovirus. The concentrated viral supemantant fim PA-3 and PA-4 was used to infect Nll{3T3. The infected NIH3T3 cells were fixed by O.5(wlv) parafoima]dehyde and stained by X-gal, those cells that expressed LacZ became blue. The result showed LzcZ gene had been expressed on NIH3T3 cell. At the same time, Nll-13T3 cells were infected to measure the viral titer of recombinant retrovirus, Results showed that the titer of concentrated viral supematant from PA-2 was the highest, and was 3 X lO6cfu/ml. 84 In order to study the gene transfer mediated by recombinant retrovims- spermatogonial stem cells, recombinant reUuvirus from PA-2, canying NeoR, together with typan blue and Polybrene, were injected into contorted seminiferous tubule of 10 mice (14-16 day) to infect spermatogonial stem cells in vivo. After 40 days, sperms were harvested from injected mice. Its qualities( living mtio. apical body intact ratio and deformation ratio) were examined, the data were collected and statistically analyzed. PCR was used to examine the integration of NeoR the genome of sperm. The results showed that injection solution doesn抰 have conlraiy effect on the development of sperm. NeaR gene were confirmed to be integrated into the genome of mature sperms from eight mice. Female mice were mated with the eight male mice respectively, and forty-eight ofipring were produced. PCR and Southern blotting were used to screen NeaR gene from the genomic DNA samples extracted from the forty-eight olpring tails. Four of them including one male and three females were demonstrated to be lransgenic. Four founders were further mated with the nontransgenic mice and fifceen offspring were produced and PCR were used to screen NeOR gene integration. Four out of five offspring from one female founder were transgenic mice. Theinteonratewas8.3%(4/48),andwasalmostastheeashaven reported. But gene transfer mediated by retrovirus-spemitogonial stem cell is much easier to manipulate than other methods and it saves time. So, gene transfer mediated by recombinant retrovinis - sperrnatogonial stem cells appears to be a promising way to construct transgenic animals, such as mice as well as large animals.更多还原