【作者】 徐焕宇；

【导师】 曾溢滔； 黄淑帧； 曾凡一； 马晴雯；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 作为一门生物高新技术,转基因动物研究既具有深远的理论意义,又在农业、医学、制药等领域中具有巨大的应用价值,因而成为近年来生物工程领域研究的热点之一。对于转基因动物而言,最为重要的是外源基因长期稳定表达。原核期胚胎的显微注射法是目前转基因动物尤其转基因小鼠的主要生产方法,但是该方法存在外源基因整合率低、表达受整合位点影响大等缺点。φC31整合酶是一种新型的重组酶,可以介导含attB位点及含外源基因的质粒在哺乳动物基因组假attP位点上呈位点特异性整合,并使其高效表达。本研究目的是构建能表达φC31整合酶的工具鼠,使其受精卵表达φC31整合酶,以解决原核显微注射法外源基因随机整合且表达水平不高的缺陷。本研究首先构建了两个系列的φC31整合酶载体,然后通过显微注射的方法分别获得了这两类载体整合的小鼠家系,一种是EF1α启动子启动全身表达φC31整合酶的小鼠(EF1α-Int,EF1α-Int-NLS);另一种是Zp3启动子启动卵母细胞特异表达φC31整合酶的小鼠(pZP3-INT,pZP3-INT-NLS)。随后对EF1α-Int小鼠进行了系统的研究,RT-PCR分析表明该类转基因小鼠的受精卵中存在φC31整合酶mRNA表达。根据pBCPB+在整合酶存在的情况下,可以发生分子内重组,产生新的杂合位点attL的机制,我们进一步应用显微注射的方法,将pBCPB+注射到表达φC31整合酶的小鼠受精卵中,PCR检测结果表明,受精卵中存在φC31整合酶并能介导attB和attP之间的位点特异性重组而产生杂合位点的attL,从而证明本研究所构建的表达φC31整合酶的工具鼠可在受精卵中发挥作用,从而有可能用于生产外源基因位点特异性整合的转基因小鼠。为了评价φC31整合酶的重组能力,根据φC31整合酶能介导同向attB和attP之间的的序列删除的特点,本研究结合φC31/att和红绿荧光蛋白系统构建了可在活细胞中检测φC31整合酶功能的双色荧光报告系统。当不存在φC31整合酶时,红色荧光表达;当存在φC31整合酶并催化重组反应时,表达绿色荧光。将该报告系统与φC31整合酶以不同比例瞬时转染NIH3T3细胞,并用流式细胞仪(FACS)测定红色和绿色荧光细胞的比例。结果发现,随着整合酶加入比例的增加,红绿荧光细胞的转换率相应地提高,并发现报告系统与φC31整合酶的比例在1:10时,可使90%的红色荧光细胞转换为绿色荧光细胞。此外,在报告系统稳定整合的细胞基因组内,同样观测到φC31整合酶催化重组而产生的红色荧光转换为绿色荧光。应用本研究中的φC31整合酶的双色荧光报告系统并结合流式细胞仪可以检测活体细胞中φC31整合酶的重组能力,从而为φC31整合酶的重组能力的检测提供了一条更加快捷的量化途径。综上所述,本研究构建了一种可表达φC31整合酶的小鼠,证明了可以将其用作生产外源基因位点特异性整合的转基因小鼠,从而改进了转基因动物制备的策略和技术。此外,本研究还构建了评价φC31整合酶的重组能力的双色荧光报告系统,为评估φC31整合酶转基因小鼠的效率提供了技术支持和材料准备。更多还原

【Abstract】 . As one of the most important research fields in modern biotechnology, transgenic animals not only has great value in basic researches on gene function and developmental studies but also has huge profit on application researches in agriculture, medicine and pharmaceutical industry. Traditionally, the main method for generating transgenic animals is pro-nuclear microinjection. However, this approach has some disadvantages such as low integration rates and poor expression for position effect of foreign genes.StreptomycesφC31 integrase is kind of recombinases, which can mediate integration of a donor plasmid containing attB sequence and foreign gene into endogenesis specific sequence(pseudo attP)in many species. Moreover, the utilization ofφC31 integrase can provide long-term and high levels foreign gene expression. To solve the obstacles caused by pro-nuclear injection, this study constructs a mouse model expressingφC31 integrase. Two kinds ofφC31 integrase expression mouse have been obtained. One is theφC31 integrase driven by EF1αpromoter, which comprises EF1α-Int and EF1α-Int-NLS and haveφC31 integrase ubiquitously expressed. Another is theφC31 integrase driven by Zp3 promoter, which comprises pZP3-INT and pZP3-INT-NLS and haveφC31 integrase expressed restrict in oocyte. EF1α-Int mice were further investigated.φC31 integrase mRNA was detected in the zygotes of EF1α-Int mice by RT-PCR analysis. Afterwards, we microinjected pBCPB+ into the zygotes of EF1α-Int mice and PCR results indicated the recombination of pBCPB+, generating a new attL site, which demonstrated thatφC31 integrase in zygotes could mediate the recombination between attB and attP sites.Additionally, a fluorescent binary switch was developed to evaluate the function ofφC31 integrase in living cells expediently. .In this switch system, the red fluorescent protein (RFP) expresses in the absence ofφC31 integrase but the green fluorescent protein (GFP) is produced whenφC31 integrase are expressed and catalyzes a recombination event. . To test the validity of this system, NIH3T3 cells were transiently transfected with different ratios of the plasmid encodingφC31 integrase and theφC31 integrase report plasmid, and the proportion of cells with red or green fluorescence were quantitatively measured by FACS. The numbers of cells converted from red to green increased with the increase of amount ofφC31 integrase encoding plasmid cotransfected. Approximately 90% of the color alternation was reached when the ratio ofφC31 integrase encoding plasmid to reporter plasmid was 10:1. This result indicated that theφC31 integrase could mediate the deletion of reporter plasmid in the context of genomic. Thus, this system may also be useful for producing transgenic mouse as“reporter”mice forφC31 integrase. We can construct a mouse model by targeting theφC31 integrase reporter into a ubiquitously expressed genomic site via homologous recombination in embryonic stem cells. Therefore, theφC31 integrase reporter mouse, which could be a fluorescent binary switch whenφC31 integrase is present, will be a useful tool for lineage-tracing studies.In conclusion, this study constructed a mouse model withφC31 integrase expression that can be used to generate transgenic mice with site-specific integration. This could offer an alternative way of producing transgenic mice with site-specific integration of foreign genes to avoid the disadvantage of the random integration in traditional pro-nuclear injection method.. Moreover, we constructed a dual fluorescent system to evaluate the function ofφC31 integrase in living cells. Meanwhile, this system provides elementary experimental data for producingφC31 integrase reporter mice.更多还原