【作者】 刘勤；

【导师】 魏泓；

【作者基本信息】 第三军医大学 ， 动物学， 2008， 硕士

【摘要】 一、研究目的及背景转基因动物模型是联系分子、细胞水平研究和整体动物研究的桥梁。自1980年Gordon[1]首次采用原核显微注射开展动物转基因研究以来,已有十多种主要的转基因动物制作方法,如原核显微注射法、逆转录病毒侵染法、精子介导法、胚胎干细胞法,体细胞核移植法等。这些转基因动物制备方法各有优缺点:原核显微注射法制作转基因动物DNA整合效率低,整合位点不定,技术难度大,应用于高等哺乳动物时的效率很低;精子介导基因转移技术技术存在随机性和不确定性;胚胎干细胞法制备的转基因首建动物一般是嵌合体,不易实现外源基因的自然繁殖传代,且建立干细胞系的难度较大,应用受到限制;体细胞核移植法操作难度大,且外源基因的表达与否受整合位点附近基因的影响。相对而言,慢病毒侵染法在可操作性、胚胎发育率、移植妊娠率、目的基因整合效率、目的基因表达率、阳性个体的选育等方面比上述几种技术均有显著的优势。1976年,Jaenish[2]首次用逆转录病毒(Retroviruses)感染胚胎制作转基因动物获得成功。逆转录病毒的RNA进入细胞后,逆转录成DNA前病毒,利用逆转录病毒的整合酶及其特异的末端核苷酸序列,将DNA前病毒整合到染色体上,从而将其携带的外源基因也插入到染色体上。慢病毒(lentivirus)属逆转录病毒的亚科,具有逆转录病毒的基本结构gag、pol和env,不同之处在于慢病毒包含4个辅助基因,vif、vpr、nef、vpu和2个调节基因tat和rev。其中调节基因tat和rev分别编码两个反式激活因子Tat蛋白和Rev蛋白, Tat蛋白在慢病毒基因组复制和转录延伸过程中发挥重要作用,Rev蛋白则可促使其基因的表达由早期向晚期转化。HIV-1 DNA前病毒的主要结构基因及其排列形式与其它逆转录病毒相同,为5’LTR-gag-pro-pol-env-3’LTR,其中gag基因编码病毒的核心蛋白,pol基因编码病毒复制所需的酶类,env基因编码病毒的包膜糖蛋白,pro基因则编码切割蛋白前体所需的蛋白酶。慢病毒载体(Lentiviral vector,LV)广泛用于体外细胞的转染和基因治疗的研究。目前用LV成功转染的细胞有神经细胞、视网膜感光细胞、肌细胞,肝细胞、早期胚胎细胞等,用LV制作的转基因动物有小鼠、大鼠、猪,牛等。LV的优势在于:1)转染效率高,能够感染分裂期和静息期细胞,能转染多种组织;2)整合到宿主基因组中的目的基因能持久稳定的表达,不易形成嵌合体,一般能自然繁殖传代;3)LV经改构后,不在宿主细胞内增殖,也不会导致寄主细胞的死亡,免疫源性极小,生物安全性高。LV用于转基因动物模型制备也有一定的局限性:1)一是制备难度较大,不易获得高滴度、高纯度的病毒;2) LV在宿主基因组上的整合一般是随机的,可能干扰插入位点及其附近基因表达;3)虽可通过控制病毒的滴度来调节转入基因的拷贝数,但是不容易实现整合拷贝数的精确控制;4)携带目的基因的容量较小(<10kb),当需要插入大片段DNA时,会限制制备LV的滴度;5) LV前病毒DNA在宿主基因上是多位点随机整合,当转基因启动子的表达需要多拷贝时应选择原核显微注射法。LV技术与siRNA结合使用,可以相对容易地在转基因动物细胞中对特定基因实现RNAi效应。RNAi效应的核心是一段20-25nt的与靶基因mRNA互补或不完全互补的短RNA,通过它和靶基因mRNA碱基配对引导沉默复合体(RISC)降解mRNA或阻碍其翻译。要实现表达如此短的一段siRNA片段,转基因载体的长度可以控制在很合适的范围内(4~5kb),对插入目的基因容量有限(<10kb)的LV来说十分理想,因而LV在制备RNA干扰转基因动物模型上的优势更加明显。本实验通过慢病毒介导高效地研制了eGFP转基因小鼠,建立了慢病毒介导的转基因小鼠制备技术体系,并利用该技术制备了树突状细胞(Dentrentic Cells,DCs)特异性干扰Rab32基因表达的RNA干扰小鼠模型,进一步验证了利用该技术体系在小鼠树突状细胞实现对特定基因进行抑制的可行性与有效性。二、研究内容及结果在第一部分,本课题开展了通过慢病毒载体介导、结合卵周隙显微注射、以eGFP为报告基因的转基因小鼠制备方法学研究,探讨了通过卵周隙显微注射重组慢病毒对胚胎发育的影响,以及通过此法研制转基因小鼠的效率。结果:1)建立并优化了三质粒慢病毒包装系统,获得了滴度达到1×10~9TU/ml以上的注射用重组慢病毒;通过卵周隙显微注射,胚胎的2-细胞卵裂率为81.8%(1189/1453),与无处理组无显著差异;在2-细胞期,每一视野下胚胎阳性率>90%(1178/1189),说明包装的病毒成功并高效地转染小鼠胚胎;2)将注射后发育至2-细胞的胚胎通过输卵管壶腹部穿刺移植法移植后,假孕母鼠妊娠率为42.8%(12/28) ,很大程度上避免了伞部移植法出血导致的胚胎存活率下降,提高了移植成功率;3)eGFP首建鼠阳性率为60.8%(73/120)、转基因小鼠的总体研制效率(转基因小鼠数/注射胚胎数)为5.0%(73/1453),说明卵周隙显微注射对胚胎2-细胞卵裂率几乎无影响,表明慢病毒载体辅以卵周隙显微注射法制作转基因动物高效可行;4)将eGFP阳性首建小鼠采用近交方法建系,至第6个世代,荧光观察发现超排获得的胚胎全部呈eGFP阳性,且新生鼠阳性率为100%,说明eGFP基因可以通过种系传代。在第二部分,采用了基于siRNA(Small interfering RNA)的RNA干扰策略、利用第一部分建立的转基因平台制作转基因小鼠模型。转基因RNA干扰载体FcdGW-Rab32的表达产物siRNA-Rab32可在小鼠树突状细胞(dendritic cells,DCs)中特异性干扰Rab32基因,报告基因为eGFP。由于DCs中并没有荧光素酶基因,构建了在DCs中特异性干扰荧光素酶基因的载体质粒FcdGW-FF3作为对照。结果:1) FcdGW-Rab32和FcdGW-FF3组的2-细胞卵裂率分别为79.8%(408/511)、75.8%(317/418),受体妊娠率分别为35.7%(5/14)、33.3%(4/12),移植胚胎体内存活率分别为2.7%(11/408)、2.8%(9/317),转基因首建鼠阳性率分别为54.5%(6/11)、66.7%(6/9),转基因小鼠总体研制效率分别为1.2%(6/511)、1.4%(6/418),除胚胎体内存活率和总体研制效率外,其他数据与第一部分的结果相当;2)经PCR检测和耳廓皮肤真皮层DCs荧光观察结果表明,FcdGW-Rab32和FcdGW-FF3转基因小鼠已被建立,目的基因已经正确整合至转基因小鼠基因组中,且能通过种系传代;3)流式细胞仪检测BMDC表明,eGFP只在小鼠的DCs内特异性表达,说明我们所采用的CD11c启动子具备较好的定向表达能力;4)通过相对荧光定量PCR(Fluorenscent Quantitative PCR,FQ-PCR)对DCs的Rab32基因表达情况进行检测,结果显示Rab32基因的表达下调78.3%,特异性地实现了对DCs的Rab32基因产生Gene knock-down的干扰效应。三、研究结论1.本课题成功建立了以慢病毒介导、结合卵周隙显微注射、以eGFP为报告基因的转基因小鼠制备技术体系,获得了可稳定传代的eGFP转基因小鼠,证实该技术体系有较高的转基因效率。2.将慢病毒介导的转基因动物技术与RNAi技术相结合,首次建立了在小鼠DCs特异性抑制Rab32基因的RNA干扰小鼠模型,验证了该转基因技术体系的在制备转基因小鼠模型的有效性,提示此技术路线在研究树突状细胞抗原交叉递呈的调控方面有一定的应用价值。更多还原

【Abstract】 1. BackgroundTransgenic technology is an important technique plateforme for in vivo investigation of gene functions. Currently, the applicable transgene techniques include micro-injection, ES cell-mediated transgenesis, sperm-mediated gene transfer and nuclear transfer. In all the techniques mentioned above, micro-injiection is most widely used and applicable to most animal species. However, this technique is inefficient, costly and requires sophisticated equipments and skills, especially when it comes to large animal species. Lentiviral vector provides an alternative method to produce transgenic animals. Lentiviral vector is a retroviral vecor. After infection of cells, the RNA genome of the viral vector can be reverse transcribed into DNA, and the DNA can be efficiently integrated into the genome of host cells. Besides, in contrast to the congene retroviral vectors, lentivral vector is capable of infecting both deviding and quiescient cells. On the basis of this point, Lois(2002) et al successfully produced transgenic mice with lentiviral vector. In this work, the transgene delivered by lentiviral vector expressed efficiently and was not subject to silence during germline transmission.Detrentic cell (DC) is the main professional antigen-presentation cells (APC) in human and mammal bodies. In addition to stimulating immune response, DC is also capable of inducing peripheral immune tolerance. Therefore, to understand the precise mechanism of antigene presentation, DC is the suitable target cells. The process of antigen presentation in DC is mediated by vestricle translocation, which is a highly organized process regulated by Rab gene farmily. On the basis of this point, to understand the detailed mechanism of antigen presentation, it is necessary to investigate the roles of Rab genes in the process of antigene presentation. In this work, we established a DC-specific Rab32 knock-down mouse model by lentiviral transgenesis which had significantly reduced Rab32 expression specificanlly in DCs, and laid down the foundation to further investigate the roles of Rab32 protein in antigen presentation.2. ResultsIn the first part, methological researches on lentiviral transgenesis was performed. The mouse eggs was collected and subjected to perivitelline space injection (PSI) with concentrated lentiviral vectors. The effects of PSI on egg development were investigated. The results were the following:1) Through the three-plasmd packgeing system, concentrated lentivial suspension of which the TU was >1×109 was prepared; after PSI of the vector, 2-cell cleavage rate of the eggs injected with lentivral vector was 81.8%(1189/1453), with no significant difference from the untreated eggs, indicating that PSI with lentiviral vector had no impactt on the development of the injected eggs; the GFP positive rate (based on counting under microscope) was more than 90%(1178/1189), indicating that the lentiviral vector was properly packaged and capable of transducing mouse eggs efficiently; 2) the 2-cell eggs which had been subjected to PSI were transferred into pseudo-pregnant female mice, and the pregnance rate of the recipients was 42.8%(12/28), which was also comparable to the untreated egg group, suggesting that in vivo development capacity of the injected eggs was not impacted by PSI process either; 3) the transgenic rate of the founder mice was 60.8%(73/120), and the overall transgenic mouse production efficiency (transgenic mice/injected eggs) was 5.0%(73/1453), further indicating that lentiviral vector-mediated transgenesis is an efficient and practicable method for transgenic mouse production; 4) based on the eGFP transgenic founder mice, a transgenic line was established by mating the transgenic founder mice with wild-type mice of the same strain. Results indicated that transgene expression remained stable at least over six generations, suggesting that the expression of transgenes delivered by lentiviral vector can be passed on through germline transmission.In the second part, based on RNA interference (RNAi) strategy, we produced transgenic knock-down mice targeting Rab32 gene in DCs by lentiviral transgenesis. The vector for expressing siRNA molecules was constructed based on the FUGW vector which was used in the first part. To express the siRNA molecules specifically in DCs, the siRNA coding sequence was cloned downstream the CD11c promoter. To trace the siRNA expression visibly, the eGFP coding sequence was placed downstream the same promoter in the same orientation. The results are the following: 1) the siRNA expression cassette was detected in the genomic DNAs of both the founder mice and their F1 offspring by PCR. Besides, eGFP expression was observed in the DCs located in the skin of ear concha under fluorescent microscope. These results indicated that transgenic mice haboring the siRNA expression cassette was produced. 2) By flow cytometry analysis of Bone Marrow Detrentic Cells (BMDC), it was shown that eGFP was specifically expressed in DCs, indicating that the used CD11c promoter drive siRNA expression specifically in DCs. 3) By Fluorenscent Quantitative PCR ( FQ-PCR) analysis, the Rab32 expression in DCs was shown to be reduced by 78.3%, indicating that the siRNA molecule delivered by lentiviral vector was expressed and knocked down the target gene expression significantly. Conclusions1. An efficient and praticable lentiviral transgenesis system, which consists of lentiviral vector package, perivitllent space injection (PSI) and mouse egg manipulation, was successfully established.2. Using the established lentiviral transgenesis system, transgenic knock-down mice targeting Rab32 specifically in DCs was produced, further indicating that the established lentiviral transgenesis can be applied to expressing both structural genes and siRNA molecules.更多还原