以生殖细胞为外源基因载体建立异种移植转基因供体动物的研究

发布时间：2018-02-16 06:52

本文关键词： 异种移植生殖细胞转基因卵巢注射精子载体 PAMAM-D α-1 2岩藻糖苷转移酶补体调节蛋白　出处：《天津医科大学》2007年博士论文　论文类型：学位论文

【摘要】： 建立转基因动物是异种移植研究中极为重要的工作基础。我们采用以生殖细胞(卵细胞和精子细胞)为外源基因载体的新型转基因技术建立转基因动物，以简化操作、降低成本、提高外源基因整合和表达的效率。第一部分卵巢注射法建立hCD59、hCD55、HT单基因转移小鼠的研究目的探讨卵巢注射法建立异种移植用转基因动物的可行性。方法采用注射法将hC955、HT、hCD59基因重组质粒分别导入雌鼠卵巢内，交配后产下原代(G_0代)小鼠。提取G_0代小鼠基因组DNA，采用PCR方法对目的基因整合进行初筛。对PCR出现特异条带的小鼠基因组DNA进行Southern印迹杂交，进一步证实目的基因整合。抽取目的基因整合阳性小鼠的外周血，用逆转录-聚合酶链反应(RT-PCR)和流式细胞术(FCM)检测目的基因表达。对FCM表达阳性的转基因小鼠肝脏和肾脏进行免疫组织化学染色，观察目的基因在器官中的表达分布情况。对表达目的基因的G_0代小鼠进行交配生产F1代小鼠，同法检测F1代目的基因的整合与表达。分离3种转基因小鼠的脾脏淋巴细胞，进行人血清溶破实验。结果对15只雌性小鼠进行卵巢注射，共产生G_0代鼠130只。其中，注射HT基因重组质粒的小鼠产仔62只，注射hCD59基因重组质粒的小鼠产仔42只，注射hCD55基因重组质粒的小鼠产仔26只。经PCR初筛后，Southern印迹杂交证实染色体中整合HT、hCD55、hCD59基因的小鼠分别有21只、4只、9只，总整合率为26.2％，高于受精卵显微注射法的整合率。经RT-PCR检测共20只G_0代小鼠阳性，包括3只hCD55小鼠、14只HT小鼠、3只hCD59小鼠。FCM检测发现外周血单核细胞表达人H抗原、hCD55、hCD59的小鼠分别有9只、2只、2只，蛋白表达的效率为10.0％，高于受精卵显微注射法的表达率。免疫组化检测显示，在转基因小鼠的肝脏、肾脏等组织中均有相应外源基因的表达。G_0代小鼠交配后产下F1代小鼠37只，PCR检测hCD55、HT、hCD59基因整合的小鼠分别为7只、6只、3只，FCM检测表达的分别为0只、1只、1只。与对照组比较，三种转基因小鼠的脾脏淋巴细胞耐受人血清溶破的能力均有所增强。结论采用卵巢注射法可以使针对异种移植的基因在小鼠体内整合，并可实现RNA水平和蛋白质水平的表达，其整合与表达的效率要高于受精卵显微注射法。三种转基因构件均可遗传给后代(F1代)，并在蛋白质水平表达。人血清溶破实验证实，通过卵巢注射法制备的三种转基因动物可以发挥抗异种排斥的作用。第二部分卵巢内共注射建立hCD59／HT双基因和hCD55／hCD59／HT三基因转移小鼠的初步研究目的探讨卵巢内多基因共注射建立多基因转移动物的可行性。方法采用hCD59／HT双基因重组质粒和hCD59／hCD55／HT三基因重组质粒共同注射卵巢的方法，得到原代小鼠。PCR和Southern印迹杂交法检测原代小鼠目的基因整合，FCM检测目的基因表达。结果双基因重组质粒共同注射4只雌鼠，获得G_0代鼠31只。Southern印迹杂交证实仅出现hCD59／HT双基因阳性鼠2只，无单基因整合鼠，仅1只鼠出现hCD59／HT共表达阳性，另1只鼠表达阴性。三基因重组质粒共同注射5只雌鼠，3胎共产生G_0代鼠137只，Southern印迹杂交证实仅出现hCD59／HT双基因阳性鼠2只，无其它整合情况，表达均为阴性。结论多基因共注射卵巢法可以得到相应的转基因动物，该方法确实可靠且周期较短。但多基因共注射后，基因之间会互相影响，整合与表达的效率比单基因注射者明显降低，，提示其中有更复杂的机制参与，需要深入研究。第三部分PAMAM-D对hCD55基因转染猪精子细胞介导作用的研究目的探讨新型纳米材料PAMAM-D对hCD55基因转染猪精子细胞的介导作用。方法将新型纳米材料PAMAM-D(G_5)和线状hCD55 DNA按照不同氮磷比(电荷比)制备PAMAM-D／hCD55复合物。取部分复合物酶切电泳。将复合物与处理后的1×10~6猪精子细胞共孵育2h，原位杂交法检测hCD55对猪精子细胞的转染效率，经精子质量检测工作站检测孵育后的精子活力和精子畸形率。结果对PAMAM-D／DNA复合物进行酶切消化后，其中的DNA分子不被限制性内切酶降解。经原位杂交法检测，加入PAMAM-D后可以明显提高各组猪精子细胞的转染效率，且基本上随着氮磷比的增加转染效率也随之增高。最高者(400ng，氮磷比20：1)可达对照组的167％(47.5％±3.5％vs 28.5％±1.5％)。经精子质量检测工作站检测PAMAM-D对精子细胞活力和精子细胞畸形率没有明显影响(p＞0.05)。结论PAMAM-D作为载体可以提高目的基因对猪精子细胞的转染效率，其对猪精子细胞无明显毒性，增强了精子载体法的实用性，为生产异种移植用转基因猪提供一种可供参考的方法。
[Abstract]:Transgenic animal is a very important basic work in xenotransplantation. We adopt germ cells (egg and sperm cells) for the establishment of transgenic animal model of transgenic technology of exogenous gene carrier, to simplify the operation, reduce the cost, improve the efficiency of exogenous gene integration and expression.
The first part of ovary injection to establish hCD59, hCD55, HT single gene transfer mice
Objective to investigate the feasibility of ovary injection method to establish transgenic animal xenograft. Methods using injection method, hC955, HT, hCD59 gene recombinant plasmids were introduced into ovaries, mating after the birth of primary (G_0) mice. G_0 mice genomic DNA extraction, using PCR method of gene integration were screened. The PCR specific band of mouse genomic DNA by Southern blot hybridization, further confirmed that the target gene integration. Peripheral blood samples were collected from the target gene integration positive mice, using reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry (FCM) to detect the expression of target gene expression. The positive transgenic mice were immunized with liver and kidney histochemical staining of FCM, observe the expression of target gene in organ distribution. The expression of target gene in G_0 generation mice were mated to produce F1 generation mice, the same method for the detection of F1 gene generation Integration and expression. Separation of 3 transgenic mouse spleen lymphocytes, human serum 1yse. Experimental results are only 15 ovarian injection on female mice, the mice produced 130 G_0 generation. The injection of recombinant plasmid of HT gene of mice born 62, injection of recombinant plasmid of hCD59 gene of mice born 42, recombinant injection of hCD55 plasmid in mice. 26 mice were born after screening by PCR, Southern blot analysis confirmed that HT, hCD55 chromosome hCD59 gene integration, the mice were 21, 4, 9, the total integration rate was 26.2%, higher than the integration rate of microinjection. Detected by RT-PCR 20 G_0 mice were positive, including 3 hCD55 mice and 14 HT mice and 3 hCD59 mice.FCM detected expression of H antigen in peripheral blood mononuclear cells hCD55, hCD59 mice were 9, 2, 2, the expression rate was 10%, higher than the zygote microinjection The rate of expression. Immunohistochemistry showed that the transgenic mice liver, kidney and other tissues have the corresponding expression of exogenous gene in.G_0 generation mice F1 generation 37 mice, PCR detection of hCD55, HT, integration of hCD59 gene in mice were 7, 6, 3, FCM. Expression of respectively 0, 1, 1. Compared with the control group, the ability of the three transgenic mouse spleen lymphocyte tolerance of human serum 1yse is enhanced. Conclusion the method of ovary injection can make for xenotransplantation gene integration in mice, and can realize the RNA expression level and protein level the efficiency of integration and expression were higher than that of microinjection. Three transgenic components can be inherited to offspring (F1), and the expression in protein level. Human serum 1yse experiments confirmed that three transgenic animal by ovary injection preparation can play against The effect of xenograft rejection.
A preliminary study on the establishment of hCD59 / HT double gene and hCD55 / hCD59 / HT three gene transfer mice in second parts of the ovary
Objective to explore the feasibility of establishing multi gene transfer animal ovary gene co injection. Method hCD59 / HT double gene recombinant plasmid and hCD59 / hCD55 / HT three gene recombinant plasmid co injection of ovarian, primary mouse.PCR and Southern blot detection of primary mouse gene integration, to detect the expression of FCM gene results. Double gene recombinant plasmid co injection of 4 rats, 31 rats received G_0.Southern blot analysis confirmed that there were only hCD59 / HT double gene positive 2 rats, no single gene integration in 1 rats, only hCD59 / HT co expression, another 1 rats of three recombinant expression. Coinjeoted into 5 rats, 3 fetal G_0 produced a total of 137 rats, Southern blot analysis confirmed that there were only hCD59 / HT double gene positive 2 rats, no other integration, expression were negative. Conclusion multi gene co injection method of ovary In order to get the corresponding transgenic animals, the method is reliable and short. But after CO injection, the genes will interact with each other. The efficiency of integration and expression is significantly lower than that of single gene injectors, suggesting that there are more complex mechanisms involved, which need further research.
Study on the mediating effect of the third part of PAMAM-D on the transfection of hCD55 gene to porcine spermatozoa
Objective to explore the new nano PAMAM-D on hCD55 gene transfected pig sperm cells mediated by a novel method of nano materials PAMAM-D (G_5) and linear hCD55 DNA according to the different ratio of nitrogen and phosphorus (charge ratio) to prepare PAMAM-D / hCD55 composites. The compounds were digested by restriction enzymes. The compound and after treatment 1 x 10~6 pig sperm cells were incubated with 2H, transfection efficiency detected by hCD55 in situ hybridization in pig sperm cells, the sperm quality inspection workstation detection after incubation of sperm motility and sperm deformity rate. The results of PAMAM-D / DNA composites were digested, DNA molecules are not restriction enzyme degradation. Detected by in situ hybridization, after joining PAMAM-D groups can significantly improve the transfection efficiency of pig sperm cells, and basically with the ratio of nitrogen and phosphorus increased transfection efficiency was also increased. The highest (400ng, N / P ratio 20:1) than the control group 167% (47.5% + 28.5% + 1.5% 3.5%vs). The sperm quality detection workstation detection of PAMAM-D on sperm viability and sperm deformity rate of cells had no significant effect (P > 0.05). Conclusion PAMAM-D as carrier can improve the gene transfection efficiency of pig sperm cells, no obvious toxicity on pig sperm cells, enhance the practical sperm vector method, provides a reference method for production of xenotransplantation transgenic pigs.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R-332

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