腺相关病毒介导的西藏小型猪酪氨酸酶基因打靶的研究

发布时间：2018-07-06 11:24

本文选题：西藏小型猪 + 腺相关病毒　；参考：《南方医科大学》2012年硕士论文

【摘要】：研究背景与目的小型猪由于它的体型较小、易于抓取固定,便于实验操作,且其解剖结构、生理特性与人类极为相似,猪的食性以及作为人类食物链的一部分,是人类重要的肉质来源等特点,使其应用是生物医药研究中,较少的伦理问题,已广泛应用于生物医药研究领域,成为了生物医药研究的重要模式动物。西藏小型猪是世界上体型较小的猪种之一,来源于青藏高原海拔2500-4300m的农区和半农牧区,因其能够适应高海拔气候和以放牧为主的特点,使其具有良好的抗逆性和较强的适应能力,同时,封闭的地理环境使西藏西藏小型猪保存了非常纯正的品种资源。本课题组于2004年,将西藏小型猪从西藏引种至广州,目前已经完成风土驯化及实验动物化研究,并开展了相关的动物模型、药物实验及转基因克隆等研究。从免疫学、遗传学的研究发现,该品系具有独特的免疫标记和遗传特征,加上其独特较小的体型,使其成为了一种优良的实验用小型猪品种。基因工程动物的制备是目前生命科学领域前沿关键技术之一,应用分子生物学相关技术和转基因或者基因敲除等操作方法,改变动物基因组的遗传组成,可从基因水平改变动物的基因性状,进而改变动物的外在表型。由于分子生物学技术的进步和转基因克隆技术核移植技术的日趋成熟,使得人们按照意愿进行各种基因改造,获得满足各种需要的基因工程动物的愿望得以实现。但是,目前的基因工程动物的制作研究,仅限于少数的几种,最为成熟的是基因工程小鼠的研究,常用的技术方法有原位显微注射法和ES细胞法。而猪的ES细胞虽然已有分离成功的报道,但其培养体系并不稳定,同时远没有达到推广应用的水平。另外猪的受精卵数量来源有限且受精卵内脂肪过多,也不利于显微操作。因此利用这些传统的方法均难以获得转基因及基因敲除的基因修饰猪。利用细胞染色体DNA可以与外源DNA的同源序列发生同源重组的性质定向修饰和改造染色体上某一基因的技术叫基因打靶(gene targeting)。是20世纪80年代发展起来的一项重要分子生物学技术。它利用基因转移的方法,将外源DNA序列导入靶细胞以后,通过外源DNA序列与靶细胞内同源DNA序列间的重组,将外源基因定点整合到靶细胞基因组上某一确定位点,从而改变细胞遗传性状的方法。该技术具有定位性强,打靶后目的片段与染色体DNA共同稳定遗传的特点,为生命科学、基因组学和疾病治疗等领域的研究提供了强有力的技术手段。随着体细胞核移植技术的出现并不断成熟,转基因克隆技术逐渐成为制备转基因及基因敲除猪的主要方法。该法应用分子生物学技术及基因转导技术实现转基因及基因敲除的目的,从而改变体外培养的体细胞基因组成,后利用体细胞核移植技术达到对动物机体的基因组的修饰,获得基因修饰克隆猪。目前,基因打靶的策略主要包括正负筛选基因打靶策略、锌指核酸酶介导的基因打靶策略以及腺相关病毒介导的基因打靶策略。外源基因导入细胞的方式是影响打靶效率的重要因素,传统的基因导入方法如：显微注射法、脂质体法、电穿孔法等在真核细胞中发生同源重组的几率非常低,而文献报道,利用腺相关病毒(adeno-associated virus, AAV)介导的基因转移策略能够克服这一缺点,实现较高的打靶效率。新近的研究表明,腺相关病毒为一种单链微小DNA病毒,具有以下优点：①该病毒对宿主无致病性,比较安全；②感染能力强,细胞谱广泛,包括分裂和非分裂细胞；③rAAV虽无整合特异性,但是插入突变的可能性也较小；④外源基因表达稳定,可以长达2年。由于猪和人的皮肤在解剖结构和生理机能方面非常相似,猪的皮肤常被用于皮肤烧伤、化妆品及药物的皮肤毒性试验和皮肤过敏反应等方面的研究,我国小型猪的皮肤或毛发多为黑色或花色,而黑色或花色系猪种的皮肤显然不利于试验的观察。白色猪种多见于家猪等大型猪种,不利于实验操作。西藏小型猪由于其体型大小,皮肤的解剖生理特性与人极为相似,其独特的生物学特性使其成为了一个优良的实验用小型猪品种。但该小型猪全身的皮肤和毛发均为黑色,因此如何将西藏小型猪的毛发和皮肤培育成白色是小型猪实验动物化迫切需要解决的问题之一哺乳动物皮肤和毛发的颜色因色素种类和含量的不同而不同。酪氨酸酶是色素合成过程中的限速酶,研究证实,酪氨酸酶基因的缺失或者突变将直接导致人、鼠、猫等动物的白化。因此为了实现TYR基因敲除获得白化西藏小型猪,结合腺相关病毒在基因转移上的优势,本研究拟应用腺相关病毒介导的基因打靶策略,制备酪氨酸酶基因敲除的白化西藏小型猪的研究。与传统的遗传育种方法相比,转基因育种技术总体效率更高,不仅大大缩短了育种时间,且所获得的品系遗传背景单一,遗传特征明确,是制备白化实验用西藏小型猪的更为有效的策略。研究方法 1.西藏小型猪胚胎成纤维细胞(PEFs)的分离培养鉴定及冻存取怀孕30-35d时的西藏小型猪,无菌剖腹手术取出胎儿。在超净台内将获得的胚胎去除头、四肢及内脏,用眼科剪将胚胎组织剪成1mm3的组织碎块,胰蛋白酶消化,待消化充分后,加入含10%胎牛血清的DMEM培养基终止消化,离心弃上清,获得细胞,加入含10%胎牛血清的DMEM培养液,重悬细胞,于培养箱内培养,待细胞长到80%汇合度后,消化细胞,离心弃上清,加入冻存液1.5-2ml,滴度降温,最后置于液氮冻存。 2.构建TYR基因打靶载体 (1)克隆TYR基因的打靶同源臂序列。根据我课题组前期的研究获得的TYR基因序列,设计针对TYR基因第二个外显子为打靶的敲除位点,在exon2的上下游,分别设计打靶短臂(750bpb)和打靶长臂(1500bp),PCR扩增获得基因打靶的长短臂。 (2)构建腺相关病毒TYR基因打靶载体。根据所获得的TYR序列,将同源重组的长短臂分别克隆入PMD-18T载体内,分别命名为Larm及Sarm,并酶切鉴定后,测序鉴定。 (3)GFP-neomycin CDS其启动子PGK,polyA的扩增：以质粒PGKneotpAlox2为模板,设计引物,PCR扩增药物抗性基因和荧光报告基因及其启动子和多聚A尾,后用1%的琼脂糖凝胶电泳对PCR产物进行鉴定,并回收目的条带,然后将目的条带克隆入PMD-18T载体内(命名为PGK—GFP—neo—PA)。 (4)融合基因的扩增：分别以质粒Larm和Sarm为模板,PCR扩增打靶长短臂,PCR产物回收(分别命名为larm和sarm)。融合基因扩增：以larm、sarm、 PGK—EGFP-neo—PA三个PCR产物为模板。扩增融合基因,将PCR产物回收(命名为融合基因),连接到pMD—18T simple载体上。 (5)融合基因与打靶载体的连接：将测序正确的融合基因质粒进行NotI酶切,将酶切产物进行凝胶回收目的片段(命名为S-neo-L)。将质粒pAAV-MCS质粒进行NotI酶切,将酶切产物进行凝胶回收目的片段(命名为AV-ITR),将凝胶回收产物AV-ITR进行去磷酸化,与S-neo-L进行连接反应,将正确的连接产物命名为AV2-TYR-KO并测序鉴定。 (6)不同血清型病毒对PEFs细胞的感染效率的研究。利用不同血清型腺相关病毒感染PEFs,观察不同血清型腺相关病毒的感染效率,获得对PEF感染效率最高的血清型病毒,以此血清型包装病毒,以期获得最高的感染效率。 (7)包装腺相关病毒。应用感染法,将辅助质粒AAV2-PDG及核心质粒共同转染293T细胞,7天后收集病毒。 (8)用包装好后的病毒,感染PEFs,观察感染效率。 3.用TYR-AV2感染成纤维细胞,阳性基因敲除猪PEFs的筛选与鉴定。将成纤维细胞培养2-3天后,加入重组腺相关病毒,病毒滴度为MOI=105,感染48小时后,观察细胞绿色荧光表达情况,后用G418进行筛选获得阳性克隆,进行传代培养,并冻存。取部分细胞,进行体外裂解,以裂解产物为模板,进行PCR鉴定。 4.以TYR基因敲除的细胞作为核供体,经体细胞克隆技术制备TYR基因敲除的西藏小型猪(TYR+/-)。研究结果 1.筛选出了AAV2这一对PEFs具有高感染效率的血清型病毒。 2.成功获得应用于TYR基因敲除的腺相关病毒TYR-AAV2病毒。 3.初步尝试了应用重组TYR-AAV2感染PEFs,显示有较高的感染效率。本研究的创新之处 1.本项目利用我国特有的小型猪品系-西藏小型猪,将体细胞基因修饰技术和克隆技术相结合,培育具有我国自主知识产权的基因修饰克隆西藏小型猪。 2.采用腺相关病毒作为基因导入的载体,比传统方法具有更高的打靶效率。 3.根据酪氨酸酶基因结构和功能特点,采用基因敲除克隆猪技术制备白化西藏小型猪。为化妆品、药品的皮肤毒性实验及过敏反应等研究提供标准化的白化西藏小型猪。此外,也为人类白化病发病机制和基因治疗研究提供大型实验动物模型。
[Abstract]:Research background and purpose
Because of its small size, small pig is easy to grab and fixed, easy to operate, and its anatomical structure, physiological characteristics are very similar to human, the feeding nature of pig and a part of human food chain are the important meat quality sources of human being, so its application is a few ethical problems in biological medicine research, and it has been widely used in life. The field of medicine and medicine has become an important model animal in the research of biological medicine. Tibet miniature pig is one of the smaller pig species in the world. It is derived from the agricultural and semi Agricultural Pastoral Areas of the Qinghai Tibet plateau at the altitude of 2500-4300m. Because of its ability to adapt to the high altitude climate and the characteristics of grazing mainly, it has good resistance and strong adaptation. In 2004, Tibet miniature pigs were introduced from Tibet to Guangzhou, and the Tibet miniature pigs were introduced from Tibet to Guangzhou. At present, the study of domestication and experimental animals has been completed, and related animal models, drug experiments and transgenic cloning have been carried out. The study of Phytophthora and genetics has found that the strain has unique immune markers and genetic characteristics, and its unique smaller size makes it a fine experimental miniature pig.
The preparation of genetically engineered animals is one of the key technologies in the field of life science. Using molecular biology related techniques and transgenic or gene knockout methods, the genetic composition of the animal genome can be changed, and the genetic characters of animals can be changed from the gene level and then the external phenotype of the animal is changed. The progress of the technique and the growing maturity of the nuclear transfer technology of transgenic cloning technology have made it possible for people to carry out various genetic modifications according to their wishes and achieve the desire to meet the needs of genetically engineered animals. However, the present research on the production of genetically engineered animals is limited to several few, and the most mature is the gene engineering mice. The common technical methods are in situ microinjection and ES cell method. While the pig's ES cells have been reported successfully, the culture system is not stable, and it is far from the level of popularization. In addition, the number of fertilized eggs in pigs is limited and the fat in the fertilized eggs is too fat, which is not conducive to micromanipulation. Some traditional methods are difficult to obtain genetically modified and gene knockout pigs. The technology called gene targeting (gene targeting) using the homologous recombination of the homologous sequence of the cell chromosome DNA with the homologous sequence of the exogenous DNA is called gene targeting (gene targeting). It is an important molecule developed in 1980s. Biological technology. It uses the method of gene transfer to import exogenous DNA sequence into target cells and recombine exogenous DNA sequence and homologous DNA sequence within target cells, and integrate foreign genes into a definite location on the target cell genome, thus changing the method of cell genetic traits. This technique has strong positioning and after targeting. The combination of target fragment and chromosome DNA has the characteristics of stable heredity, which provides a powerful technical means for the research of life science, genomics and disease treatment. With the emergence and maturation of somatic cell nuclear transplantation technology, transgenic cloning technology has gradually become the main method to prepare transgenic and gene knockout pigs. Molecular biology technology and gene transduction technology realize the purpose of transgenic and gene knockout, so as to change the gene composition of somatic cells in vitro, and then use somatic cell nuclear transplantation to modify the genome of animal body, and obtain gene modification to clone pigs.
At present, the strategies of gene targeting include positive and negative screening gene targeting strategy, zinc finger nuclease mediated gene targeting strategy and adeno-related virus mediated gene targeting strategy. Exogenous gene introduction to cells is an important factor affecting target efficiency. The traditional methods of gene introduction, such as microinjection, liposome, electricity, etc. It is reported that the gene transfer strategy mediated by adeno-associated virus (AAV) can overcome this shortcoming and achieve high target efficiency. Recent studies have shown that adenosine is a single strand small DNA virus, with the following advantages : (1) the virus has no pathogenicity to the host, it is safer, the infection ability is strong, the cell spectrum is wide, including the split and non split cells; 3. Although rAAV has no integration specificity, the possibility of inserting mutation is also small; (4) the expression of foreign gene is stable for up to 2 years.
As pigs and human skin are very similar in anatomical structure and physiological function, the skin of pigs is often used in skin burns, skin toxicity tests of cosmetics and drugs and skin allergies. The skin or hair of small pigs of our country are mostly black or color, and the skin of black or flower and color pigs is obviously disadvantageous to test. The white pig species is mostly found in large pig species such as domestic pigs, which is not conducive to experimental operation. Because of its size and size, Tibet miniature pig is similar to human anatomy and physiology. Its unique biological characteristics make it a fine experimental miniature pig breed. But the skin and hair of the miniature pig are black, because of its skin and hair. How to breed white hair and skin of Tibet miniature pigs is one of the urgent problems to be solved in the laboratory animal experiment of miniature pigs.
The color of the skin and hair of mammals varies depending on the variety and content of the pigment. Tyrosinase is a speed limiting enzyme in the process of pigment synthesis. Studies have shown that the deletion or mutation of the tyrosinase gene will directly cause the albino of animals, such as humans, mice and cats.
Therefore, in order to achieve TYR knockout in Tibet miniature pigs and to combine the advantages of adeno related viruses in gene transfer, this study intends to use adeno-related virus mediated gene targeting strategy to prepare albino Tibet miniature pigs with tyrosinase gene knockout. Compared with traditional genetic breeding methods, transgenic breeding technology is general. Higher efficiency, not only greatly shortens the breeding time, but also has a single genetic background and a clear genetic feature. It is a more effective strategy for the preparation of Tibet miniature pigs in albino experiment.
research method
Isolation, culture, identification and cryopreservation of 1. Tibet miniature pig embryo fibroblasts (PEFs)
The Tibet miniature pig, who was pregnant with 30-35d, was taken out of the fetus by aseptic laparotomy. The embryos were removed in the super net, the embryos were removed, the limbs and the viscera were removed. The embryo tissue was cut into 1mm3 tissue fragments by the eye scissors, trypsin digestion, and after digestion, the digestion was added to the DMEM medium containing 10% fetal bovine serum, and the cells were centrifuged to get the cells. The DMEM culture medium containing 10% fetal bovine serum was added, and the cells were suspended in the incubator and cultured in the incubator. After the cells grew to 80% confluence, the digested cells, centrifuged and supernatant, were added to the cryopreservation liquid 1.5-2ml, and the titer was cooled down and finally stored in liquid nitrogen.
2. construction of TYR gene targeting carrier
(1) clone TYR gene target homologous arm sequence. According to the TYR gene sequence obtained by our previous research group, we designed the knockout loci for the target of the second exons of the TYR gene, and designed the short arm (750bpb) and the target long arm (1500bp) in the upper and lower reaches of the Exon2, and obtained the long and short arm of the gene targeting by PCR amplification.
(2) the gene targeting vector of adeno-associated virus (TYR) was constructed. According to the TYR sequence obtained, the long and short arms of the homologous recombinant were cloned into the PMD-18T carrying body respectively, named Larm and Sarm respectively, and identified by enzyme digestion.
(3) GFP-neomycin CDS promoter PGK, polyA amplification: using plasmid PGKneotpAlox2 as a template, designing primers, PCR amplification of drug resistance gene and fluorescent reporter gene and its promoter and A tail, then using 1% agarose gel electrophoresis to identify PCR products, and reclaim target bands, and then clone the target bands into the PMD-18T carrier body. (named PGK - GFP - neo - PA).
(4) amplification of fusion gene: plasmid Larm and Sarm as templates, PCR amplification of long and short arms, and recovery of PCR products (named larm and SARM respectively). Fusion gene amplification: larm, SARM, PGK EGFP-neo and PA three PCR products as templates. On the carrier.
(5) the connection between the fusion gene and the target carrier: the correct fusion gene plasmid was sequenced to cut the NotI enzyme, and the enzyme cut product was reclaimed by the gel. The plasmid pAAV-MCS plasmid was cut through the NotI enzyme, and the enzyme cut product was recycled by the gel to recover the target segment (named AV-ITR), and the gel recovery product AV-ITR was carried out. Phosphorylation was carried out to connect with S-neo-L. The correct linkage product was named AV2-TYR-KO and identified by sequencing.
(6) study on the infection efficiency of different serotype viruses to PEFs cells. Using different serotype adeno-related viruses to infect PEFs, observe the infection efficiency of different serotype adeno-related viruses, obtain the serotype virus with the highest efficiency of PEF infection, and use this serotype package virus to obtain the highest infection efficiency.
(7) packaging adeno-associated virus. Using the infection method, the plasmid AAV2-PDG and core plasmid were co transfected into 293T cells, and the virus was collected 7 days later.
(8) virus infection after packaging was used to infect PEFs and observe the infection efficiency.
3. screening and identification of PEFs knockout pigs with TYR-AV2 infection of fibroblasts.
After 2-3 days of fibroblast culture, the recombinant adeno-related virus was added to the virus, the titer of the virus was MOI=105. After 48 hours of infection, the expression of green fluorescence of the cells was observed. Then the positive clones were screened by G418, and then cultured and frozen. Some cells were frozen in vitro, and the pyrolysis products were used as the template for PCR identification.
4. TYR knockout Tibet miniature pig (TYR+/-) was prepared by somatic cell cloning technique using TYR gene knockout cells as nuclear donors.
Research results
1. screened out the AAV2 serotype PEFs with high infection efficiency.
2. the adeno-associated virus TYR-AAV2 virus was successfully obtained from TYR gene knockout.
3. a preliminary attempt was made to infect PEFs with recombinant TYR-AAV2, showing a high infection efficiency.
The innovation of this study
1. in this project, the small pig of our country, Tibet miniature pig, is used to combine the somatic cell gene modification and cloning technology to cultivate the gene modification of Tibet miniature pig with our own intellectual property rights.
2. adeno-associated virus is used as a vector for gene delivery, which has higher targeting efficiency than traditional methods.
3. according to the structural and functional characteristics of tyrosinase gene, albino Tibet miniature pigs were prepared by gene knockout cloning pigs. The study provided standardized albino Tibet miniature pigs for cosmetics, drug skin toxicity experiments and allergic reactions. In addition, it also provided large experimental animals for the pathogenesis and gene therapy of human albinism. Model.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332;Q78

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