本文選題：西藏小型豬 + 腺相關(guān)病毒�。� 參考：《南方醫(yī)科大學(xué)》2012年碩士論文

【摘要】：研究背景與目的 小型豬由于它的體型較小、易于抓取固定,便于實驗操作,且其解剖結(jié)構(gòu)、生理特性與人類極為相似,豬的食性以及作為人類食物鏈的一部分,是人類重要的肉質(zhì)來源等特點,使其應(yīng)用是生物醫(yī)藥研究中,較少的倫理問題,已廣泛應(yīng)用于生物醫(yī)藥研究領(lǐng)域,成為了生物醫(yī)藥研究的重要模式動物。西藏小型豬是世界上體型較小的豬種之一,來源于青藏高原海拔2500-4300m的農(nóng)區(qū)和半農(nóng)牧區(qū),因其能夠適應(yīng)高海拔氣候和以放牧為主的特點,使其具有良好的抗逆性和較強(qiáng)的適應(yīng)能力,同時,封閉的地理環(huán)境使西藏西藏小型豬保存了非常純正的品種資源。本課題組于2004年,將西藏小型豬從西藏引種至廣州,目前已經(jīng)完成風(fēng)土馴化及實驗動物化研究,并開展了相關(guān)的動物模型、藥物實驗及轉(zhuǎn)基因克隆等研究。從免疫學(xué)、遺傳學(xué)的研究發(fā)現(xiàn),該品系具有獨特的免疫標(biāo)記和遺傳特征,加上其獨特較小的體型,使其成為了一種優(yōu)良的實驗用小型豬品種。 基因工程動物的制備是目前生命科學(xué)領(lǐng)域前沿關(guān)鍵技術(shù)之一,應(yīng)用分子生物學(xué)相關(guān)技術(shù)和轉(zhuǎn)基因或者基因敲除等操作方法,改變動物基因組的遺傳組成,可從基因水平改變動物的基因性狀,進(jìn)而改變動物的外在表型。由于分子生物學(xué)技術(shù)的進(jìn)步和轉(zhuǎn)基因克隆技術(shù)核移植技術(shù)的日趨成熟,使得人們按照意愿進(jìn)行各種基因改造,獲得滿足各種需要的基因工程動物的愿望得以實現(xiàn)。但是,目前的基因工程動物的制作研究,僅限于少數(shù)的幾種,最為成熟的是基因工程小鼠的研究,常用的技術(shù)方法有原位顯微注射法和ES細(xì)胞法。而豬的ES細(xì)胞雖然已有分離成功的報道,但其培養(yǎng)體系并不穩(wěn)定,同時遠(yuǎn)沒有達(dá)到推廣應(yīng)用的水平。另外豬的受精卵數(shù)量來源有限且受精卵內(nèi)脂肪過多,也不利于顯微操作。因此利用這些傳統(tǒng)的方法均難以獲得轉(zhuǎn)基因及基因敲除的基因修飾豬。利用細(xì)胞染色體DNA可以與外源DNA的同源序列發(fā)生同源重組的性質(zhì)定向修飾和改造染色體上某一基因的技術(shù)叫基因打靶(gene targeting)。是20世紀(jì)80年代發(fā)展起來的一項重要分子生物學(xué)技術(shù)。它利用基因轉(zhuǎn)移的方法,將外源DNA序列導(dǎo)入靶細(xì)胞以后,通過外源DNA序列與靶細(xì)胞內(nèi)同源DNA序列間的重組,將外源基因定點整合到靶細(xì)胞基因組上某一確定位點,從而改變細(xì)胞遺傳性狀的方法。該技術(shù)具有定位性強(qiáng),打靶后目的片段與染色體DNA共同穩(wěn)定遺傳的特點,為生命科學(xué)、基因組學(xué)和疾病治療等領(lǐng)域的研究提供了強(qiáng)有力的技術(shù)手段。隨著體細(xì)胞核移植技術(shù)的出現(xiàn)并不斷成熟,轉(zhuǎn)基因克隆技術(shù)逐漸成為制備轉(zhuǎn)基因及基因敲除豬的主要方法。該法應(yīng)用分子生物學(xué)技術(shù)及基因轉(zhuǎn)導(dǎo)技術(shù)實現(xiàn)轉(zhuǎn)基因及基因敲除的目的,從而改變體外培養(yǎng)的體細(xì)胞基因組成,后利用體細(xì)胞核移植技術(shù)達(dá)到對動物機(jī)體的基因組的修飾,獲得基因修飾克隆豬。 目前,基因打靶的策略主要包括正負(fù)篩選基因打靶策略、鋅指核酸酶介導(dǎo)的基因打靶策略以及腺相關(guān)病毒介導(dǎo)的基因打靶策略。外源基因?qū)爰?xì)胞的方式是影響打靶效率的重要因素,傳統(tǒng)的基因?qū)敕椒ㄈ纾猴@微注射法、脂質(zhì)體法、電穿孔法等在真核細(xì)胞中發(fā)生同源重組的幾率非常低,而文獻(xiàn)報道,利用腺相關(guān)病毒(adeno-associated virus, AAV)介導(dǎo)的基因轉(zhuǎn)移策略能夠克服這一缺點,實現(xiàn)較高的打靶效率。新近的研究表明,腺相關(guān)病毒為一種單鏈微小DNA病毒,具有以下優(yōu)點：①該病毒對宿主無致病性,比較安全；②感染能力強(qiáng),細(xì)胞譜廣泛,包括分裂和非分裂細(xì)胞；③rAAV雖無整合特異性,但是插入突變的可能性也較�。虎芡庠椿虮磉_(dá)穩(wěn)定,可以長達(dá)2年。 由于豬和人的皮膚在解剖結(jié)構(gòu)和生理機(jī)能方面非常相似,豬的皮膚常被用于皮膚燒傷、化妝品及藥物的皮膚毒性試驗和皮膚過敏反應(yīng)等方面的研究,我國小型豬的皮膚或毛發(fā)多為黑色或花色,而黑色或花色系豬種的皮膚顯然不利于試驗的觀察。白色豬種多見于家豬等大型豬種,不利于實驗操作。西藏小型豬由于其體型大小,皮膚的解剖生理特性與人極為相似,其獨特的生物學(xué)特性使其成為了一個優(yōu)良的實驗用小型豬品種。但該小型豬全身的皮膚和毛發(fā)均為黑色,因此如何將西藏小型豬的毛發(fā)和皮膚培育成白色是小型豬實驗動物化迫切需要解決的問題之一 哺乳動物皮膚和毛發(fā)的顏色因色素種類和含量的不同而不同。酪氨酸酶是色素合成過程中的限速酶,研究證實,酪氨酸酶基因的缺失或者突變將直接導(dǎo)致人、鼠、貓等動物的白化。 因此為了實現(xiàn)TYR基因敲除獲得白化西藏小型豬,結(jié)合腺相關(guān)病毒在基因轉(zhuǎn)移上的優(yōu)勢,本研究擬應(yīng)用腺相關(guān)病毒介導(dǎo)的基因打靶策略,制備酪氨酸酶基因敲除的白化西藏小型豬的研究。與傳統(tǒng)的遺傳育種方法相比,轉(zhuǎn)基因育種技術(shù)總體效率更高,不僅大大縮短了育種時間,且所獲得的品系遺傳背景單一,遺傳特征明確,是制備白化實驗用西藏小型豬的更為有效的策略。 研究方法 1.西藏小型豬胚胎成纖維細(xì)胞(PEFs)的分離培養(yǎng)鑒定及凍存 取懷孕30-35d時的西藏小型豬,無菌剖腹手術(shù)取出胎兒。在超凈臺內(nèi)將獲得的胚胎去除頭、四肢及內(nèi)臟,用眼科剪將胚胎組織剪成1mm3的組織碎塊,胰蛋白酶消化,待消化充分后,加入含10%胎牛血清的DMEM培養(yǎng)基終止消化,離心棄上清,獲得細(xì)胞,加入含10%胎牛血清的DMEM培養(yǎng)液,重懸細(xì)胞,于培養(yǎng)箱內(nèi)培養(yǎng),待細(xì)胞長到80%匯合度后,消化細(xì)胞,離心棄上清,加入凍存液1.5-2ml,滴度降溫,最后置于液氮凍存。 2.構(gòu)建TYR基因打靶載體 (1)克隆TYR基因的打靶同源臂序列。根據(jù)我課題組前期的研究獲得的TYR基因序列,設(shè)計針對TYR基因第二個外顯子為打靶的敲除位點,在exon2的上下游,分別設(shè)計打靶短臂(750bpb)和打靶長臂(1500bp),PCR擴(kuò)增獲得基因打靶的長短臂。 (2)構(gòu)建腺相關(guān)病毒TYR基因打靶載體。根據(jù)所獲得的TYR序列,將同源重組的長短臂分別克隆入PMD-18T載體內(nèi),分別命名為Larm及Sarm,并酶切鑒定后,測序鑒定。 (3)GFP-neomycin CDS其啟動子PGK,polyA的擴(kuò)增：以質(zhì)粒PGKneotpAlox2為模板,設(shè)計引物,PCR擴(kuò)增藥物抗性基因和熒光報告基因及其啟動子和多聚A尾,后用1%的瓊脂糖凝膠電泳對PCR產(chǎn)物進(jìn)行鑒定,并回收目的條帶,然后將目的條帶克隆入PMD-18T載體內(nèi)(命名為PGK—GFP—neo—PA)。 (4)融合基因的擴(kuò)增：分別以質(zhì)粒Larm和Sarm為模板,PCR擴(kuò)增打靶長短臂,PCR產(chǎn)物回收(分別命名為larm和sarm)。融合基因擴(kuò)增：以larm、sarm、 PGK—EGFP-neo—PA三個PCR產(chǎn)物為模板。擴(kuò)增融合基因,將PCR產(chǎn)物回收(命名為融合基因),連接到pMD—18T simple載體上。 (5)融合基因與打靶載體的連接：將測序正確的融合基因質(zhì)粒進(jìn)行NotI酶切,將酶切產(chǎn)物進(jìn)行凝膠回收目的片段(命名為S-neo-L)。將質(zhì)粒pAAV-MCS質(zhì)粒進(jìn)行NotI酶切,將酶切產(chǎn)物進(jìn)行凝膠回收目的片段(命名為AV-ITR),將凝膠回收產(chǎn)物AV-ITR進(jìn)行去磷酸化,與S-neo-L進(jìn)行連接反應(yīng),將正確的連接產(chǎn)物命名為AV2-TYR-KO并測序鑒定。 (6)不同血清型病毒對PEFs細(xì)胞的感染效率的研究。利用不同血清型腺相關(guān)病毒感染PEFs,觀察不同血清型腺相關(guān)病毒的感染效率,獲得對PEF感染效率最高的血清型病毒,以此血清型包裝病毒,以期獲得最高的感染效率。 (7)包裝腺相關(guān)病毒。應(yīng)用感染法,將輔助質(zhì)粒AAV2-PDG及核心質(zhì)粒共同轉(zhuǎn)染293T細(xì)胞,7天后收集病毒。 (8)用包裝好后的病毒,感染PEFs,觀察感染效率。 3.用TYR-AV2感染成纖維細(xì)胞,陽性基因敲除豬PEFs的篩選與鑒定。 將成纖維細(xì)胞培養(yǎng)2-3天后,加入重組腺相關(guān)病毒,病毒滴度為MOI=105,感染48小時后,觀察細(xì)胞綠色熒光表達(dá)情況,后用G418進(jìn)行篩選獲得陽性克隆,進(jìn)行傳代培養(yǎng),并凍存。取部分細(xì)胞,進(jìn)行體外裂解,以裂解產(chǎn)物為模板,進(jìn)行PCR鑒定。 4.以TYR基因敲除的細(xì)胞作為核供體,經(jīng)體細(xì)胞克隆技術(shù)制備TYR基因敲除的西藏小型豬(TYR+/-)。 研究結(jié)果 1.篩選出了AAV2這一對PEFs具有高感染效率的血清型病毒。 2.成功獲得應(yīng)用于TYR基因敲除的腺相關(guān)病毒TYR-AAV2病毒。 3.初步嘗試了應(yīng)用重組TYR-AAV2感染PEFs,顯示有較高的感染效率。 本研究的創(chuàng)新之處 1.本項目利用我國特有的小型豬品系-西藏小型豬,將體細(xì)胞基因修飾技術(shù)和克隆技術(shù)相結(jié)合,培育具有我國自主知識產(chǎn)權(quán)的基因修飾克隆西藏小型豬。 2.采用腺相關(guān)病毒作為基因?qū)氲妮d體,比傳統(tǒng)方法具有更高的打靶效率。 3.根據(jù)酪氨酸酶基因結(jié)構(gòu)和功能特點,采用基因敲除克隆豬技術(shù)制備白化西藏小型豬。為化妝品、藥品的皮膚毒性實驗及過敏反應(yīng)等研究提供標(biāo)準(zhǔn)化的白化西藏小型豬。此外,也為人類白化病發(fā)病機(jī)制和基因治療研究提供大型實驗動物模型。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R-332;Q78

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 雷鐵池;酪氨酸酶基因家族與皮膚黑素生成[J];國外醫(yī)學(xué)(皮膚性病學(xué)分冊);1998年02期

2 曾昭智;劉運忠;任麗華;趙樂;龔寶勇;朱少璇;顧為望;;西藏小型豬在廣州地區(qū)生長繁殖性能的研究[J];豬業(yè)科學(xué);2006年08期

，

本文編號：2102627