多位点基因打靶技术的鳜鱼体内实验研究

在转基因动物的研究中,一般采用随机插入的方法。基因在受体动物的基因组中随机整合,严重制约了转基因动物外源基因的稳定遗传,是转基因动物研究亟待解决的问题。转基因定点整合技术,即基因打靶技术是解决上述问题的重要途径,但是基因打靶存在打靶效率太低的问题。本研究小组已建立了体外动物细胞的多位点基因打靶技术,并构建了用于鳜鱼的体内多位点基因打靶的打靶载体,本文开展多位点基因打靶技术的鳜鱼体内实验研究。以鳜鱼rDNA基因(编码rRNA的基因)及其间隔序列作为同源重组引导序列,构建含干扰素基因的打靶载体,以其间隔序列为靶位点,针对鱼类胚胎发育特点,采用精子介导技术进行多位点基因打靶,最终获得定点整合干扰素基因的鳜鱼,并建立一套适用于动物的基因定点敲入的体内基因打靶技术。主要研究内容如下:(1)采用组织培养方法,获得鳜鱼头肾淋巴细胞和脾淋巴细胞,采用脂质体介导法使多位点基因打靶载体在鳜鱼的细胞上实行基因转移。采用正负筛选方法,利用筛选药物G418和GCV(更昔洛韦)筛选转染后的细胞两周后,对存活细胞进行DNA水平和RNA水平鉴定。结果证明了干扰素基因在鳜鱼细胞上实现了定点整合和稳定表达。(2)利用精子介导法将经线性化处理后的多位点基因打靶载体在鳜鱼的胚胎上实行基因转移。(3)根据靶位点定点整合的正负筛选原理,利用筛选药物G418和GCV筛选与富集定点整合后的阳性鱼胚或鱼苗,结果成功孵化出106尾转基因鳜鱼鱼苗。(4)培育6个月后,取20尾转基因鳜鱼进行鉴定。采用PCR、RT-PCR、测序等方法进行定点整合的鉴定。结果表明:6尾检测到已转入的基因——干扰素基因,其中有3尾实现了定点整合并表达。本研究率先将多位点基因打靶技术应用到转基因鱼的研究中,建立一套适用于鱼类的高效、安全的多位点基因打靶技术,并获得外源基因定点整合并表达的鳜鱼。

Research of multiple locus gene targeting in vivo of Siniperca

The transgenic technique of random integration was used generally in the study of transgenic animals.The random integration in receptor genome has restricted heredity stabilization of target gene in transgenic animals.It's the key problem which should be solved in the study of transgenic animals.The techniques of the site-directed gene integration or the gene targeting techniques are the effective ways to solve the above mentioned problems.But,there were extremely low efficiency of gene integration.The authors have established the techniques of multiple locus gene targeting and have constructed the vector of multiple locus gene targeting for Siniperca.This study will complete the research of multiple locus genes targeting in vivo.Using the DNA sequences of rDNA repeat gene and its internal transcribed spacer of Siniperca as homologous recombination directed sequences(HRDS),the vector of gene targeting which contains interferon gene has been constructed.The internal transcribed spacer was used as target locus.The sperm mediate method was used for gene transfer.The Sinipercaes integrated interferon gene in desired loci were gotten.Finally,the method of gene targeting in desired loci in animal was established.The principal researches were as follow:(1) The lymphocyte of spleen and the head kidney of Siniperca were obtained by the tissue cultural method.The liner vectors of multiple locus gene targeting were transferred into the Siniperca cells by lipofectin method.After selected by G418 and GCV(ganciclovir) for more than 2 weeks the survival cells were identified in the level of DNA and RNA.Therefore,it was confirmed that the interferon gene can be site-directed integration and expression.(2) The liner vectors of multiple locus gene targeting were transferred into the Siniperca embryoes by the sperm mediate method.(3) According to the principle of positive-negative selection,the positive embryoes were selected by G418 and GCV.106 transgenic Sinipercaes were obtained.(4) After cultured for more than 6 months,20 transgenic Sinipercaes were identified by the PCR,RT-PCR and sequencing.It was confirmed that there were 6 fishes were detected to have the interferon gene.3 fishes were site-directed gene integration.The research firstly applied the techniques of multiple locus gene targeting to study the transgenic fish and established a set of gene targeting technique with high efficiency and safe for fish.Finally the fish integrating gene in desired loci and expressing the interferon gene were obtained.