基因打靶技术的原理及操作

基因打靶是近几年发展起来的一种通过同源重组定点改变小鼠基因组特定位点的技术,其诞生是分子生物学与实验胚胎学方法相结合的产物,它的出现又导致了体内研究与体外研究、分子生物学与临床病理学的有机结合,为研究基因的体内功能和疾病的致病机理提供了一种有力的实验手段。本文以基因打靶的实验过程为主线,介绍该技术的原理、操作、进展和应用。     

在小鼠胚胎干细胞进行基因打靶的策略

基因打靶技术是一种通过同源重组按预期方式改变生物活体的遗传信息的实验手段,与小鼠胚胎干细胞培养系统相结合,使得人们可以方便地将各种突变引入小鼠体内,得以从生物整体水平上研究高等真核生物基因的表达、调控及其生理功能.扼要介绍了近年来在小鼠胚胎干细胞进行基因打靶的研究进展.     

基因打靶突变小鼠的早期T细胞发育

早期Ｔ细胞的发育是一个受到多种分子精确调控的过程,基因打靶技术的建立和发展 内研究上述分子的作用提供了有效的手段。对ＴＣＲ、ＣＤ３基因打靶小鼠的研究表明,ＣＤ４４－ＣＤ２５阶段是早期Ｔ细胞发育的重要调控点,在此发育阶段,由ＴＣＲβ、ＴＣＲα和ＣＤ３成分组成的ｐｒｅ－ＴＣＲ复俣体的表达或其与未知配体的结合通过ｐ５６ｌｃｋ传递信号,介导ＣＤ４４－ＣＤ２５细胞的进一上发育,该复全体任何成分的缺失都将使Ｔ     

基因打靶及其应用

用活细胞染色体DNA可与外源性DNA同源序列发生同源重组的性质,达到定点修饰改造染色体某基因的目的,此法称基因打靶．基因的同源重组是较普遍的生物现象,其分子机理尚未阐明,但活细胞内确有一酶系可使DNA的同源序列在细胞内发生重组,这一事实已无可争辨．此事实为基因打靶的理论基础．基因打靶技术操作的关键是建立一含筛选基因的重组载体,并有效地把它转入细胞核内．基因打靶命中的细胞可稳定遗传．基因打靶在改造生物品种,一些复杂生命现象（如发育的分子机制等）及临床理论研究均有广阔的前景．     

微生物基因打靶系统的研究进展

基因打靶技术是微生物功能基因组学研究的有力工具之一,通过定向改变微生物的遗传信息可以对目的基因进行有效的功能分析。在大肠杆菌中研究较多的是转座子突变系统、RecBCD^-sbcB重组系统、RecA依赖的重组打靶系统、Chi位点刺激的重组、利用单链DNA进行的重组工程。在酵母中进行基因打靶的策略主要是转座子标记的突变、基于PCR方法的基因删除和转化相关重组。在其他微生物中主要应用转座子突变和自杀载体进行基因打靶。近年来,噬菌体重组系统的发展更使对微生物基因打靶系统的研究进入了新的阶段,主要包括Rac编码的RecET系统、Red重组系统和噬菌体退火蛋白介导的单链寡核苷酸重组系统。     

基因打靶技术的研究进展

基因打靶技术是20世纪80年代发展起来的新技术,是一种利用DNA同源重组原理和胚胎干细胞(ES细胞)技术按定向组合的方式改变生物活体遗传信息的试验手段,具有广阔的应用前景。对基因打靶技术原理、步骤、条件性基因打靶以及应用进行综述。     

基因打靶技术的研究进展

基因打靶在人类遗传病动物模型的构建,基因治疗和基因功能研究等方面都有重要的作用。本文综述了一些常用的基因打靶策略,并对这些技术的研究进展作一介绍。     

植物基因打靶技术

基因打靶是反向遗传学的基础工具,它通过同源重组置换染色体内的基因用于复杂基因组的基因功能分析。但是,在植物中,外源DNA的插入主要是非序列依赖的非同源末端连接方式,基因打靶频率很低,只有10-5～10-4的水平。综述了近年来为了提高植物基因打靶频率,研究人员的工作和最新进展 。     

基于Cre/LoxP系统的Smad2条件基因打靶小鼠的建立

Smads家族是转化生长因子TGF-b信号转导途径中一个重要的新基因家族.SMAD2属于受体激活的SMADs.Smad2基因在多种肿瘤中发生突变,是一种可能的肿瘤抑制基因. Smad2基因完全剔除导致小鼠在胚胎期6.5d死亡.为了研究Smad2在脊椎动物成体各组织器官及肿瘤发生中的可能作用,利用Cre/LoxP系统构建了Smad2条件基因打靶载体,将两个方向相同的LoxP序列置于编码SMAD2蛋白C末端功能域序列两侧的内含子中,并在组成型表达Cre重组酶的大肠杆菌中检测了LoxP位点的功能;用打靶载体电击转染小鼠胚胎干细胞,经Southern杂交筛选到3株发生正确同源重组的中靶ES细胞克隆;通过囊胚显微注射将中靶ES细胞导入受体小鼠囊胚腔,获得了ES细胞种系嵌合体;对高嵌合度小鼠与C57BL/6J的子代小鼠进行基因型鉴定,成功地获得了Smad2条件基因打靶小鼠.     

Disruption of the adenosine deaminase (ADA) gene using a dicistronic promoterless construct: Production of an ADA-deficient homozygote ES cell line

In man, deficiency of ADA activity is associated with an autosomal recessive form of severe combined immunodeficiency (SCID), a disease with profound defects of both cellular and humoral immunity. Current treatments of ADA deficient patients include bone marrow transplantation, enzyme replancement and somatic gene therapy. The mechanism of the selective immune cell pathogenesis in ADA-SCIDS is, however, still poorly understood. Thus, the generation of an ADA deficient mouse model will be of considerable benefit to understand better the pathophysiology of the disorder and to improve the gene therapy treatments.We have disrupted the adenosime deaminase (ADA) gene in embryonic stem cells using a new efficient promoter trap gene-targeting approach. To this end, a dicistronic targeting construct containing a promoterless IRES geo cassette was used. This cassette allows, via the internal ribosomal entry site (IRES), the direct cap-independent translation of the geo reporter gene which encodes a protein with both -galactosidase and neomycin activities. After indentification of targeted clones by Southern blot, successful inactivation of the ADA gene was first confirmed by producing, from our heterozygote clones, an homozygote cell line. This line shows no ADA activity as judged by zymogram analysis. Second, we have been able to detect in the targeted clones, a specific galactosidase activity using a sensitive fluorogenic assay. The targeted ES cell clones are currently being injected into blastocysts to create an ADA deficient mouse model.     

利用细菌人工染色体同源重组对小鼠基因进行条件型敲除

目的:探索通过细菌人工染色体(BAC)同源重组系统构建条件基因敲除载体的高效率方法,提高条件基因敲除小鼠(Flox小鼠)的构建效率。方法:利用作者自己构建的噬菌体重组酶系统,通过BAC同源重组进行条件型基因敲除载体构建工作。首先通过亚克隆构建了一系列载体含有同源臂的靶向质粒,线性化后,打靶片段经电穿孔法转入大肠杆菌内,与相应的BAC同源重组,再经过三步同源重组和一步位点特异性重组,构建小鼠条件型基因敲除载体。结果:高效率构建了小鼠基因的最终条件基因敲除载体。结论:通过BAC同源重组高效构建条件基因敲除载体,为条件基因敲除载体的构建提供了全新思路,并为FLox小鼠的建立,及相应基因在发育、生理、致病机制等方面的功能研究奠定了基础。     

基因打靶技术的研究进展

基因打靶技术是一项新兴的分子生物学技术,是利用外源DNA与受体细胞染色体DNA上的同源序列之间发生重组,并整合在预定位点上,从而改变细胞遗传特性的方法。它的产生是遗传工程领域的一次革命,为发育生物学、分子遗传学、免疫学及医学等学科提供了一个全新的、强有力的研究手段。目前基因打靶技术在研究基因的结构和功能、表达与调控,转基因及基因治疗等方面均取得了进展。但基因打靶技术仍存在一些问题,主要是打靶的效率太低。本文综述了基因打靶技术的原理、操作程序并对提高基因打靶效率的可能途径进行了探讨。 Progress on Gene Targeting LIU Hong-quan1,DAI Ji-xun1,YU Wen-gong2,YANG Kun-feng1 1.Ocean University of Qingdao,College of Marine Life Sciences,Qingdao 266003,China; 2.Institute of Marine Drugs and Foods,Qingdao 266003,China Abstract:Gene targeting is a rising technology in molecular biology,which is defined as the introduction of exogeneous DNA to specific site in genome by homologous recombination,and consequently change the hereditary character of the cell.This technology provides a new and powerful means for research in developmental biology,molecular genetics,immunology and medicine.Progresses have been made in exploring gene structure and function,gene expression and regulation,transgene and gene therapy with the application of gene targeting.But there are some problems in gene targeting,especially for the low efficiency.This article just provided a review of the principle and program of gene targeting,and discussed the possible approaches to increase the efficiency of gene targeting. Key words：gene targeting;homologous recombination;targeting vector;targeting efficiency     

Targeted gene modification in mouse ES cells using integrase‐defective lentiviral vectors

Lentiviral vectors efficiently integrate into the host genome of both dividing and nondividing cells, and so they have been used for stable transgene expression in biological and biomedical studies. However, recent studies have highlighted the risk of insertional mutagenesis and subsequent oncogenesis. Here, we used an integrase‐defective lentiviral (IDLV) vector to decrease the chance of random integration and examined the feasibility of lentiviral vector‐mediated gene targeting into murine embryonic stem (ES) cells. After transduction with wild‐type lentiviral vectors, none of the 512 G418 resistant clones were found to be homologous recombinant clones. Although the transduction efficiency was lower with the IDLV vectors (5.9% of wild‐type), successful homologous recombination was observed in nine out of the 941 G418 resistant clones (0.83 ± 1.32%). Pluripotency of the homologous recombinant ES cells was confirmed by the production of chimeric mice and subsequent germ line transmission. Because lentiviral vectors can efficiently transduce a variety of stem cell types, our strategy has potential relevance for secure gene‐manipulation in therapeutic applications. genesis 47:217–223, 2009. © 2009 Wiley‐Liss, Inc.     

2N一4N嵌合体小鼠及其在基因功能研究上的应用

2N-4N嵌合体是由二倍体(2N)的胚胎细胞(或ES细胞)与四倍体(4N)的胚胎细胞组构成的一种拯救型嵌合体。这种嵌合体由于4N胚胎细胞独特的胚外组织发育特性,从而可获得完全源自2N细胞成份的仔鼠(或胎儿)。这一特性最终在ES细胞转基因的基因功能研究中极具重要价值。综述了2N-4N嵌合体研究进展及其在基因功能研究领域的应用。     

小鼠胚胎干细胞hprt基因的定位致变

利用DNA的同源重组原理,通过基因打靶技术,在小鼠胚胎干细胞(ES细胞）中将pMC1-neo导人hprt座位,实现了基因组内指定基因的定位致变．通过电穿孔导人质粒pRV4.0线性化D^A,分别用G418与6-TG筛选HPRT^-突变子．经抗性检验及DNA印迹分析,证明得到了一株预期的定位转化细胞,转化效率为1.32 x10^-8载体的非同源序列对定位致变的效率和整合方式没有影响,由于采取了有效的措施,所获HPRT^- ES细胞株仍维持了未分化和二倍体状态,保留了胚胎干细胞的特性．     

Gene targeting in Arabidopsis

Precise modification by gene targeting (GT) provides an important tool for studies of gene function in vivo. Although routine with many organisms, only isolated examples of GT events have been reported for flowering plants. These were at low frequencies precluding reliable estimation of targeting efficiency and evaluation of GT mechanisms. Here we present an unambiguous and straightforward system for detection of GT events in Arabidopsis using an endogenous nuclear gene encoding protoporphyrinogen oxidase (PPO), involved in chlorophyll and heme syntheses. Inhibition of PPO by the herbicide Butafenacil results in rapid plant death. However, the combination of two particular mutations renders PPO highly resistant to Butafenacil. We exploited this feature for selection of GT events by introducing the mutations into the PPO gene by homologous recombination. We have estimated the basal GT frequency to be 2.4 x 10(-3). Approximately one-third of events were true GT (TGT) leading to the anticipated modification of the chromosomal PPO copy. The remaining events could be classified as ectopic GT (EGT) arising by modification of vector DNA by the chromosomal template and its random integration into the Arabidopsis genome. Thus the TGT frequency in our experimental setup is 0.72 x 10(-3). In view of the high efficiency of Arabidopsis transformation, GT experiments of a reasonable size followed by a PCR screen for GT events should also allow for modification of non-selectable targets. Moreover, the system presented here should contribute significantly to future improvement of GT technology in plants.