含FLP“交换盒”结构的打靶载体构建及ES细胞打靶研究

利用小鼠HPRT基因组DNA片段和人工合成的含有FLP重组酶识别位点变异体FRT和F3RT序列的寡核苷酸 ,构建了针对小鼠HPRT基因位点的置换型打靶载体pSP HPRT Fneo F₃。经过限制酶酶切及部分测序鉴定其结构正确后 ,将线性化了的打靶载体以电穿孔法导入ES细胞内 ,经G418和 6 -TG双药筛选和分子鉴定 ,得到了 2个在HPRT位点整合有FLP重组酶“交换盒”F Neo F3结构的双交换重组ES细胞克隆 ,为建立基于FLP重组酶介导的盒式交换的高效、定点转基因体系创造了条件.     

重组酶介导的盒式交换及其应用

重组酶介导的盒式交换 (ｒｅｃｏｍｂｉｎａｓｅｍｅｄｉａｔｅｄｃａｓｓｅｔｔｅｅｘｃｈａｎｇｅ ,ＲＭＣＥ)是近年发展起来的一种基因定点整合新方法。与同类方法相比 ,它具有定点、高效、简单而又可对基因组进行反复修饰等特点 ,因而在研究基因功能、分析顺式作用成分以及构建各种转基因动物、建立人类疾病动物模型等方面具有很大的应用价值。本文拟就ＲＭＣＥ的原理、策略、特点及应用等方面作一综述。1 .ＲＭＣＥ的原理1 .1　重组酶及其介导的位点特异性重组　　重组酶又称位点特异性重组酶 ,是一类源于微生物[1、2 ] 能识别特异的Ｄ…     

ΦC31整合酶介导猪基因组定点修饰的探讨

高效与特异的基因组定点修饰是基因工程动物研究的前沿与难点.链霉菌噬菌体ΦC31整合酶能介导含attB位点的外源基因定点整合于多种真核生物基因组的假attP位点,可维持外源基因的正常结构及高效表达.本文探讨ΦC31整合酶介导外源基因在猪基因组内定点整合的分子基础.构建含attB位点的报告载体pEGFP-N1-attB,与ΦC31整合酶表达载体pCMV-INT共转染猪肾PK15细胞,G418筛选获得单克隆细胞系.实时荧光定量PCR筛选出单拷贝整合的转基因细胞系.TAIL-PCR鉴定出1个猪基因组假attP位点,位于猪1号染色体,watson链,坐标114220087-114220126,命名为pig-attP-1.测序结果显示,pEGFP-N1-attB在attB位点处断开插入到pig-attP-1.用荧光计测定细胞培养基上清EGFP含量发现,该转基因细胞系EGFP的表达水平是本底的50倍(13500 AU vs.280 AU),表明pig-attP-1是利于外源基因高效表达的"友好位点".该研究不仅为实现外源基因在猪基因组内的定点整合提供了新策略,也为创制基因工程猪、建立动物生物反应器等研究注入了新思路.     

利用Red重组系统对大肠杆菌ClpP基因的敲除

利用含有质粒pKD4 6的菌株BW2 5 113,在阿拉伯糖诱导后 ,表达λ噬菌体的 3个重组蛋白 ,宿主菌就具有了同源重组的能力 .设计的引物 5′端有 5 0bp的拟敲除基因的同源臂 ,3′端为扩增引物 ,以pKD3为模板 ,扩增两侧含FRT位点的氯霉素抗性基因 ,将此线性片段电转入具重组功能的感受态细胞 ,利用氯霉素平板就可以筛选到阳性转化体 .再利用表达Flp重组酶的质粒pCP2 0 ,可将FRT位点之间的氯霉素抗性基因删除 .利用该重组系统 ,构建了ClpP蛋白酶缺失的大肠杆菌工程菌株 ,可望在减少外源蛋白的降解方面发挥一定的作用 .     

软骨组织特异性表达Cre重组酶转基因小鼠的研制和鉴定

构建了含有软骨组织特异性Ⅱ型胶原A1启动子和Cre重组酶基因的转基因载体pcol2Al-Cre。323枚小鼠受精卵经显微注射引入转基因片段后,分别移植至14只假孕母鼠的输卵管使其发育。共得到仔鼠52只,PCR结果显示其中10只小鼠基因组上有Cre基因的整合,整合率为19．2％。用整合有Cre基因的转基因小鼠与基因组上携带LoxP位点的条件基因打靶小鼠交配,以检测Cre酶介导的重组及其组织特异性。PCR结果表明：col2Al-Cre转基因小鼠软骨组织中表达的Cre重组酶成功地介导了LoxP之间的重组。此结果通过Southern杂交得到了进一步的证实。     

利用热诱导的位点专一性重组系统在烟草中控制基因表达

采用热激启动子Gmhsp17．5C控制Cre定位重组酶介导的DNA删除系统。在这个系统中,在热激启动子控制下的Cre重组酶的表达导致两侧带有相同方向loxp位点的CaMV35S—GUS片段从转基因烟草(Nicotiana tabacum L.cv.W38)的基因组中删除。通过定量PCR的方法鉴定这个转基因系统,显示了这个系统的重组效率。结果显示在两个小时热激处理后转基因烟草中有41％的CaMV35S—Gus片段被删除。由于热激诱导的定点重组系统有容易操作、对热敏感和无背景表达等优点,因此有利于采用这个系统在转基因植物中进行可诱导的基因操作。     

卵母细胞特异表达φC31整合酶载体pZP3-INT的构建及其在小鼠卵母细胞中的表达

链霉菌噬菌体φC31整合酶是一种位点特异性重组酶(Site-specific recombinase,SSR),可介导链霉菌噬菌体attP位点(Phage attachment site)和链霉菌基因组attB位点(Bacterial attachment site)间的单向重组.为探讨它能否应用于卵母细胞特定基因的重组,文章采用卵巢针刺取卵法采集生发泡(GV)期小鼠卵母细胞,将卵透明带糖蛋白3(ZP3)启动子驱动φC31整合酶表达载体pZP3-INT和检测φC31整合酶位点特异性重组功能的重组质粒载体pBCPB+,通过显微注射导入到小鼠卵母细胞中.培养48 h后,RT-PCR检测φC31整合酶mRNA表达以及PCR检测pBCPB+载体发生重组的情况.结果表明:载体pzP3-INT在卵母细胞中表达φC31整合酶mRNA;并且pBCPB+载体发生了位点特异性重组,提示φC31整合酶在卵母细胞中可以介导位点特异性重组反应.     

利用热诱导的位点专一性重组系统在烟草中控制基因表达

采用热激启动子Gmhsp17.5C控制Cre定位重组酶介导的DNA删除系统.在这个系统中,在热激启动子控制下的Cre重组酶的表达导致两侧带有相同方向loxp位点的CaMV35S-GUS片段从转基因烟草(Nicotiana tabacum L.cv.W38)的基因组中删除.通过定量PCR的方法鉴定这个转基因系统,显示了这个系统的重组效率.结果显示在两个小时热激处理后转基因烟草中有41%的CaMV35S-GUS片段被删除.由于热激诱导的定点重组系统有容易操作、对热敏感和无背景表达等优点,因此有利于采用这个系统在转基因植物中进行可诱导的基因操作.     

重组酶介导的定点整合及在构建重组CHO细胞中的应用

中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)是生产治疗性重组蛋白最常用的细胞。随机整合(random integration,RI)工艺是目前构建重组CHO细胞株的主要策略,由于CHO细胞基因组缺乏稳定性,为得到产量高、品质好且适应特定工艺的细胞株,通常需要1~2轮高通量筛选,不仅工作量大、耗时长且批次稳定性差。定点整合(site-specific integration,SSI)基因编辑技术将外源基因整合至细胞基因组的特定位点,经一轮筛选得到稳定、高产且适应特定生产工艺的细胞株,从而缩短细胞株构建周期。近年来,不断有将定点整合策略应用于构建重组CHO细胞株的报道。基因编辑技术的发展是实现细胞外源基因定点整合工艺的基础,常用的基因编辑技术包括核酸酶技术、转座子技术和重组酶技术。比较这三种基因编辑技术在构建流程、整合效率和专利等方面的不同特点,重点讨论重组酶介导的定点整合及其在CHO细胞株构建中的应用。     

应用接头PCR的方法扩增假attP位点

φC31整合酶可高效介导外源基因特异、稳定地与哺乳动物基因组发生重组反应.基因组中被整合的住点为假attP位点.运用接头PCR的方法对φC31整合酶介导的含有attB序列及表达绿色荧光蛋白(GFP)的载体在牛基因组中的一个新的特异整合位点(假attP位点)进行扩增.并且显示接头PCR技术在克隆与已知序列相邻的未知旁侧序列上其具有高效、特异、灵敏等特点.     

基于Flp重组酶介导的盒式交换HPRT位点定点转基因研究

Exchange plasmid pF-HPRT-F3 and Flp expression plasmid pCMV-Flp were constructed and then introduced using electroporation system into F18 ES cell line, which have an exchange cassette F-Neo-F3 at HPRT locus. After HAT selection, HAT resistant clones were obtained. Then G418 sensitivity test and Southern blotting were carried out to screen RMCE recombinants. The results indicated that RMCE had taken place in three of 12 HAT resistant clones. The frequency is 25%. The result demonstrates that it is realizable to introduce transgene to HPRT locus by using Flp recombinase mediated cassette exchange reaction.     

Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange

Elk-1 is a member of the TCF subfamily of Ets proteins. TCFs interact with SRF at serum response elements (SREs) of immediate early genes (IEGs), such as c-fos and Egr-1, thereby mediating IEG induction upon extracellular stimulation. We previously generated an Elk-1 null allele (Elk1-137) in murine embryonic stem (ES) cells by homologous recombination. In Elk1-137, the Elk-1 gene was replaced by a Hygromycin B phosphotransferase - Thymidine Kinase (HygTk) fusion gene, flanked by two nonidentical Flp recombinase recognition (FRT) sites (Cesari et al., [2004] Mol Cell Biol, in press) to allow for the subsequent generation of alternative alleles of interest by recombinase-mediated cassette exchange (RMCE). Elk1-deficient mice derived from Elk-1((137/0)) ES cells are viable and do not reveal strong phenotypical abnormalities, apart from male sterility. However, the Elk-1 locus contains the Tk cassette, which has previously been related to this defect. Therefore, in our first experiment involving the technique of Flp RMCE we chose to remove the HygTk cassette in Elk-1((137/0)) ES cells and to generate Elk-1((RMCE16/0)) and Elk-1((RMCE16/RMCE16)) mice. In so doing, we provide evidence that the sterility of Elk1((137/0)) mice was not due to the absence of Elk-1 but rather the presence of HygTk. This is the first report of mice derived from ES cells which were subjected to Flp RMCE and thus proves that RMCE is a powerful tool for the genetic engineering of previously tagged loci in the mouse genome.     

Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges

Traditional DNA transduction routes used for the modification of cellular genomes are subject to unpredictable alterations, as the cell-intrinsic repair machinery may affect both the integrity of the transgene and the recipient locus. These problems are overcome by recombinase-mediated cassette exchange (RMCE) approaches enabling predictable expression patterns by the nondisruptive insertion of a gene cassette at a pre-characterized genomic locus. The destination is marked by a “tag” consisting of two heterospecific recombination target sites (RTs) at the flanks of a selection marker. Provided on a circular donor vector, an analogous cassette encoding the gene of interest can cleanly replace the resident cassette under the influence of a site-specific recombinase. RMCE was first based on the yeast integrase Flp but had to give way to the originally more active phage-derived Cre enzyme. To be effective, both Tyr-recombinases have to be applied at a considerable concentration, which, in the case of Cre, triggers endonucleolytic activities and therefore cellular toxicity. This review addresses the particularities of both recombination routes depending on the structure of the synaptic complex and on improved integrase and RT variants. While the performance of Flp-RMCE can now firmly rely on optimized Flp variants and multiple sets of functional target sites (FRTs), the Cre system suffers from the promiscuity of its RT mutants, which is explained in molecular terms. At present, RMCE enters applications in the stem cell field. Remarkable efforts are noted in the framework of various mouse mutagenesis programs, which, in their first phase, have targeted virtually all genes and now start to shift their emphasis from gene trapping to gene modification.     

Efficient DNA cassette exchange in mouse embryonic stem cells by staggered positive-negative selection

Recombinase-mediated cassette exchange (RMCE), when applied to mouse embryonic stem (ES) cells, promises to increase the ease with which genetic alterations can be introduced into targeted genomic loci in the mouse. However, existing selection strategies for identifying ES cells in which replacement DNA cassettes from a carrier plasmid have been exchanged correctly into a defined locus are suboptimal. Here, we report the generation in mouse ES cells of a loxed cassette acceptor (LCA) allele within the glucokinase (gk) gene locus. Using the gkLCA as a test allele, we developed a staggered positive-negative selection strategy that facilitates efficient identification of ES cell clones in which a DNA replacement cassette from a carrier plasmid has been exchanged correctly into the gkLCA allele. This selection strategy, by facilitating more efficient production of ES cell clones with various replacement DNA cassettes, should accelerate targeted repetitive introduction of gene modifications into the mouse.     

Sequential genetic modification of the hprt locus in human ESCs combining gene targeting and recombinase-mediated cassette exchange

Genetic modification of human embryonic stem cells (hESCs) will be an essential tool to allow full exploitation of these cells in regenerative medicine and in the study of hESC biology. Here we report multiple sequential modifications of an endogenous gene (hprt) in hESCs. A selectable marker flanked by heterospecific lox sites was first introduced by homologous recombination (HR) into the hprt gene. In a subsequent step, exchange of the selectable marker with another cassette was achieved by recombinase-mediated cassette exchange (RMCE). We show that 100% of the recovered clones were the result of RMCE using a promoter trap strategy at the hprt locus. hprt-targeted H1 cells maintained a diploid karyotype and expressed hESC surface markers before and after RMCE. Finally, we report a double replacement strategy using two sequential gene targeting steps resulting in the targeted correction of an hprt-mutated hESC line.     

High efficiency of a sequential recombinase-mediated cassette exchange reaction in Escherichia coli

A comparison between the efficiency of recombinase-mediated cassette exchange (RMCE) reactions catalyzed in Escherichia coli by the site-specific recombinases Flp of yeast and Int of coliphage HK022 has revealed that an Flp-catalyzed RMCE reaction is more efficient than an Int-HK022 catalyzed reaction. In contrast, an RMCE reaction with 1 pair of frt sites and 1 pair of att sites catalyzed in the presence of both recombinases is very inefficient. However, the same reaction catalyzed by each recombinase individually supplied in a sequential order is very efficient, regardless of the order. Atomic force microscopy images of Flp with its DNA substrates show that only 1 pair of recombination sites forms a synaptic complex with the recombinase. The results suggest that the RMCE reaction is sequential.     

Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology

The availability of site-specific recombinases has revolutionized the rational construction of cell lines with predictable properties. Early efforts were directed to providing pre-characterized genomic loci with a single recombinase target site that served as an address for the integration of vectors carrying a compatible tag. Efficient procedures of this type had to await recombinases like ΦC31, which recombine attP and attB target sites in a one-way reaction – at least in the cellular environment of the higher eukaryotic cell. Still these procedures lead to the co-introduction of prokaryotic vector sequences that are known to cause epigenetic silencing. This review illuminates the actual status of the more advanced recombinase-mediated cassette exchange (RMCE) techniques that have been developed for the major members of site-specific recombinases (SR), Flp, Cre and ΦC31. In RMCE the genomic address consists of a set of heterospecific recombinase target (RT-) sites permitting the exchange of the intervening sequence for the gene of interest (GOI), as part of a similar cassette. This process locks the GOI in place and it is ‘clean’ in the sense that it does not co-introduce prokaryotic vector parts nor does it leave behind a selection marker.     

Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe

Cre/lox site-specific recombination systems provide important tools for genetic manipulation. Here we present an efficient method for gene tagging and gene replacement using Cre recombinase-mediated cassette exchange (RMCE). The cassette consists of the S. pombe ura4(+) selectable marker flanked by a wild-type loxP site at one end and by a modified heterospecific lox site (loxM3) at the other. The cassette is stable because the flanking lox sites cannot recombine with each other. Following integration of the cassette at the chosen chromosomal locus, exchange is achieved by introducing a Cre-expression plasmid containing an equivalent cassette containing the required tag or gene sequence. Recombinants are selected by uracil prototrophy using the reagent 5-fluoroorotic acid (5-FOA). The cassette exchange system provides for repetitive integrations at the same locus, allowing different protein tags or gene sequences to be integrated quickly and efficiently. We have established a range of reagents and verified utility by C-terminally tagging the S. pombe rad4 and swi1 genes with yEGFP and the yEGFP derivatives yECFP and yECitrine and by transferring the coding sequence for both genes.     

Cre recombinase‐mediated site‐specific modification of a cellular genome using an integrase‐defective retroviral vector

Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase‐mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild‐type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase‐defective retroviral vectors (IDRV), which contains an ATG‐deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418‐resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site‐specific recombination. The successful substitution of the original viral integration machinery with a non‐viral mechanism could expand the application of retroviral vectors. Biotechnol. Bioeng. 2010;107:717–729. © 2010 Wiley Periodicals, Inc.