一种基于线性DNA片段同源重组的嗜盐古菌高效基因敲除系统

随着功能基因组学研究的深入发展,基因敲除技术日益成为基因功能研究的重要手段。嗜盐古菌Haloferax volcanii易于培养,是研究古菌基因功能的良好模式菌株。虽然现已开发了多种嗜盐古菌的遗传操作系统,但基因敲除成功率不十分理想。这些遗传操作方法基于pyrE筛选标记,利用携带同源片段的环状质粒与基因组同源片段间的两次同源重组,敲除目的基因。由于基于环状质粒和pyrE筛选标记的经典同源重组敲除方法在二次重组时,普遍存在回复到野生型菌株的可能,导致二次重组子中敲除目的基因的阳性菌株比例较低。为了克服传统同源重组技术的上述缺陷,文章建立了基于线性DNA片段的同源重组技术。该方法通过一次重组在目标基因的下游引入一段上游同源片段和pyrE标记,从而限定二次重组的发生部位只能在两段上游同源片段之间,发生二次重组的重组子理论上都敲除了目标基因。利用该方法,文章成功敲除了嗜盐古菌Haloferax volcanii的xpd2基因,阳性克隆率达65%。这种线性DNA片段重组法为嗜盐古菌的基因敲除提供了一种高效策略,便于嗜盐古菌的基因改造。     

A targeted gene knockout method using a newly constructed temperature-sensitive plasmid mediated homologous recombination in Bifidobacterium longum

Bifidobacteria are the main component of the human microflora. We constructed a temperature-sensitive (Ts) plasmid by random mutagenesis of the Bifidobacterium-Escherichia coli shuttle vector pKKT427 using error-prone PCR. Mutant plasmids were introduced into Bifidobacterium longum 105-A and, after screening approximately 3,000 colonies, candidate clones that grew at 30?°C but not at 42?°C were selected. According to DNA sequence analysis of the Ts plasmid, five silent and one missense mutations were found in the repB region. The site-directed mutagenesis showed only the missense mutation to be relevant to the Ts phenotype. We designated this plasmid pKO403. The Ts phenotype was also observed in B. longum NCC2705 and Bifidobacterium adolescentis ATCC15703. Single-crossover homologous-recombination experiments were carried out to determine the relationship between the length of homologous sequences encoded on the plasmid and recombination frequency: fragments greater than 1?kb gave an efficiency of more than 10(3) integrations per cell. We performed gene knockout experiments using this Ts plasmid. We obtained gene knockout mutants of the pyrE region of B. longum 105-A, and determined that double-crossover homologous recombination occurred at an efficiency of 1.8?%. This knockout method also worked for the BL0033 gene in B. longum NCC2705.     

Non-homologous end joining pathway of the human pathogen Cryptococcus neoformans influences homologous integration efficiency but not virulence

The efficiency of gene targeting by integration through homologous recombination (homologous integration, HI) in the human pathogen Cryptococcus neoformans remains unsatisfactory. In order to achieve a much more efficient gene targeting system in C. neoformans, a new double knockout strain in genes involved in the non-homologous end joining (NHEJ) pathway was constructed. HI frequency was elevated by as much as approximately fivefold in the single or double knockout strains in NHEJ genes, and the frequency depended on the gene targeted. None of the NHEJ gene knockouts showed significant differences in regular growth, sensitivity to DNA-damaging drugs or UV, and virulence compared to the wild-type control, suggesting that the NHEJ pathway does not play a significant role in these biological stresses in C. neoformans. It was also suggested that the genes analyzed in this study are components of a single NHEJ pathway, as the mutants (including the double mutant) displayed the same phenotypes.     

一种两步基因同源重组敲除酵母靶标蛋白基因的方法

目的:建立一种在代谢工程改造的毕赤酵母菌中高效敲除靶标蛋白基因的方法。方法:构建敲除载体,以粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因为靶基因,以尿嘧啶合成关键酶URA3基因为营养缺陷型筛选标记,第一步重组利用ura3正筛选获得敲除载体在酵母染色体中的定点整合,第二步通过5-氟乳清酸(5-FOA)筛选到表型为ura3-的克隆,ura3基因被剔除的同时,目的基因GM-CSF也随之丢失,实现第二次重组;利用基因组PCR和蛋白电泳进行鉴定。结果:通过两步基因同源重组,敲除了毕赤酵母GJK01的报告蛋白GM-CSF的编码基因388 bp,PCR结果显示该基因已完全丢失,SDS-PAGE分析无GM-CSF表达。结论:仅通过2周时间的筛选、鉴定,在毕赤酵母GJK01中敲除了报告蛋白GM-CSF的编码基因,突变菌株的阳性率达到50%,最终建立了以URA3为筛选标记的两步基因同源重组敲除目的基因的方法。     

Intragenic recombination at the white locus of Drosophila hydei.

A fine-structure analysis of the white locus in Drosophila hydei was carried out by means of allele recombination. Four mutants, derived from wild type, mapped at three subloci. These are possibly homologous to the main subloci 2, 3, and 4 of D. melanogaster. Three secondary mutants, derived from the primary wiv allele, were located in the proximal part of the gene. One of them appeared as a homoallele of the original wiv, whereas the remaining two are better explained either as double mutants or as mutants which facilitate irregular exchange. Intragenic recombination at the white locus seems to be more frequent in D. hydei than in D. melanogaster. The comparatively high incidence is probably a general characteristic, common to intragenic and intergenic recombination in D. hydei.     

基因敲除技术在微生物中的应用

随着分子生物学技术的发展,基因敲除技术越来越广泛地应用于动植物、微生物领域,成为研究生物基因功能最有力的工具之一。基因敲除技术在改造动植物、微生物基因组、研究发育生物学、鉴定新基因新功能、育种以及医疗领域都有应用价值。针对微生物方面,对实现基因敲除的各种原理方法,RecA系统同源重组法, Red系统同源重组法,基于自杀载体的同源重组法,基于温敏型质粒的同源重组法, CRISPR/Cas系统介导的基因敲除方法进行了总结,比较各自的优缺点,并提供一些成功案例以及各种方法相关的发明专利,以期对了解基因敲除技术的方法与发展提供参考。     

Mosaic analysis with double markers in mice

We describe a method termed MADM (mosaic analysis with double markers) in mice that allows simultaneous labeling and gene knockout in clones of somatic cells or isolated single cells in vivo. Two reciprocally chimeric genes, each containing the N terminus of one marker and the C terminus of the other marker interrupted by a loxP-containing intron, are knocked in at identical locations on homologous chromosomes. Functional expression of markers requires Cre-mediated interchromosomal recombination. MADM reveals that interchromosomal recombination can be induced efficiently in vivo in both mitotic and postmitotic cells in all tissues examined. It can be used to create conditional knockouts in small populations of labeled cells, to determine cell lineage, and to trace neuronal connections. To illustrate the utility of MADM, we show that cerebellar granule cell progenitors are fated at an early stage to produce granule cells with axonal projections limited to specific sublayers of the cerebellar cortex.     

工业微生物代谢途径调控的基因敲除策略

基因敲除技术是一项重要的分子生物学技术,在工业微生物代谢工程中具有广泛应用。以下从基因敲除技术的遗传重组原理出发,总结了基因敲除策略的类型、特征和应用,重点介绍了采用线性双链DNA的λRed同源重组系统、使用环状质粒载体介导的单交换或双交换同源重组策略以及采用转座酶介导的转座重组等几种主要的基因敲除方法,进一步展望了基因敲除技术的发展前沿和应用前景。     

sacB-5-Fluoroorotic acid-pyrE-based bidirectional selection for integration of unmarked alleles into the chromosome of Rhodobacter capsulatus

The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus is a widely used model organism and has well-developed molecular genetics. In particular, interposon mutagenesis using selectable gene cartridges is frequently employed for construction of a variety of chromosomal knockout mutants. However, as the gene cartridges are often derived from antibiotic resistance-conferring genes, their numbers are limited, which restricts the construction of multiple knockout mutants. In this report, sacB-5-fluoroorotic acid (5FOA)--pyrE-based bidirectional selection that facilitates construction of unmarked chromosomal knockout mutations is described. The R. capsulatus pyrE gene encoding orotate phosphoribosyl transferase, a key enzyme of the de novo pyrimidine nucleotide biosynthesis pathway, was used as an interposon in a genetic background that is auxotrophic for uracil (Ura-) and hence resistant to 5FOA (5FOA(r)). Although Ura+ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOA(r) to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOA(r) selection was combined with sucrose tolerance selection using a suicide plasmid carrying the Bacillus subtilis sacB gene encoding levansucrase that induces lethality upon exposure to 5% (wt/vol) sucrose in the growth medium. Sucrose-tolerant, 5FOA(r) colonies that were obtained carried chromosomal unmarked mutant alleles of the target gene via double crossovers between the resident pyrE-marked and incoming unmarked alleles. The effectiveness of this double selection was proven by seeking insertion and deletion alleles of helC involved in R. capsulatus cytochrome c biogenesis, which illustrated the usefulness of this system as a genetic means for facile construction of R. capsulatus unmarked chromosomal mutants.     

BPOZ基因剔除小鼠模型的建立

BPOZ是在卵巢癌等肿瘤组织中表达下调的细胞生长抑制基因,建立BPOZ基因剔除小鼠模型,可以为在体研究BPOZ基因的生物学功能及其与肿瘤发生的关系创造条件.运用生物信息学手段确定小鼠BPOZ基因组序列,设计基因剔除策略,构建完成了基因剔除载体XpPNT-BPOZ.以电穿孔方法将基因剔除载体导入ES细胞,用G418和Ganciclovoir进行正负筛选,获得抵抗克隆,PCR和DNA印迹鉴定出正确同源重组的ES细胞克隆.将同源重组的ES细胞注入小鼠囊胚,获得嵌合体小鼠.嵌合体小鼠与C57BL/6J小鼠交配后获得Aguoti毛色的小鼠30只,其中15只为BPOZ基因剔除杂合子小鼠,阳性率为50%.在雌雄杂合子交配的后代中获得纯合子小鼠.初步的表型观察发现BPOZ基因剔除小鼠发育正常,有繁殖能力,进一步的表型分析工作正在进行之中.     

Myostatin基因打靶载体的构建及猪胎儿成纤维细胞Myostatin基因的敲除

目的:用缺口修复等技术构建Myostatin(肌肉生长抑制素,MSTN)基因打靶载体,并对大白猪胎儿成纤维体细胞进行转染,获得基因敲除细胞。方法与结果:首先构建用于MSTN基因同源长臂(LA)的抓捕载体,然后在大肠杆菌内利用Red同源重组系统介导的缺口修复,从含大白猪MSTN基因座的细菌人工染色体上亚克隆9.9 kb的LA到抓捕载体上,经过部分序列测定,同源性为100%;通过PCR获得1.4 kb的同源短臂(SA);将LA和SA连入载体pLOXP,构建含有neo和tk正负筛选标记基因的MSTN基因打靶载体pLOXP-MSTN-KO;将线性化的pLOXP-MSTN-KO通过电转染整合到大白猪胎儿成纤维细胞基因组中,利用G418和丙氧鸟苷进行药物筛选,获得抗性细胞克隆890个,通过PCR和DNA测序鉴定获得基因敲除的细胞克隆4个。结论:构建了有效的MSTN基因打靶载体,通过转染获得基因敲除细胞,为利用体细胞核移植制备MSTN基因敲除猪奠定了基础。     

Gene trap and gene inversion methods for conditional gene inactivation in the mouse

Conditional inactivation of individual genes in mice using site-specific recombinases is an extremely powerful method for determining the complex roles of mammalian genes in developmental and tissue-specific contexts, a major goal of post-genomic research. However, the process of generating mice with recombinase recognition sequences placed at specific locations within a gene, while maintaining a functional allele, is time consuming, expensive and technically challenging. We describe a system that combines gene trap and site-specific DNA inversion to generate mouse embryonic stem (ES) cell clones for the rapid production of conditional knockout mice, and the use of this system in an initial gene trap screen. Gene trapping should allow the selection of thousands of ES cell clones with defined insertions that can be used to generate conditional knockout mice, thereby providing extensive parallelism that eliminates the time-consuming steps of targeting vector construction and homologous recombination for each gene.     

A targeted gene knockout in Drosophila

We previously described a method for targeted homologous recombination at the yellow gene of Drosophila melanogaster. Because only a single gene was targeted, further work was required to show whether the method could be extended to become generally useful for gene modification in Drosophila. We have now used this method to produce a knockout of the autosomal pugilist gene by homologous recombination between the endogenous locus and a 2.5-kb DNA fragment. This was accomplished solely by tracking the altered genetic linkage of an arbitrary marker gene as the targeting DNA moved from chromosome X or 2 to chromosome 3. The results indicate that this method of homologous recombination is likely to be generally useful for Drosophila gene targeting.     

Mechanisms for recombination between stably integrated vector sequences in CHO cells

Possible mechanisms for homologous recombination in CHO cells have been investigated using a stably integrated vector, pIII-14gpt. The vector contains 2 inactive neo gene fragments in tandem arrangement. Functional neo gene activity can be restored by recombination between homologous regions in the 2 fragments. Cells in which this event has taken place become resistant to the antibiotic G418. Possible mechanisms for neo gene reactivation in this system are unequal exchange between chromatids, intra-chromatidal deletion and gene conversion.

DNA from a total of 74 G418-resistant cell clones have been isolated, and analyzed on Southern blots using neo-specific probes. Rearrangements of neo-specific restriction fragments were found to have occurred in all cell clones. In 50% of the revertants, these rearrangements can be explained by a deletion which brings the complementary regions in the 2 neo gene gragments together.

One single revertant (1.3%) shows a possible gene conversion event. The other isolated revertants (about 48%) contain more complex rearrangements. These results indicate that the predominating recombination mechanism for reactivation of the neo gene in this system is either a deletion within a chromatid or an unequal exchange between sister chromatids.     

High-frequency homologous recombination between duplicate chromosomal immunoglobulin mu heavy-chain constant regions.

Homologous recombination was used in a previous study to correct a 2-base-pair deletion in the third constant domain (Cmu3) of the haploid chromosomal mu gene in a mutant hybridoma cell line by transfer of a pSV2neo vector bearing a subfragment of the normal Cmu region (M.D. Baker, N. Pennell, L. Bosnoyan, and M.J. Shulman, Proc. Natl. Acad. Sci. USA 85:6432-6436, 1988). In these experiments, both gene replacement and single reciprocal crossover events were found to restore normal, cytolytic 2,4,6-trinitrophenyl-specific immunoglobulin M production to the mutant cells. In the cases of single reciprocal recombination, the structure of the recombinant mu gene is such that the normal Cmu region, in its correct position 3' of the expressed 2,4,6-trinitrophenyl-specific heavy-chain variable region, is separated from the mutant Cmu region by the integrated vector sequences. I report here that homologous recombination occurs with high frequency between the duplicate Cmu regions in mitotically growing hybridoma cells. The homologous recombination events were easily detected since they generated hybridomas that were phenotypically different from the parental cells. Analysis of the recombinant cells suggests that gene conversion is the most frequent event, occurring between 60 and 73% of the time. The remaining events consisted of single reciprocal crossovers. Intrachromatid double reciprocal recombination was not detected. The high frequency of recombination, the ability to isolate and analyze the participants in the recombination reactions, and the capacity to generate specific modifications in the immunoglobulin Cmu regions by gene targeting suggest that this system will be useful for studying mammalian chromosomal homologous recombination. Moreover, the ability to specifically modify the chromosomal immunoglobulin genes by homologous recombination should facilitate studies of immunoglobulin gene regulation and expression and provide a more convenient of engineering specifically modified antibody.     

Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7×10⁻⁶. In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F₁ generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P₀ generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism. ^★Equal contributions to this article.     

基因编辑技术对大肠杆菌yjjW基因点突变的两步法策略

[目的]为了实现对大肠杆菌靶基因的点突变,本研究将同源重组系统与CRISPR-Cas9技术相结合,探索一种高效、简捷的两步法策略.[方法]将靶基因的上下游同源臂和标记基因(amp)与pKOV质粒连接,获得pKOV-HR重组质粒.将pKOV-HR转化至大肠杆菌,借助其自身RecA重组系统,介导DNA发生同源重组,获得靶基...     

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non-motile mutants deficient in endoflagella

Leptospira spp. offer many advantages as model bacteria for the study of spirochaetes. However, homologous recombination between introduced DNA and the corresponding chromosomal loci has never been demonstrated. A unique feature of spirochaetes is the presence of endoflagella between the outer membrane sheath and the cell cylinder. We chose the flaB flagellin gene, constituting the flagellar core, as a target for gene inactivation in the saprophyte Leptospira biflexa. The amino acid sequence of the FlaB protein of L. biflexa was most similar to those of spirochaetes Brachyspira hyodysenteriae (agent of swine dysentery), Leptospira interrogans (agent of leptospirosis) and Treponema pallidum (agent of syphilis). A suicide vector containing the L. biflexa flaB gene disrupted by a kanamycin marker was UV irradiated or alkali denatured before electroporation. This methodology allowed the selection of many kanamycin-resistant colonies resulting from single and double cross-over events at the flaB locus. The double recombinant mutants are non-motile, as visualized in both liquid and semi-solid media. In addition, a flaB mutant selected for further analysis was shown to be deficient in endoflagella by electron microscopy. However, most of the transformants had resulted from a single homologous recombination event, giving rise to the integration of the suicide vector. We evaluated the effect of the sacB and rpsL genes in L. biflexa as potential counterselectable markers for allelic exchange, and then used the rpsL system for the positive selection of flaB double recombinants in a streptomycin-resistant strain. Like the flaB mutant studied above, the Strr double cross-over mutant was non-motile and deficient in endoflagella. Our results demonstrate that FlaB is involved in flagella assembly and motility. They also show the feasibility of performing allelic replacement in Leptospira spp. by homologous recombination.     

介绍一种鉴定胚胎干细胞基因打靶快速、经济的筛选新方法

小鼠基因剔除动物模型越来越广泛地应用于哺乳动物基因功能与疾病的研究。然而每当胚胎干细胞同源重组的效率过低时,鉴定与分离带有定位变异的阳性克隆就会既费力又昂贵。本工作以类固醇受体共激活子基因为例,研究出一种快速鉴定阳性克隆的新方法。在构造重组载体时,将一段编码半乳糖苷酶的DNA序列整合到共激活子基因的蛋白起始码后面。于是,在干细胞内同源重组发生以后,半乳糖苷酶的表达就会受控于内源性共激活子基因的启动子。在载体与半乳糖苷酶DNA随机整合的大多数非特异克隆中,因为缺少启动子或由于不正确的氨基酸编码连接,导致合成半乳糖苷酶的可能性较小。因此,在半乳糖苷酶染色阳性的克隆中,具有特异突变的阳性克隆可以富集30倍以上。从半乳糖苷酶的阳性克隆中,再用Southern Blot方法进一步确认带有基因剔除的阳性克隆就大大减少了工作量。因为半乳糖苷酶的细胞化学染色法简便而可靠,所以在重组效率低时,可以用这种方法在短期内筛选大量克隆。但是应该注意,应用该方法的前提条件是所研究的基因必须在胚胎干细胞内表达。这些方法更为重要的意义在于当带有基因剔除的胚胎干细胞发育成小鼠后,半乳糖苷酶的组化染色法可以轻而易举地用来揭示所研究基因在动物不同组织与细胞中的表达水平。     

Complete Knockout of the Lactate Dehydrogenase A Gene is Lethal in Pyruvate Dehydrogenase Kinase 1, 2, 3 Down-Regulated CHO Cells

Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3.