PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome

Acquisition of flanking sequence adjacent to a known DNA site is an important task in microbial genome-related research. In this study, we developed a new method containing two rounds of PCR followed by cloning and sequencing. Firstly, specific primer (SP) is added into the reaction system for primary locus-specific linear amplification, and then a complex long primer (CLP) is added into the cooled reaction system for only one cycle. Amplification products from the first round of PCR are directly purified without electrophoresis, diluted, and used as the templates of the second PCR. Secondly, one long specific primer (LSP) and one long base-fixed primer (LFP) are adopted. The amplicons are purified for cloning and sequencing. The achievement of specific amplification for long flanking region mainly depends on ingenious and precise settings of PCR programs, structure design of CLP primer, adding of CLP primer after specific linear amplification, concentration ratio of CLP and SP primer, applying long primers, etc. Through this method, we successfully performed the long PCR walkings (>1.5 Kb) on rpoB gene of Vibrio vulnificus, transposon-like gene of V. alginolyticus, and sto gene of V. cholerae. The method provides a robust and simple strategy for rapid amplification of long unknown DNA fragments from microbes.     

Red/ET重组在基因打靶载体快速构建中的应用

通过合理应用Red/ET重组技术实现基因打靶载体的快速构建。在Red/ET重组介导下,首先从基因组DNA中将靶基因片段亚克隆至打靶质粒载体中,随后将两端带有50 bp同源臂的抗性筛选基因插入并替换靶基因上的目标序列,如此两步操作即可完成一个传统型基因敲除打靶载体的构建;结合Cre-loxP系统,在传统型基因敲除打靶载体的基础上,经过再一轮的Red/ET重组就能够成功实现条件性基因敲除打靶载体的构建。整个实验过程不需要PCR扩增长、短臂序列,也不涉及酶切、连接反应,因此,不仅省时、省力,而且所构建的基因打靶载体序列准确,无突变。此实验方法的建立为加速后基因组时代的基因功能研究提供了一条捷径。     

博尔纳病病毒pEGFP-p24基因重组质粒的构建及鉴定

构建博尔纳病病毒pEGFP-p24基因重组表达质粒。通过PCR方法扩增获得博尔纳病痛毒p24基因的完整序列,将此片段定向克隆到pEGFP-N1载体多克隆位点区,筛选重组阳性菌株,提取重组质粒,利用PCR方法和核酸序列测定验证重组质粒构建的正确性。PCR及核酸序列测定证明博尔纳病病毒pEGFP-p24基因重组表达质粒构建成功。构建的重组质粒将为研究博尔纳病病毒p24基因在真核细胞中的功能和作用提供实验依据。     

Open reading frame analysis by selective PCR-mediated deletion mutagenesis

Recently, we demonstrated that a nested set of DNA fragments can be obtained by using one specific primer and one semirandom primer in a polymerase chain reaction (PCR). We now describe a strategy for selective deletion mutagenesis that is based on this observation. The gene of interest is cloned as a fusion construct with a selectable marker in a small vector, allowing for PCR amplification of the entire recombinant plasmid. The specific primer is complementary to the vector sequence beyond the gene of interest and is oriented downstream. The 3′ end of the semirandom primer is complementary to a triplet (GAT) that is scattered over the entire open reading frame (ORF). It is shown by nucleotide sequence analysis that deletion mutants result exclusively from annealing of the semirandom primer at different GAT triplets. PCR products resulting fro from annealing to GAT triplets elsewhere in the plasmid are counterselected by the need for replication functions and for the expression of the selectable marker. This technique is demonstrated on the Saccharomyces cerevisiae ORF YCL56C.     

Establishment of Smad2 conditional gene targeting mice based on the Cre-LoxP system

Smads is a new gene family in transforming growth factor-β (TGF- β) signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electroporated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targeting mouse has laid a solid foundation for producing the tissue specific Smad2 gene knockout mice.     

A novel method for gene-targeting vector construction—Red/ET recombination

The study of the function of novel human genes has become an increasingly active academic field with the gene-knockout (KO) mouse model forming the basis of this study. Because of the low efficiency of recombination of targeting vector constructed using the traditional method, the KO mouse model has become the key step in the construction of a targeting vectors, both economically and efficiently. To study the function of a novel gene (Resp18), a novel DNA engineering platform, Red/ET recombination, was introduced to construct the Resp18 targeting vector. Red/ET recombineering differs from the conventional methods of vector construction (e.g., PCR, restriction enzyme digestion, and ligation), and genetic modification is accomplished by acquisition, insertion, fusion, or replacement of the target gene through small fragments-mediated homologous recombination. At present, Resp18 targeting vectors constructed using three strategies mentioned above were successfully released through two homologous recombination processes of retrieval and neo-targeting. Red/ET recombination has the advantage of producing genes with longer homology regions without mutation.     

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Function studies of many proteins are waited to develop after genome sequencing. High‐throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation‐independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate‐modified primers and the digestion of PCR products by λ exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3′ overhangs, which are designed to form complementary double‐stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3′ overhangs is a better cohesive end for LIC than 5′ overhang and the existence of 5′phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.     

Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

In contrast to the high accumulation in sequence data for hyperthermophilic archaea, methodology for genetically manipulating these strains is still at an early stage. This study aimed to develop a gene disruption system for the hyperthermophilic euryarchaeon Thermococcus kodakaraensis KOD1. Uracil-auxotrophic mutants with mutations in the orotidine-5'-monophosphate decarboxylase gene (pyrF) were isolated by positive selection using 5-fluoroorotic acid (5-FOA) and used as hosts for further transformation experiments. We then attempted targeted disruption of the trpE locus in the host strain by homologous recombination, as disruption of trpE was expected to result in tryptophan auxotrophy, an easily detectable phenotype. A disruption vector harboring the pyrF marker within trpE was constructed for double-crossover recombination. The host cells were transformed with the exogenous DNA using the CaCl(2) method, and several transformants could be selected based on genetic complementation. Genotypic and phenotypic analyses of a transformant revealed the unique occurrence of targeted disruption, as well as a phenotypic change of auxotrophy from uracil to tryptophan caused by integration of the wild-type pyrF into the host chromosome at trpE. As with the circular plasmid, gene disruption with linear DNA was also possible when the homologous regions were relatively long. Shortening these regions led to predominant recombination between the pyrF marker in the exogenous DNA and the mutated allele on the host chromosome. In contrast, we could not obtain trpE disruptants by insertional inactivation using a vector designed for single-crossover recombination. The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms.     

转录因子DREB1A基因的克隆与Gateway克隆技术构建植物表达载体

目的:研究转录因子DREB1A在植物抗渗透胁迫反应中的作用,并探讨利用Gateway克隆技术构建植物表达载体的方法。方法:根据GenBank中登录的DREB1A基因的全长mRNA序列设计引物,克隆了拟南芥的转录因子DREBIA基因。根据Gateway克隆技术的要求,设计含有attB接头的引物,利用高保真的PlatinumpfxDNA聚合酶,通过PCR方法在克隆基因的两端加上B序列。通过BP反应将包含有attB接头的PCR产物克隆到含有attP的donor载体上以产生Entry克隆,通过LR反应将已经重组入Entry载体的DREB1A基因再克隆到pH2GW7双元载体。结果:对重组载体pH2GW7-DREB1A的鉴定结果表明成功构建了DREB1A基因的植物表达载体。结论:利用Gateway克隆技术构建植物表达载体简便易行,该结果为遗传转化研究奠定了基础。     

一步法快速构建多片段连接的同源臂载体

目的：建立一种简便、高效,可一步完成多个片段连接,从而构建含同源臂的载体的方法。方法：按照酶切后可产生前后片段相匹配的粘性末端接头的原则设计PCR引物,在目的片段两端均引入BsaⅠ酶切位点。以G160基因为例,PCR扩增打靶用左右同源臂片段、示踪基因CMV-EGFP片段、载体骨架pMD19－T等4个片段,纯化后一起加入一个反应管中,并加入BsaⅠ限制性内切酶和T7DNA连接酶及相应缓冲液,进行酶切、酶连接共10～50个循环反应,一步构建含同源臂载体的质粒;产物经高温处理后,直接转化感受态细胞,并进行重组子PCR鉴定;对pMD19－T载体进行优化,突变载体上的BsaⅠ酶切位点,把示踪基因CMV-EGFP片段引入pHSG298－T载体,再选择不同的G160基因同源臂片段组合对构建系统进行验证。结果：重组质粒酶切和PCR结果表明,应用一步法可成功连接多个片段来构建含同源臂及示踪基因的克隆载体;用优化后的pMD19－T－O载体体系,在2d内即完成了6种各含4个片段的载体的构建。结论：多个基因片段一步无缝连接的方法简便、易行、可靠,不仅可快速构建某类载体系统,还可对基因进行精确的点突变,该系统可用于快速构建基因打靶载体。     

迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白在蓝藻中的表达

在蓝藻中表达迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白。提取迟缓爱德华氏菌基因组DNA为模板,用PCR技术分别扩增两个已知具有较强免疫原性的基因eta1和gapdh,再采用重叠延伸PCR将这两个基因融合,获得目的融合基因eta1-L-gapdh。将目的基因连接到表达载体pRL489的两个Bam H I酶切位点之间构建表达载体,用质粒提取、PCR、酶切、测序等手段对表达载体进行验证。验证正确的表达载体通过三亲接合转化野生鱼腥藻PCC7120,用新霉素抗性筛选出转基因藻落,通过质粒提取和PCR验证转基因藻。用RT-PCR和Western-blot分别从转录水平和翻译水平对转基因藻中融合基因的表达进行了检测。结果表明,含目的基因的表达载体构建成功,目的基因在蓝藻中转录并表达蛋白,该蛋白在蓝藻中的表达量为2.46%。     

Transposon mutagenesis: Cloning of chromosomal DNA from the site of Tn916 insertion using polymerase chain reaction

A protocol that allows fast recovery and further analyses of chromosomal DNA adjacent to the Tn916 site of insertion is described. The procedure is based on single specific primer PCR amplification using restricted chromosomal DNA ligated into a suitable vector. Two primers, one Tn916-specific and the second vector-specific, allow amplification of the chromosomal DNA flanking the site of insertion.     

蛋白质起始的DNA病毒复制机制

DNA聚合酶在DNA合成过程中需要的引物包括RNA引物、DNA自我引物和蛋白质引物3种类型。新DNA链(如冈崎片段)的复制多是在DNA模板上合成一段RNA引物,细小病毒利用其基因组末端的反向末端重复序列(ITRs)自我折叠成DNA引物,而一些DNA、RNA病毒及真菌质粒起始复制反应的引物则是蛋白质。以感染原核生物的噬菌体Phi29和真核DNA病毒腺病毒为例,从复制过程所涉及的蛋白质、对复制原点的识别、复制起始反应、新链的延伸、复制终止过程等方面详细阐述DNA病毒由蛋白质引发的复制机制,并对已商品化的Phi29 DNA聚合酶产品多重置换扩增及单细胞测序等的应用以及基于噬菌体Phi29蛋白质起始的最小复制系统体外扩增异源DNA等最新的应用研究作相关总结介绍。     

TECHNICAL REPORT: Rapid confirmation of gene targeting in embryonic stem cells using two long-range PCR techniques

Gene targeting in mouse embryonic stem (ES) cells generally includes the analysis of numerous colonies to identify a few with mutations resulting from homologous recombination with a targeting vector. Thus, simple and efficient screening methods are needed to identify targeted clones. Optimal screening approaches require probes from outside of the region included in the targeting vector to avoid detection of the more common random insertions. However, the use of large genomic fragments in targeting vectors can limit the availability of cloned DNA, thus necessitating a strategy to obtain unique flanking sequences. We describe a rapid method to identify sequences adjacent to cloned DNA using long-range polymerase chain reaction (PCR) amplification from a genomic DNA library, followed by direct nucleotide sequencing of the amplified fragment. We have used this technique in two independent gene targeting experiments to obtain genomic DNA sequences flanking the mouse cholecystokinin (CCK) and gastrin genes. The sequences were then used to design primers to characterize ES cell lines with CCK or gastrin targeted gene mutations, employing a second long-range PCR approach. Our results show that these two long-range PCR methods are generally useful to rapidly and accurately characterize allele structures in ES cells     

Establishment of Smad2 conditional gene targeting mice based on the Cre-LoxP system

Smads is a new gene family in transforming growth factor-β (TGF- β signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electropo-rated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targetin     

A simple strategy for generation of gene knockdown constructs with convergent H1 and U6 promoters

RNA interference (RNAi) is a powerful tool for functional genetic studies in model organisms and mammalian cells. To facilitate rapid construction of gene knockdown constructs and RNAi libraries for known genes of mammalian cells, a new and simple strategy to produce small interfering RNA (siRNA) expression vectors with two opposing polymerase III promoters was developed. The design involved a one-step PCR amplification and single cloning procedure to construct a dual promoter siRNA expression vector. The forward primer is identical for all PCR reactions, only a single reverse primer that contains the siRNA targeting sequence has to be synthesized in the construction of each individual vector. This single primer design is cost-effective and it reduces the risk of sequence errors during synthesis of long oligos. Sense and antisense strands of siRNA duplexes were transcribed from the same template and this eliminated the need to synthesize long hairpin-forming oligonucleotides. Our study demonstrated that this vector design could mediate potent inhibition of expression of both exogenous and endogenous genes in mammalian cells.     

Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines

Targeted gene disruption is a powerful tool for studying gene function in cells and animals. In addition, this technology includes a potential to correct disease-causing mutations. However, constructing targeting vectors is a laborious step in the gene-targeting strategy, even apart from the low efficiency of homologous recombination in mammals. Here, we introduce a quick and simplified method to construct targeting vectors. This method is based on the commercially available MultiSite Gateway technology. The sole critical step is to design primers to PCR amplify genomic fragments for homologous DNA arms, after which neither ligation reaction nor extensive restriction mapping is necessary at all. The method therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms whose genome has been sequenced. Recently, we and others have shown that the human pre-B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line has enabled rapid disruption of virtually any locus of the human genome within one month, and homozygous knockout clones lacking a human gene of interest can be created within 2-3 months. Thus, our system greatly facilitates reverse genetic studies of mammalian--particularly human--genes.     

Targeted cloning of a subfamily of LINE-1 elements by subfamily-specific LINE-1-PCR

Subfamily-specific LINE-1 PCR (SSL1-PCR) is the targeted amplification and cloning of defined subfamilies of LINE-1 elements and their flanking sequences. The targeting is accomplished by incorporating a subfamily-specific sequence difference at the 3 end of a LINE-1 PCR primer and pairing it with a primer to an anchor ligated within the flanking region. SSL1-PCR was demonstrated by targeting amplification of a Mus spretus-specific LINE-1 subfamily. The amplified fragments were cloned to make an SSL1-PCR library, which was found to be 100-fold enriched for the targeted elements. PCR primers were synthesized based on the sequence flanking the LINE-1 element of four different clones. Three of the clones were recovered from Mus spretus DNA. A fourth clone was recovered from a congenic mouse containing both Mus spretus and Mus domesticus DNA. Amplification between these flanking primers and LINE-1 PCR primers produced a product in Mus spretus and not in Mus domesticus. These dimorphisms were further verified to be due to insertion of Mus spretus-specific LINE-1 elements into Mus spretus DNA and not into Mus domesticus DNA.