In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.     

Parallel In Vivo DNA Assembly by Recombination: Experimental Demonstration and Theoretical Approaches

The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration.     

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。     

应用细菌内同源重组机制快速克隆腺病毒基因组的研究

以犬2型腺病毒为载体进行疫苗和基因治疗的研究,是近年的研究热点.目前的焦点问题是新型载体的构建,经典的方法是体外连接,费时、费力.     

应用细菌内同源重组机制快速克隆腺病毒基因组的研究

A novel procedure was used for cloning large adenovirus genome fragment by the homologous recombination in E.coli strain BJ5183. The 11.2Kb downstream fragment of the CAV-2 strain YCA18 genome was cloned by homologous recombination, the 1029bp left end and the 970bp fight end of this fragment were separately amplified by PCR. They were then cloned into plasmid pPoly2 with direction from left fragment to fight fragment, obtaining a “rescue” plasmid pT615. The pT615 was liberalized by Hind Ⅲ and PstⅠ digestion and was cotransformed with the purified CAV-2 genome which was cut by BstBI into competent E.coli strain BJ5183. Recombinant plasmids harboring the 11.2Kb downstream fragment of CAV-2 genome were obtained after bacterial intermolecular homologous recombination. The recombinant efficiency of all E.coli strains tested was 78.3%. One of the recombinant plasmids, pT618, was further identified by enzyme digestion analysis and PCR amplification. The results showed the plasmids contained the 11.2kb fragment downstream the genome of CAV-2.     

Gene Knockout of the Intracellular Amylase Gene by Homologous Recombination in Streptococcus bovis

Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular α-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15–20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium. Received: 26 August 1996 / Accepted: 18 December 1996     

Strategy for Cloning Large Gene Assemblages as Illustrated Using the Phenylacetate and Polyhydroxyalkanoate Gene Clusters

We report an easy procedure for isolating chromosome-clustered genes. By following this methodology, the entire set of genes belonging to the phenylacetic acid (PhAc; 18-kb) pathway as well as those required for the synthesis and mobilization of different polyhydroxyalkanoates (PHAs; 6.4 kb) in Pseudomonas putida U were recovered directly from the bacterial chromosome and cloned into a plasmid for the first time. The transformation of different bacteria with these genetic constructions conferred on them the ability to either degrade PhAc or synthesize bioplastics (PHAs).     

p53 Gene Targeting by Homologous Recombination in Fish ES Cells

Background

Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes).

Methodology and Principal Findings

Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1∼MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by ∼12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo.

Conclusions

Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.     

Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli

PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 5′ termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biology-oriented laboratories.     

利用同源重组改变家蚕丝心蛋白重链基因

在家蚕丝心蛋白重链基因5‘和3‘端序列之间插入以绿色荧光蛋白（green fluorescent protein,GFP）基因（gfp）与人工合成丝心蛋白样基因的融合基因,利用电穿孔方法导入蚕卵中,卵孵化、发育和结茧后,用紫外灯检查,在约5400个茧中有73个“亮茧”,茧蛋白在ELISA应中可以与GFP的多克隆抗体反应。“亮茧”对应的蚕蛾进行交配、制种。对其后代进行了基因鉴定,Southern杂交的结果表明,gfp基因和人工合成丝心蛋白样基因都存在于家蚕基因组DNA中且发生了预期的同源重组事件。上述结果说明“亮茧”这一表型能用于筛选转基因蚕,融合基因已通过同源重组进入家蚕基因组。     

细菌内同源重组法构建靶向Survivin 的腺病毒载体的研究

目的:研究细菌内同源重组法构建靶向Survivin的腺病毒载体及其体外扩增表达。方法:将survivin基因克隆至穿梭质粒载体中,特异性酶切后回收、连接、转化,构建负载survivin片段的重组腺病毒载体。提取重组病毒基因酶切鉴定后,包装成病毒,并扩增到所需滴度。行Western blotting鉴定,观察重组腺病毒载体在真核细胞的表达。结果:(1)重组穿梭质粒的Mlu I酶切鉴定结果显示,酶切结果均与相应的载体及目的片段的大小相符合,基因测序结果基本一致。(2)凝胶电泳产生了两条大约15 kb和8.5kb的片段,由图2可知重组腺病毒质粒酶切充分完全,且回收率较高。(3)重组腺病毒载体转染AD293细胞24 h后已出现细胞病变效应,病变细胞细胞核变大。(4)D260/OD280的值为1.92,表明重组腺病毒纯度较高。(5)AD293细胞病毒上清中有能与抗Survivin单抗反应的蛋白,其相对分子质量与理论值相吻合,阴性对照组无对应条带出现。结论:本方法成功构建表达了含Survivin的重组腺病毒载体,为进一步进行Survivin基因功能的研究提供了实验基础和理论支持。     

Restoration of Gene Function by Homologous Recombination: from PCR to Gene Expression in One Step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete λP_R or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage λ Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding β-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.     

Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant.     

Construction of Double-Copy Glucose Isomerase Gene Engineering Strain of Streptomyces diastaticus by Homologous Recombination

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance (tsr ^R) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr^R transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI (GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI. Received: 9 July 2001 / Accepted: 8 August 2001     

The Population and Evolutionary Dynamics of Homologous Gene Recombination in Bacteria

In bacteria, recombination is a rare event, not a part of the reproductive process. Nevertheless, recombination—broadly defined to include the acquisition of genes from external sources, i.e., horizontal gene transfer (HGT)—plays a central role as a source of variation for adaptive evolution in many species of bacteria. Much of niche expansion, resistance to antibiotics and other environmental stresses, virulence, and other characteristics that make bacteria interesting and problematic, is achieved through the expression of genes and genetic elements obtained from other populations of bacteria of the same and different species, as well as from eukaryotes and archaea. While recombination of homologous genes among members of the same species has played a central role in the development of the genetics and molecular biology of bacteria, the contribution of homologous gene recombination (HGR) to bacterial evolution is not at all clear. Also, not so clear are the selective pressures responsible for the evolution and maintenance of transformation, the only bacteria-encoded form of HGR. Using a semi-stochastic simulation of mutation, recombination, and selection within bacterial populations and competition between populations, we explore (1) the contribution of HGR to the rate of adaptive evolution in these populations and (2) the conditions under which HGR will provide a bacterial population a selective advantage over non-recombining or more slowly recombining populations. The results of our simulation indicate that, under broad conditions: (1) HGR occurring at rates in the range anticipated for bacteria like Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, and Bacillus subtilis will accelerate the rate at which a population adapts to environmental conditions; (2) once established in a population, selection for this capacity to increase rates of adaptive evolution can maintain bacteria-encoded mechanisms of recombination and prevent invasion of non-recombining populations, even when recombination engenders a modest fitness cost; and (3) because of the density- and frequency-dependent nature of HGR in bacteria, this capacity to increase rates of adaptive evolution is not sufficient as a selective force to provide a recombining population a selective advantage when it is rare. Under realistic conditions, homologous gene recombination will increase the rate of adaptive evolution in bacterial populations and, once established, selection for higher rates of evolution will promote the maintenance of bacteria-encoded mechanisms for HGR. On the other hand, increasing rates of adaptive evolution by HGR is unlikely to be the sole or even a dominant selective pressure responsible for the original evolution of transformation.     

Variables Affecting In Vivo Performance of High-Capacity Adenovirus Vectors

In high-capacity adenovirus (HC-Ad) vectors the size and/or composition of the vector genome influences vector stability during production and the expression profile following gene transfer. Typically, an HC-Ad vector will contain both a gene or an expression cassette and stuffer DNA that is required to balance the final vector genome to a size of between 27 and 36 kb. To gain an improved understanding of factors that may influence gene expression from HC-Ad vectors, we have generated a series of vectors that carry different combinations of human alpha-1 antitrypsin (hAAT) expression constructs and stuffer DNAs. Expression in vitro did not predict in vivo performance: all vectors expressed hAAT at similar levels when tested in cell culture. Hepatic expression was evaluated following in vivo gene transfer in C57BL/6J mice. hAAT levels obtained from genomic DNA were significantly higher than levels achieved with small cDNA expression cassettes. Expression was independent of the orientation and only marginally influenced by the location of the expression cassette within the vector genome. The use of lambda stuffer DNA resulted in low-level but stable expression for at least 3 months when higher doses were applied. A potential matrix attachment region element was identified within the hAAT gene and caused a 10-fold increase in expression when introduced in an HC-Ad vector genome carrying a phosphoglycerate kinase (pgk) hAAT cDNA construct. We also illustrate the influence of the promoter on anti-hAAT antibody formation in C57BL/6J mice: a human cytomegalovirus but not a pgk promoter resulted in an anti-hAAT antibody response. Thus, the overall design of HC-Ad vectors may significantly influence amounts and duration of gene expression at different levels.     

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.     

大片段克隆载体研究进展

DNA克隆技术是分子生物学研究中一项重要的技术手段。自第一个质粒载体pSC1 0 1作为克隆载体以来 ,随着分子生物学技术的发展 ,克隆载体的整体结构、容载能力和转化效率都有了很大的改善。尤其是人类基因组计划的实施 ,产生了YAC和BAC克隆体系。随着植物基因组计划的进行 ,又产生了既能够克隆大片段DNA又能够将候选克隆直接通过农杆菌介导进行功能互补实验的载体。综述了几种常用大片段克隆载体YAC、BAC、BIBAC、PAC和TAC的特点及其应用 ,并对克隆载体的发展作了展望。