Tissue-specific expression of a BAC transgene targeted to the Hprt locus in mouse embryonic stem cells

The hypoxanthine phosphoribosyltransferase (Hprt) locus has been shown to have minimal influence on transgene expression when used as a surrogate site in the mouse genome. We have developed a method to transfer bacterial artificial chromosomes (BACs) as a single copy into the partially deleted Hprt locus of embryonic stem cells. BACs were modified by Cre/loxP recombination to contain the sequences necessary for homologous recombination into and complementation of the partially deleted Hprt locus. Modified BACs were shown to undergo homologous recombination into the genome intact, to be stably transmitted through the germ line of transgenic mice, and to be expressed in the proper tissue-specific manner. This technology will facilitate many studies in which correct interpretation of data depends on developmentally appropriate transgene expression in the absence of rearrangements or deletions of endogenous DNA.     

The evolution of IncP catabolic plasmids

The recent adoption of whole plasmid genome sequencing as a routine analytical technique has provided the basis for cataloging the historical events through which plasmids are assembled from the available families of modular plasmid components. Horizontal gene transfer mediated by plasmids plays an important role in the adaptation of bacteria to the presence of specific metabolizable compounds, including man-made chemicals, in the surrounding environment. Bacterial plasmid genome sequence comparisons indicate that plasmids have complex genetic histories resulting from transposition, homologous recombination, and illegitimate recombinational events. Evidence from IncP plasmid genome sequences indicates that cryptic plasmid backbones acquire diverse catabolic pathways through gene capture and horizontal gene transfer.     

Gene targeting,genome editing: from Dolly to editors

One of the most powerful strategies to investigate biology we have as scientists, is the ability to transfer genetic material in a controlled and deliberate manner between organisms. When applied to livestock, applications worthy of commercial venture can be devised. Although initial methods used to generate transgenic livestock resulted in random transgene insertion, the development of SCNT technology enabled homologous recombination gene targeting strategies to be used in livestock. Much has been accomplished using this approach. However, now we have the ability to change a specific base in the genome without leaving any other DNA mark, with no need for a transgene. With the advent of the genome editors this is now possible and like other significant technological leaps, the result is an even greater diversity of possible applications. Indeed, in merely 5 years, these ‘molecular scissors’ have enabled the production of more than 300 differently edited pigs, cattle, sheep and goats. The advent of genome editors has brought genetic engineering of livestock to a position where industry, the public and politicians are all eager to see real use of genetically engineered livestock to address societal needs. Since the first transgenic livestock reported just over three decades ago the field of livestock biotechnology has come a long way—but the most exciting period is just starting.     

Molecular mechanisms of exon shuffling: illegitimate recombination

Illegitimate recombination (IR) is a process that takes place far more often than homologous recombination and is characterized by the recombination between non-homologous or short homologous sequences. The consequences of IR frequently emerge after the introduction of DNA in cell lines because it more frequently integrates in non-homologous than in homologous regions of the host genome. As a result, unexpected truncated or elongated products may be found. By not discarding those products as transfection artifacts, but by studying how they are generated, it might elucidate a possible molecular mechanism of IR. Here we review the current literature describing different mechanisms by which non-homologous DNA recombination can be induced.     

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.     

芥菜型油菜与埃塞俄比亚芥杂种基因组原位杂交分析

原产于非洲的埃塞俄比亚芥(Brassica carinata,2n=34,BBCC)具有适应于炎热干旱地区种植等特点,是改良我国芥菜型油菜(B.juncea,2n=36,AABB)的重要种质资源。本研究用基因组原位杂交方法(GISH,Genomic in situ hybridization)分析了芥菜型油菜与埃塞俄比亚芥种间杂种花粉母细胞的染色体分离,发现在后期I染色体主要以17∶18类型分离,其次是16∶19,染色体落后现象偶然发生,其中B染色体组以8∶8的分离比率较高,表明不同来源的B染色体可正常配对分离。本研究表明,芥菜型油菜与埃塞俄比亚芥远缘杂交,通过染色体同源重组(B染色体间),以及部分同源染色体配对交换的方式(A、B、C基因组间),有可能将埃塞俄比亚芥优良遗传成分转移到芥菜型油菜中。     

Targeted modification of mammalian genomes

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination.

Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or ΦC31 integrase) are usually 30–50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.     

Rapid construction of adenoviral vectors by lambda phage genetics

Continued improvements of adenoviral vectors require the investigation of novel genome configurations. Since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid DNA, it is possible to separate genome construction from virus production. In this way failure to generate a virus is not associated with an inability to generate the desired genome. We have developed a novel lambda-based system that allows rapid modification of the viral genome by double homologous recombination in Escherichia coli. The recombination reaction and newly generated genome may reside in a recombination-deficient bacterial host for enhanced plasmid stability. Furthermore, the process is independent of any restriction endonucleases. The strategy relies on four main steps: (i) homologous recombination between an adenovirus cosmid and a donor plasmid (the donor plasmid carries the desired modification[s] and flanking regions of homology to direct its recombination into the viral genome); (ii) in vivo packaging of the recombinant adenoviral cosmids during a productive lambda infection; (iii) transducing a recombination-deficient E. coli lambda lysogen with the generated lysate (the lysogen inhibits the helper phage used to package the recombinant andenoviral cosmid from productively infecting and destroying the host bacteria); (iv) effectively selecting for the desired double-recombinant cosmid. Approximately 10,000 double-recombinant cosmids are recovered per reaction with essentially all of them being the correct double-recombinant molecule. This system was used to generate quickly and efficiently adenoviral genomes deficient in the E1/E3 and E1/E3/E4 regions. The basis of this technology allows any region of the viral genome to be readily modified for investigation of novel configurations.     

Mammalian cell function mediating recombination of genetic elements

Recombination of segments of the SV40 genome by a variety of mechanisms is described. These include the faithful joining of linear segments that have flush termini as opposed to previously described cohesive or resected termini. Lack of involvement of viral proteins has been demonstrated for recombination of segments with homologous overlapping termini, but probably applies also to the other joining reactions. Segments of the genome that have been cleaved in such a manner as to be unable to manufacture any known viral proteins are neutral elements of genetic information, incapable of selection by replication or biological function until recombined. These recombination functions presumably are available to the host cell and any element of genetic information that can be generated in that cell.     

An<Emphasis Type="Italic"> Arabidopsis</Emphasis> homologue of bacterial RecA that complements an<Emphasis Type="Italic"> E. coli recA</Emphasis> deletion is targeted to plant mitochondria

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mt recA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mt recA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mt recA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mt recA in homologous recombination and/or repair in Arabidopsis mitochondria.     

Multiple pathways for Cre/lox-mediated recombination in plastids

Plastid transformation technology involves the insertion by homologous recombination and subsequent amplification of plastid transgenes to approximately 10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subsequent role in the transformed line. We report here a feasibility study in the model plant tobacco, to test the heterologous Cre/lox recombination system for antibiotic marker gene removal from plastids. To study its efficiency, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molecular assays, we show that transgene excision occurs completely from all plastid genomes early in plant development. Selectable marker-free transplastomic plants are obtained in the first seed generation, indicating a potential application of the Cre/lox system in plastid transformation technology. In addition to the predicted transgene excision event, two alternative pathways of Cre-mediated recombination were also observed. In one alternative pathway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in the plastid genome. The other alternative pathway uncovered a plastid genome 'hot spot' of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, the resultant plastid mutations are silent, and their study provides the first insights into tRNA accumulation and trans-splicing events in higher plant plastids.     

Targeted Genome Editing by Recombinant Adeno-Associated Virus （rAAV） Vectors for Generating Genetically Modified Pigs

Recombinant adeno-associated virus(rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases.Several advantages,such as simple vector construction,high targeting frequency by homologous recombination,and applicability to many cell types,make rAAV an attractive approach for targeted genome editing.Combined with cloning by somatic cell nuclear transfer(SCNT),this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1,and breast cancer.This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination.We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts,which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.     

Progress of targeted genome modification approaches in higher plants

Transgene integration in plants is based on illegitimate recombination between non-homologous sequences. The low control of integration site and number of (trans/cis)gene copies might have negative consequences on the expression of transferred genes and their insertion within endogenous coding sequences. The first experiments conducted to use precise homologous recombination for gene integration commenced soon after the first demonstration that transgenic plants could be produced. Modern transgene targeting categories used in plant biology are: (a) homologous recombination-dependent gene targeting; (b) recombinase-mediated site-specific gene integration; (c) oligonucleotide-directed mutagenesis; (d) nuclease-mediated site-specific genome modifications. New tools enable precise gene replacement or stacking with exogenous sequences and targeted mutagenesis of endogeneous sequences. The possibility to engineer chimeric designer nucleases, which are able to target virtually any genomic site, and use them for inducing double-strand breaks in host DNA create new opportunities for both applied plant breeding and functional genomics. CRISPR is the most recent technology available for precise genome editing. Its rapid adoption in biological research is based on its inherent simplicity and efficacy. Its utilization, however, depends on available sequence information, especially for genome-wide analysis. We will review the approaches used for genome modification, specifically those for affecting gene integration and modification in higher plants. For each approach, the advantages and limitations will be noted. We also will speculate on how their actual commercial development and implementation in plant breeding will be affected by governmental regulations.     

High-frequency homologous recombination in plants mediated by zinc-finger nucleases

Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.     

Homologous recombination efficiency enhanced by inhibition of MEK and GSK3β

Homologous recombination in embryonic stem cells (ESCs) is widely utilized in genome engineering, particularly in the generation of gene targeted mice. However, genome engineering is often plagued by the problem of low homologous recombination efficiency. In this study, we developed a novel method to increase the efficiency of homologous recombination in ESCs by changing its culture conditions. By comparing the efficiency of different ESCs in various culture conditions, we determined that chemicals that inhibit the MEK and GSK3β pathways (2i condition) enhance homologous recombination and eliminate differences in efficiencies among cell lines. Analysis of gene expression patterns in ESCs maintained in different culture conditions has identified several homologous recombination‐related candidates, including the pluripotent markers Eras and Tbx3. The results of this study suggest that homologous recombination is associated with ESC pluripotency. genesis 52:889–896, 2014. © 2014 Wiley Periodicals, Inc.     

影响动物细胞同源重组发生与基因打靶效率的分子机制

真核细胞的基因打靶是基因结构与功能研究的一种非常有价值的技术,也是可应用于基因治疗的具有潜力的工具。有2个限制因素束缚真核细胞基因打靶的发展,即同源重组(HR)率非常低而随机整合率非常高。通过特定基因的过表达或表达干涉,使一些参与DNA重组的蛋白表达水平瞬间改变,可能会增加HR率,降低随机整合率。本文列举了一些与HR相关的候选基因,详细介绍了其中的Rad52上位簇基因,还讨论了打靶载体的设计与修饰、DNA转染方法的有效性等。     

Homologous and illegitimate recombination in developing Xenopus oocytes and eggs.

Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-->3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.     

Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12

In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F’-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10^-12 CFU/recipient per hour.     

Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates

The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.     

Horizontal gene transfer regulation in bacteria as a "spandrel" of DNA repair mechanisms

Horizontal gene transfer (HGT) is recognized as the major force for bacterial genome evolution. Yet, numerous questions remain about the transferred genes, their function, quantity and frequency. The extent to which genetic transformation by exogenous DNA has occurred over evolutionary time was initially addressed by an in silico approach using the complete genome sequence of the Ralstonia solanacearum GMI1000 strain. Methods based on phylogenetic reconstruction of prokaryote homologous genes families detected 151 genes (13.3%) of foreign origin in the R. solanacearum genome and tentatively identified their bacterial origin. These putative transfers were analyzed in comparison to experimental transformation tests involving 18 different genomic DNA positions in the genome as sites for homologous or homeologous recombination. Significant transformation frequency differences were observed among these positions tested regardless of the overall genomic divergence of the R. solanacearum strains tested as recipients. The genomic positions containing the putative exogenous DNA were not systematically transformed at the highest frequencies. The two genomic "hot spots", which contain recA and mutS genes, exhibited transformation frequencies from 2 to more than 4 orders of magnitude higher than positions associated with other genes depending on the recipient strain. These results support the notion that the bacterial cell is equipped with active mechanisms to modulate acquisition of new DNA in different genomic positions. Bio-informatics study correlated recombination "hot-spots" to the presence of Chi-like signature sequences with which recombination might be preferentially initiated. The fundamental role of HGT is certainly not limited to the critical impact that the very rare foreign genes acquired mainly by chance can have on the bacterial adaptation potential. The frequency to which HGT with homologous and homeologous DNA happens in the environment might have led the bacteria to hijack DNA repair mechanisms in order to generate genetic diversity without losing too much genomic stability.