Replacement Vector Research Articles

A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1

Here we report an approach to generate a knock-in mouse model using an 'ends-out' gene replacement vector to substitute the murine Parp-1 (mParp-1) coding sequence (32 kb) with its human orthologous sequence (46 kb). Unexpectedly, examination of mutant ES cell clones and mice revealed that site-specific homologous recombination was mimicked in three independently generated ES cell clones by bidirectional extension of the vector homology arms using the endogenous mParp-1-flanking sequences as templates. This was followed by adjacent integration of the targeting vector, thus leaving the endogenous mParp-1 locus functional. A related phenomenon termed 'ectopic gene targeting' has so far only been described for 'ends-in' integration-type vectors in non-ES cell gene targeting. We provide reliable techniques to detect such ectopic gene targeting which represents an unexpected caveat in mouse genetic engineering that should be considered in the design and validation strategy of future gene knock-in approaches.

Iron-Sulfur Cluster N5 Is Coordinated by an HXXXCXXCXXXXXC Motif in the NuoG Subunit of Escherichia coli NADH:Quinone Oxidoreductase (Complex I)

NADH:quinone oxidoreductase (complex I) plays a central role in cellular energy metabolism, and its dysfunction is found in numerous human mitochondrial diseases. Although the understanding of its structure and function has been limited, the x-ray crystal structure of the hydrophilic part of Thermus thermophilus complex I recently became available. It revealed the localization of all redox centers, including 9 iron-sulfur clusters and their coordinating ligands, and confirmed the predictions mostly made by Ohnishi et al. (Ohnishi, T., and Nakamaru-Ogiso, E. (2008) Biochim. Biophys. Acta 1777, 703-710) based on various EPR studies. Recently, Yakovlev et al. (Yakovlev, G., Reda, T., and Hirst, J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 12720-12725) claimed that the EPR signals from clusters N4, N5, and N6b were misassigned. Here we identified and characterized cluster N5 in the Escherichia coli complex I whose EPR signals had never been detected by any group. Using homologous recombination, we constructed mutant strains of H101A, H101C, H101A/C114A, and cluster N5 knock-out. Although mutant NuoEFG subcomplexes were dissociated from complex I, we successfully recovered these mutant NuoCDEFG subcomplexes by expressing the His-tagged NuoCD subunit, which had a high affinity to NuoG. The W221A mutant was used as a control subcomplex carrying wild-type clusters. By lowering temperatures to around 3 K, we finally succeeded in detecting cluster N5 signals in the control for the first time. However, no cluster N5 signals were found in any of the N5 mutants, whereas EPR signals from all other clusters were detected. These data confirmed that, contrary to the misassignment claim, cluster N5 has a unique coordination with His(Cys)(3) ligands in NuoG.

Genetic tools for allelic replacement in Burkholderia species.

Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the alpha-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Tel(r)) and trimethoprim (Tp(r)) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Tel(r)-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tp(r)), which is one of two constructed flp-containing plasmids, pFLP-AB4 (Tel(r)) and pFLP-AB5 (Tp(r)). These flp-containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p-chlorophenylalanine after Flp-FRT excision. The characterization of the Delta betBA::Tel(r)-FRT and Delta betBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources.

Comparison of gene targeting efficiencies in two mosses suggests that it is a conserved feature of Bryophyte transformation

The moss, Physcomitrella patens, is a novel tool in plant functional genomics due to its exceptionally high gene targeting efficiency that is so far unique for plants. To determine if this high gene targeting efficiency is exclusive to P. patens or if it is a common feature to mosses, we estimated gene-targeting efficiency in another moss, Ceratodon purpureus. We transformed both mosses with replacement vectors corresponding to the adenine phosphoribosyl transferase (APT) reporter gene. We achieved a gene targeting efficiency of 20.8% for P. patens and 1.05% for C. purpureus. Our findings support the hypothesis that efficient gene targeting could be a general mechanism of Bryophyte transformation.

Quantitative Analysis of Efficient Endogenous Gene Silencing in Nicotiana benthamiana Plants Using Tomato bushy stunt virus Vectors That Retain the Capsid Protein Gene

Tomato bushy stunt virus (TBSV) coat protein (CP) replacement vectors have been used previously to silence transgenes (e.g., the green fluorescent protein gene) but have not been effective for silencing endogenous plant genes. New TBSV vectors which retained the CP gene were developed by engineering an XhoI restriction site in three positions (3f, CEB, and CEA) of the pTBSV-100 infectious clone. Magnesium chelatase (ChlH) and phytoene desaturase (PDS) were chosen as targets for endogenous gene silencing. Initial experiments using CP replacement vectors with a 230-bp sense or antisense ChlH insert gave a silencing phenotype prominent only in the first new leaves above those inoculated. No silencing phenotype was apparent beyond these leaves whereas, for PDS, no silencing phenotype was observed. When plants were inoculated with the XhoI insert vectors containing ChlH and PDS sequences, plants showed a silencing phenotype extensively throughout the challenged plant, indicating an improved ability for virus movement and silencing in Nicotiana benthamiana host plants. Silencing efficiencies were quantified using realtime reverse-transcription polymerase chain reaction, indicating specific silencing effects of each individual silencing vector. Only one recombinant vector (pPD-3f5), where the XhoI insert was at the 3' end of the CP gene, failed to give effective silencing. Here, we show that our new CP-retaining TBSV vectors (CEA-CEB) form typical TBSV virions, retain silencing inserts of variable lengths (110 to 260 nucleotides), and can systemically silence endogenous genes in N. benthamiana.

Construction of a replacement vector to disrupt pksCT gene for the mycotoxin citrinin biosynthesis in Monascus aurantiacus and maintain food red pigment production.

More and more people pay attention to citrinin produced by Monascus, which has nephrotoxic activity in mammals. It was reported that pksCT gene is responsible for citrinin biosynthesis in Monascus purpureus. In this paper, two DNA fragments in both ends of pksCT were amplified by genomic PCR from fourteen Monascus spp. strains. The PCR products were gained from all of the strains. It is suggested that pksCT gene was highly conserved in different citrinin-producing Monascus strains. A pksCT-replacement vector (pHD106) was constructed to disrupt pksCT with a hygromycin resistance gene as the selection marker, and was transformed into M. aurantiacus Li AS3.4384. Three transformants (M. aurantiacus PHDS18, PHDS26, PHDS31) were selected from transformant selective plates. The targeting fragment D was gained by genomic PCR from PHDS18 and PHDS26 except PHDS31. The expressing citrinin capacities of PHDS26 was decreased by about 98%, while PHDS18 was reserved the high capacity of producing citrinin, after 10 days of growth on YM medium. The results indicated that PHDS26 is a pksCT-disrupted strain. There are maybe other genes besides pksCT responsible for citrinin biosynthesis in M. aurantiacus. It is the effective way to solve the problem of citrinin in M. aurantiacus products by constructing replacement vectors to disrupt the genes responsible for citrinin biosynthesis to reduce the capacity of expressing citrinin.

In Celebration of Dr. Mario R. Capecchi's Nobel Prize

In Celebration of Dr. Mario R. Capecchi's Nobel Prize

Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

BackgroundTargeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome.ResultsHere we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction.ConclusionTogether, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

Placental Thrombosis and Spontaneous Fetal Death in Mice Deficient in Ethanolamine Kinase 2

Ethanolamine kinase catalyzes the first step in the CDP-ethanolamine pathway for the formation of the major membrane phospholipid phosphatidylethanolamine (PtdEtn). In this work, the predicted Etnk2 cDNA was established as a soluble protein with ethanolamine-specific kinase activity that was most highly expressed in liver. Mice with an inactivated Etnk2 gene were derived, and its absence reduced the rate of PtdEtn synthesis from exogenous ethanolamine in hepatocytes. PtdEtn is a major precursor to phosphatidylcholine in liver; however, Etnk2(-/-) mice did not have reduced amounts of either PtdEtn or phosphatidylcholine or an altered phospholipid molecular species distribution. The knock-out animals were able to adapt to a choline-deficient diet. The Etnk2(-/-) mice exhibited a maternal-specific intrauterine growth retardation phenotype that resulted in a 33% reduction in litter size and frequent perinatal death. Histological analysis of pregnant Etnk2(-/-) females showed that fetal development failed at the late stage of pregnancy in a significant percentage of embryos because of the appearance of extensive placental thrombosis. These results illustrate a non-redundant role for EtnK2 expression in regulating placental hemostasis.

Transformation of Tetrahymena thermophila by Electroporation

INTRODUCTIONThis protocol describes a method for transformation of the Tetrahymena using electroporation. The vector is electroporated into cells after mating, where it is incorporated into the DNA of developing macronuclei. Because T. thermophila can be propagated indefinitely without conjugation, transformation of the macronucleus provides a way to obtain stable somatic transformants. DNA vectors transformed using this protocol include those containing drug-resistant versions of Tetrahymena genes (which replace endogenous genes via homologous recombination) as well as those containing rDNA replication origins. Cotransformation using these two vector types is also possible: Electroporated cells are selected for drug resistance conferred by the replicating vector for 10-15 generations, then screened for the gene replacement using another drug-resistant marker. Usually a few percent of the cells that are transformed by the replicating vector are cotransformed by the gene replacement vector.

Construction of ivermectin producer by domain swaps of avermectin polyketide synthase in Streptomyces avermitilis

Ivermectin, 22, 23-dihydroavermectin B1, is commercially important in human, veterinary medicine, and pesticides. It is currently synthesized by chemical reduction of the double bond between C22 and C23 of avermectins B1, which are a mixture of B1a (>80%) and B1b (<20%) produced by fermentation of Streptomyces avermitilis. The cost of ivermectin is much higher than that of avermectins B1 owing to the necessity of region-specific hydrogenation at C22-C23 of avermectins B1 with rhodium chloride as the catalyst for producing ivermectin. Here we report that ivermectin can be produced directly by fermentation of recombinant strains constructed through targeted genetic engineering of the avermectin polyketide synthase (PKS) in S. avermitilis Olm73-12, which produces only avermectins B and not avermectins A and oligomycin. The DNA region encoding the dehydratase (DH) and ketoreductase (KR) domains of module 2 from the avermectin PKS in S. avermitilis Olm73-12 was replaced by the DNA fragment encoding the DH, enoylreductase, and KR domains from module 4 of the pikromycin PKS of Streptomyces venezuelae ATCC 15439 using a gene replacement vector pXL211. Twenty-seven of mutants were found to produce a small amount of 22, 23-dihydroavermectin B1a and avermectin B1a and B2a by high performance liquid chromatography and liquid chromatography mass spectrometry analysis. This study might provide a route to the low-cost production of ivermectin by fermentation.

A combined gate replacement and input vector control approach for leakage current reduction

Input vector control (IVC) is a popular technique for leakage power reduction. It utilizes the transistor stack effect in CMOS gates by applying a minimum leakage vector (MLV) to the primary inputs of combinational circuits during the standby mode. However, the IVC technique becomes less effective for circuits of large logic depth because the input vector at primary inputs has little impact on leakage of internal gates at high logic levels. In this paper, we propose a technique to overcome this limitation by replacing those internal gates in their worst leakage states by other library gates while maintaining the circuit's correct functionality during the active mode. This modification of the circuit does not require changes of the design flow, but it opens the door for further leakage reduction when the MLV is not effective. We then present a divide-and-conquer approach that integrates gate replacement, an optimal MLV searching algorithm for tree circuits, and a genetic algorithm to connect the tree circuits. Our experimental results on all the MCNC91 benchmark circuits reveal that 1) the gate replacement technique alone can achieve 10% leakage current reduction over the best known IVC methods with no delay penalty and little area increase; 2) the divide-and-conquer approach outperforms the best pure IVC method by 24% and the existing control point insertion method by 12%; and 3) compared with the leakage achieved by optimal MLV in small circuits, the gate replacement heuristic and the divide-and-conquer approach can reduce on average 13% and 17% leakage, respectively.

Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens.

In the moss Physcomitrella patens, transforming DNA containing homologous sequences integrates predominantly by homologous recombination with its genomic target. A systematic investigation of the parameters that determine gene targeting efficiency shows a direct relationship between homology length and targeting frequency for replacement vectors (a selectable marker flanked by homologous DNA). Overall homology of only 1 kb is sufficient to achieve a 50% yield of targeted transformants. Targeting may occur through homologous recombination in one arm, accompanied by non-homologous end-joining by the other arm of the vector, or by allele replacement following two homologous recombination events. Allele replacement frequency depends on the symmetry of the targeting vector, being proportional to the length of the shorter arm. Allele replacement may involve insertion of multiple copies of the transforming DNA, accompanied by ectopic insertions at non-homologous sites. Single-copy and single insertions at targeted loci (targeted gene replacements, ‘TGR’) occur with a frequency of 7–20% of all transformants when the minimum requirements for allele replacement are met. Homologous recombination in Physcomitrella is substantially more efficient than in any multicellular eukaryote, recommending it as the outstanding model for the study of homologous recombination in plants.

An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

BackgroundTraditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process.ResultsWe describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.ConclusionWhile maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.

Vectors that facilitate the replacement of transcriptional lacZ fusions in Streptococcus mutans and Bacillus subtilis with fusions to gfp or gusA

Plasmid vectors have been constructed for Streptococcus mutans and Bacillus subtilis that make possible rapid replacement of the widely used reporter gene lacZ (encoding β-galactosidase) with either gfp (encoding green fluorescent protein) or gusA (encoding β-glucuronidase). The lacZ → gfp replacement vectors greatly facilitate the analysis of the spatial location of gene expression in biofilms of S. mutans and in sporulating B. subtilis. The lacZ → gusA replacement vectors facilitate the comparison of two promoters within the same organism. A vector is also described that enables gusA to be replaced with gfp in B. subtilis.

Post-entrapment genome engineering: First exon size does not affect the expression of fusion transcripts generated by gene entrapment

Gene trap mutagenesis in mouse embryonic stem cells has been widely used for genome-wide studies of mammalian gene function. However, while large numbers of genes can be disrupted, individual mutations may suffer from limitations due to the structure and/or placement of targeting vector. To extend the utility of gene trap mutagenesis, replaceable 3' [or poly(A)] gene trap vectors were developed that permit sequences inserted in individual entrapment clones to be engineered by Cre-mediated recombination. 3' traps incorporating different drug resistance genes could be readily exchanged, simply by selecting for the drug-resistance gene of the replacement vector. By substituting different 3' traps, we show that otherwise identical fusion genes containing a large first exon (804 nt) are not expressed at appreciably lower levels than genes expressing small first exons (384 and 151 nt). Thus, size appears to have less effect on the expression and processing of first exons than has been reported for internal exons. Finally, a retroviral poly(A) trap (consisting of a RNA polymerase II promoter, a neomycin-resistance gene, and 5'-splice site) typically produced mutagenized clones in which vector sequences spliced to the 3'-terminal exons of cellular transcription units, suggesting strong selection for fusion transcripts that evade nonsense-mediated decay. The efficient exchange of poly(A) traps should greatly extend the utility of mutant libraries generated by gene entrapment and provides new strategies to study the rules that govern the expression of exons inserted throughout the genome.

Transposon-mediated generation of targeting vectors for the production of gene knockouts

Vectors used for gene targeting experiments usually consist of a selectable marker flanked by two regions of homology to the targeted gene. In a homologous recombination event, the selectable marker replaces an essential element of the target gene rendering it inactive. Other applications of gene targeting technology include gene replacement (knockins) and conditional vectors which allow for the generation of inducible or tissue-specific gene-targeting events. The assembly of gene-targeting vectors is generally a laborious process requiring considerable technical skill. The procedures presented here report the application of transposons as tools for the construction of targeting vectors. Two mini-Mu transposons were sequentially inserted by in vitro transposition at each side of the region targeted for deletion. One such transposon carries an antibiotic resistance marker suitable for selection in mammalian cells. A deletion is then generated between the two transposons either by LoxP-induced recombination or by restriction digestion followed by ligation. This deletion removes part of both transposons plus the targeted region in between, leaving a transposon carrying the selectable marker flanked by two arms which are homologous to the targeted gene. Targeting vectors constructed using these transposons were electroporated into embryonic stem cells and shown to be effective in gene-targeting events.

Instability of Chimaeric Antibody Secretion by Anti-Carcinoembryonic Antigen Producing Hybridoma Cells after Gene Targeting

Objective: To produce a chimaeric version of the 11-285-14 anti-carcinoembryonic antigen (CEA) monoclonal antibody using a gene targeting approach. Materials and Methods: A replacement vector was constructed to insert the human constant γ1 gene within the mouse heavy chain locus of 11-285-14 hybridoma cells. The mouse constant γ1 gene (1.5 kb) and the mouse µ intron fragment (2.2 kb) were amplified by PCR and cloned into a pKO Scrambler vector. The human constant γ1 gene fragment (2.2 kb) was cloned next to the intron fragment. Resistant colonies were screened by ELISA for the presence of the human isotype in their supernatants. Results: Of the 4,370 resistant colonies obtained, 87 colonies showed secretion of the human isotype at levels between 4 and 32 ng/ml. PCR and Southern blot results confirmed the correct integration of the human gene by homologous recombination within the heavy chain locus. Most of the producers ceased to express the human isotype within a few weeks after the initial positive ELISA results. Instability of secretion could not be explained by genetic instability in all the clones, which suggests the presence of other undefined epigenetic or physiologic mechanisms. Conclusion: Gene targeting resulted in transformants with unstable and low production rates of chimaeric anti-CEA antibody.

Improving gene replacement by intracellular formation of linear homologous DNA

Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids. Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target. Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease. Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.

Cancer Gene Therapy through Autonomous Parvovirus - Mediated Gene Transfer

Parvoviruses are small nuclear replicating DNA viruses. The rodent parvoviruses are usually weakly pathogenic in adult animals, bind to cell surface receptors which are fairly ubiquitously expressed on cells, and do not appear to integrate into host chromosomes during either lytic or persistent infection. The closely related rodent parvoviruses MVM, H-1 and LuIII efficiently infect human cell lines. Most interesting, malignant transformation of human and rodent cells was often found to correlate with a greater susceptibility to parvovirus-induced killing (oncolysis) and with an increase in the cellular capacity for amplifying and / or expressing the incoming parvoviral DNA. These and other interesting properties make these autonomous rodent parvoviruses and recombinant derivatives promising candidate antitumor vectors. Capsid replacement vectors have been produced from MVM or H-1 virus that carry transgenes encoding either therapeutic products (cytokines/chemokines, Apoptin, herpes simplex virus thymidine kinase) or marker proteins (green fluorescent protein, chloramphenicolacetyl transferase, luciferase). This review describes the current state of the art regarding the potential application of wild-type parvoviruses and derived vectors for the treatment of cancer. In particular, recent successes with the development of replication-competent virus-free vector stocks are discussed and results from pre-clinical studies using recombinant parvoviruses transducing various cytokines/chemokines are presented.