Bacterial Artificial Chromosome Recombineering Research Articles

Tn7-mediated introduction of DNA into bacmid-cloned pseudorabies virus genome for rapid construction of recombinant viruses

lacZα-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome (BAC) by homologous recombination in E. coli. The resulting recombinant BAC (pBeckerZF1) was confirmed by PCR and sequencing. Green fluorescent protein (GFP) gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect (CPE) observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3. vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.

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.

Evaluation of the Lytic Origins of Replication of Kaposi's Sarcoma-Associated Virus/Human Herpesvirus 8 in the Context of the Viral Genome

The lytic origins of DNA replication for human herpesvirus 8 (HHV8), oriLyt-L and oriLyt-R, are located between open reading frames K4.2 and K5 and ORF69 and vFLIP, respectively. These lytic origins were elucidated using a transient replication assay. Although this assay is a powerful tool for identifying many herpesvirus lytic origins, it is limited in its ability to evaluate the activity of replication origins in the context of the viral genome. To this end, we investigated the ability of a recombinant HHV8 bacterial artificial chromosome (BAC) to replicate in the absence of oriLyt-R, oriLyt-L, or both oriLyt regions. We generated the HHV8 BAC recombinants (BAC36-DeltaOri-R, BAC36-DeltaOri-L, and BAC36-DeltaOri-RL), which removed one or all of the identified lytic origins. An evaluation of these recombinant BACs revealed that oriLyt-L was sufficient to propagate the viral genome, whereas oriLyt-R alone failed to direct the amplification of viral DNA.

Promoter analysis in ES cell-derived neural cells

Neural cells derived from ES cells in cell culture (ESNCs) have many of the properties of normal neural cells and provide a model of “neurogenesis-in-a-dish.” Here we show that ESNCs provide a powerful system for analyzing neural gene transcription. ES cells are transfected with bacterial artificial chromosomes (BACs) containing Olig2, a gene with a key role in neural fate choice. One BAC is modified by recombineering to insert a reporter gene and a gene for selecting stably transfected clones. Another BAC contains a deletion of a suspected Olig2 promoter. Stable transgenic clones of ES cells are isolated, differentiated in culture, and the expression of transgenes is assayed. Differentiated cells dramatically up-regulate transgene expression and a deletion analysis reveals a basal promoter for Olig2. The combination of ESNCs and BAC recombineering will have broad application for analyzing gene transcription in the nervous system and will be applicable to human ES cells. The general approach should also be applicable to the many other cell lineages that can now be derived from mouse and human ES cells in culture.

DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.

Acid assisted consolidation of silver alloy powders : M.M.Wray et al. J. of Research of NIST, USA, vol 103, no 5, 1998, 511–516

Previously we have shown that insertion of a LacZ reporter gene into the Col10a1 gene in the context of a bacterial artificial chromosome (BAC) drives strong and specific expression of LacZ in hypertrophic cartilage of transgenic mice [Gebhard S., Hattori T., Bauer E., Bosl M.R., Schlund B., Poschl E., Adam N., de Crombrugghe B., von der Mark K., 2007 Histochem. Cell Biol. 19 127:183–194]. BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter. Here we report on the generation of Col10a1-specific Cre deleter mice using a BAC recombineering technique based on homologous recombination in E. coli. Sixteen BAC-Col10-Cre transgenic lines were generated containing between 1 and 5 copies of the BAC-Col10-Cre gene. All lines tested so far expressed Cre specifically in hypertrophic chondrocytes of E16.5 and P1 growth plates of long bones, ribs, vertebrae and sternum as examined by crossing with ROSA26 reporter mice. Cre activity was detected as early as E13.5 when hypertrophic cartilage develops in the diaphysis of femur and humerus. The data confirm that expression of Cre under the control of the complete BAC-Col10a1 promoter occurs with high efficiency and specificity in hypertrophic chondrocytes. The BAC-Col10-Cre lines should thus provide a valuable tool to get further insight into the role of genes involved in endochondral ossification by allowing their specific deletion in the hypertrophic zone of the growth plate.