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Use of recombination-mediated genetic engineering for construction of rescue human cytomegalovirus bacterial artificial chromosome clones.

Dulal K, Silver B, Zhu H - J. Biomed. Biotechnol. (2012)

Bottom Line: Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones.Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions.This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, UMDNJ-NJ Medical School, Newark, New Jersey 07101-1709, USA.

ABSTRACT
Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

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Related in: MedlinePlus

1% Agarose gel electrophoresis of targeted amplification reactions in WT Toledo BAC (WT) and two pUC-19-IV capture plasmids. Lane 1: 1 kb Plus DNA Ladder. Lanes 2–4: amplification of UL 132 (~850 bases) with UL 132 F and R primers. Lanes 5–7: amplification of UL 150 (~1.9 kb) with UL 150 F and R primers. Lanes 8–10: amplification of UL 147 (negative control, not within region IV) (~500 bases) with UL 147 F and R primers. Lanes 11–13: amplification of zeocin marker (positive control for capture only) (~550 bases) with Zeocin F and R primers.
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fig5: 1% Agarose gel electrophoresis of targeted amplification reactions in WT Toledo BAC (WT) and two pUC-19-IV capture plasmids. Lane 1: 1 kb Plus DNA Ladder. Lanes 2–4: amplification of UL 132 (~850 bases) with UL 132 F and R primers. Lanes 5–7: amplification of UL 150 (~1.9 kb) with UL 150 F and R primers. Lanes 8–10: amplification of UL 147 (negative control, not within region IV) (~500 bases) with UL 147 F and R primers. Lanes 11–13: amplification of zeocin marker (positive control for capture only) (~550 bases) with Zeocin F and R primers.

Mentions: Region IV was cloned into a linearized rescue vector (pUC19) with flanking homology arms, as previously described by gene capture, and the captured fragment was then put back into the region IV-Δ BAC from the rescue vector. In order to facilitate screening of rescue clones, a zeocin resistance gene flanked with loxP sites was inserted between ORF UL149 and UL130 region, which is included in the region IV ROI, by homologous recombination. The zeocin resistance gene, along with the flanking loxP sites, was amplified from pGEM-lox-zeo plasmid by PCR. The primers contained 40-bp homology flanking the BAC DNA locus where the gene was inserted (Table 1). The PCR product was treated with DpnI, as mentioned before, to remove the template plasmid and then gel purified using Qiagen's gel purification kit (Qiagen Company, CA). ~300 ng of the purified PCR product was electroporated into recombination-activated electrocompetent DY380 cells harboring WT Toledo BAC. The colonies were grown on LB agar with zeocin (50 μg/mL) and the insertion of the zeocin resistance gene was confirmed by PCR (Figure 5).


Use of recombination-mediated genetic engineering for construction of rescue human cytomegalovirus bacterial artificial chromosome clones.

Dulal K, Silver B, Zhu H - J. Biomed. Biotechnol. (2012)

1% Agarose gel electrophoresis of targeted amplification reactions in WT Toledo BAC (WT) and two pUC-19-IV capture plasmids. Lane 1: 1 kb Plus DNA Ladder. Lanes 2–4: amplification of UL 132 (~850 bases) with UL 132 F and R primers. Lanes 5–7: amplification of UL 150 (~1.9 kb) with UL 150 F and R primers. Lanes 8–10: amplification of UL 147 (negative control, not within region IV) (~500 bases) with UL 147 F and R primers. Lanes 11–13: amplification of zeocin marker (positive control for capture only) (~550 bases) with Zeocin F and R primers.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303630&req=5

fig5: 1% Agarose gel electrophoresis of targeted amplification reactions in WT Toledo BAC (WT) and two pUC-19-IV capture plasmids. Lane 1: 1 kb Plus DNA Ladder. Lanes 2–4: amplification of UL 132 (~850 bases) with UL 132 F and R primers. Lanes 5–7: amplification of UL 150 (~1.9 kb) with UL 150 F and R primers. Lanes 8–10: amplification of UL 147 (negative control, not within region IV) (~500 bases) with UL 147 F and R primers. Lanes 11–13: amplification of zeocin marker (positive control for capture only) (~550 bases) with Zeocin F and R primers.
Mentions: Region IV was cloned into a linearized rescue vector (pUC19) with flanking homology arms, as previously described by gene capture, and the captured fragment was then put back into the region IV-Δ BAC from the rescue vector. In order to facilitate screening of rescue clones, a zeocin resistance gene flanked with loxP sites was inserted between ORF UL149 and UL130 region, which is included in the region IV ROI, by homologous recombination. The zeocin resistance gene, along with the flanking loxP sites, was amplified from pGEM-lox-zeo plasmid by PCR. The primers contained 40-bp homology flanking the BAC DNA locus where the gene was inserted (Table 1). The PCR product was treated with DpnI, as mentioned before, to remove the template plasmid and then gel purified using Qiagen's gel purification kit (Qiagen Company, CA). ~300 ng of the purified PCR product was electroporated into recombination-activated electrocompetent DY380 cells harboring WT Toledo BAC. The colonies were grown on LB agar with zeocin (50 μg/mL) and the insertion of the zeocin resistance gene was confirmed by PCR (Figure 5).

Bottom Line: Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones.Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions.This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, UMDNJ-NJ Medical School, Newark, New Jersey 07101-1709, USA.

ABSTRACT
Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

Show MeSH
Related in: MedlinePlus