Limits...
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.

Show MeSH

Related in: MedlinePlus

Gene Capture Method. (a) Linearized plasmid vector (b) is amplified by primers containing homology arms to the sequences flanking the region of interest (ROI) in Toledo to create the plasmid vector cassette (c). Electrocompetent and recombination-activated DY380 harboring WT Toledo BAC (d) are used for electroporation with the plasmid vector cassette (e). Upon homologous recombination and circularization, the ROI is captured, creating the ROI rescue plasmid (f).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3303630&req=5

fig2: Gene Capture Method. (a) Linearized plasmid vector (b) is amplified by primers containing homology arms to the sequences flanking the region of interest (ROI) in Toledo to create the plasmid vector cassette (c). Electrocompetent and recombination-activated DY380 harboring WT Toledo BAC (d) are used for electroporation with the plasmid vector cassette (e). Upon homologous recombination and circularization, the ROI is captured, creating the ROI rescue plasmid (f).

Mentions: When making rescue clones by this gene capture method using DY380 containing the wild-type virus BAC (Figure 2), first the region of interest (ROI) is deleted by the above-mentioned method in DY380, generating an ROI deletion mutant. Next, a linearized plasmid (a) containing a positive selection marker is amplified by PCR with primers containing homologous sequences flanking the ROI in the BAC (b). In order to facilitate screening of rescue clones, a gene providing resistance to an antibiotic different than the antibiotic markers in both the BAC and rescue plasmid, flanked with loxP sites, is inserted within the captured ROI by homologous recombination. The PCR-amplified cassette (c) is transformed into electrocompetent and recombination-activated DY380 harboring the virus BAC (d). By homologous recombination, the ROI will be captured into the plasmid due to gap repair cloning (e). These recombinant clones are selected using the drug resistance marker gene on the plasmid (Note: the plasmid antibiotic resistance should be separate than the antibiotic resistance of the BAC). This capture can be further verified by amplifying an open reading frame within the ROI by PCR and also by digesting the plasmid with restriction enzymes to check for the presence of the captured region.


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)

Gene Capture Method. (a) Linearized plasmid vector (b) is amplified by primers containing homology arms to the sequences flanking the region of interest (ROI) in Toledo to create the plasmid vector cassette (c). Electrocompetent and recombination-activated DY380 harboring WT Toledo BAC (d) are used for electroporation with the plasmid vector cassette (e). Upon homologous recombination and circularization, the ROI is captured, creating the ROI rescue plasmid (f).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Gene Capture Method. (a) Linearized plasmid vector (b) is amplified by primers containing homology arms to the sequences flanking the region of interest (ROI) in Toledo to create the plasmid vector cassette (c). Electrocompetent and recombination-activated DY380 harboring WT Toledo BAC (d) are used for electroporation with the plasmid vector cassette (e). Upon homologous recombination and circularization, the ROI is captured, creating the ROI rescue plasmid (f).
Mentions: When making rescue clones by this gene capture method using DY380 containing the wild-type virus BAC (Figure 2), first the region of interest (ROI) is deleted by the above-mentioned method in DY380, generating an ROI deletion mutant. Next, a linearized plasmid (a) containing a positive selection marker is amplified by PCR with primers containing homologous sequences flanking the ROI in the BAC (b). In order to facilitate screening of rescue clones, a gene providing resistance to an antibiotic different than the antibiotic markers in both the BAC and rescue plasmid, flanked with loxP sites, is inserted within the captured ROI by homologous recombination. The PCR-amplified cassette (c) is transformed into electrocompetent and recombination-activated DY380 harboring the virus BAC (d). By homologous recombination, the ROI will be captured into the plasmid due to gap repair cloning (e). These recombinant clones are selected using the drug resistance marker gene on the plasmid (Note: the plasmid antibiotic resistance should be separate than the antibiotic resistance of the BAC). This capture can be further verified by amplifying an open reading frame within the ROI by PCR and also by digesting the plasmid with restriction enzymes to check for the presence of the captured region.

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