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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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1% Agarose gel electrophoresis of ORF targeted amplification reactions in WT Toledo BAC (WT), Region IV Deletion Mutant BAC (IV-Δ), and Region IV Rescued BAC (IV-R). 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 151 (~1100 kb) with UL 151 F and R primers. Lanes 8–10: amplification of UL 148 (~1100 bases) with UL 148 F and R primers (positive control outside of region for deletion).
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fig6: 1% Agarose gel electrophoresis of ORF targeted amplification reactions in WT Toledo BAC (WT), Region IV Deletion Mutant BAC (IV-Δ), and Region IV Rescued BAC (IV-R). 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 151 (~1100 kb) with UL 151 F and R primers. Lanes 8–10: amplification of UL 148 (~1100 bases) with UL 148 F and R primers (positive control outside of region for deletion).

Mentions: Region IV was captured into a rescue vector, pUC19, along with an extra 150 bp sequences at each end that were used as homology arms. To capture the region IV DNA fragment, the rescue plasmid was first linearized by BamHI digestion and amplified using primers containing homology flanking region IV (Table 1). Since the large size of the region being captured did not possess adequate restriction enzyme sites for digestion, unique restriction sites for AscI (upstream) and FseI (downstream) were added to the ends of the region via these primers. The PCR product with the homology arms was purified and 300 ng of the purified PCR cassette was electroporated into DY380 carrying WT Toledo BAC with zeocin inserted into region IV. The transformed cells were grown overnight at 32°C on LB agar with zeocin and ampicillin. The colonies grown were picked from the LB agar plate and cultured overnight in 5 mL of LB with zeocin and ampicillin. Miniprep of pUC19-IV capture plasmid isolated and purified the DNA from the overnight culture using Qiagen's miniprep kit (Qiagen, CA). The captured region IV DNA fragment was confirmed by antibiotic selection and verified by PCR amplification of the first (UL132) ORF, the second to last (UL150) ORF, another ORF outside of region IV as a negative control (UL147), and zeocin resistance gene was verified as a positive control for capture (Figure 5). The plasmid was then digested with AscI and FseI restriction enzymes to separate the vector sequence from the region IV ROI. 500 ng of the purified digestion product (the rescue cassette) was transformed into DY380 carrying the Toledo IV-Δ mutant BAC by electroporation and the transformants were cultured on LB agar with zeocin and hygromycin (50 μg/mL). The resulting colonies were restreaked onto LB agar with either kanamycin or zeocin. The colonies that grew only on zeocin, and not on kanamycin plates, were used for miniprep. Region IV rescue (IV-R) BAC DNA was used for PCR verification along with WT and IV-Δ BACs (Figure 6). The IV-R BAC was also digested by EcoRI to check the integrity of the BAC DNA.


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 ORF targeted amplification reactions in WT Toledo BAC (WT), Region IV Deletion Mutant BAC (IV-Δ), and Region IV Rescued BAC (IV-R). 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 151 (~1100 kb) with UL 151 F and R primers. Lanes 8–10: amplification of UL 148 (~1100 bases) with UL 148 F and R primers (positive control outside of region for deletion).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: 1% Agarose gel electrophoresis of ORF targeted amplification reactions in WT Toledo BAC (WT), Region IV Deletion Mutant BAC (IV-Δ), and Region IV Rescued BAC (IV-R). 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 151 (~1100 kb) with UL 151 F and R primers. Lanes 8–10: amplification of UL 148 (~1100 bases) with UL 148 F and R primers (positive control outside of region for deletion).
Mentions: Region IV was captured into a rescue vector, pUC19, along with an extra 150 bp sequences at each end that were used as homology arms. To capture the region IV DNA fragment, the rescue plasmid was first linearized by BamHI digestion and amplified using primers containing homology flanking region IV (Table 1). Since the large size of the region being captured did not possess adequate restriction enzyme sites for digestion, unique restriction sites for AscI (upstream) and FseI (downstream) were added to the ends of the region via these primers. The PCR product with the homology arms was purified and 300 ng of the purified PCR cassette was electroporated into DY380 carrying WT Toledo BAC with zeocin inserted into region IV. The transformed cells were grown overnight at 32°C on LB agar with zeocin and ampicillin. The colonies grown were picked from the LB agar plate and cultured overnight in 5 mL of LB with zeocin and ampicillin. Miniprep of pUC19-IV capture plasmid isolated and purified the DNA from the overnight culture using Qiagen's miniprep kit (Qiagen, CA). The captured region IV DNA fragment was confirmed by antibiotic selection and verified by PCR amplification of the first (UL132) ORF, the second to last (UL150) ORF, another ORF outside of region IV as a negative control (UL147), and zeocin resistance gene was verified as a positive control for capture (Figure 5). The plasmid was then digested with AscI and FseI restriction enzymes to separate the vector sequence from the region IV ROI. 500 ng of the purified digestion product (the rescue cassette) was transformed into DY380 carrying the Toledo IV-Δ mutant BAC by electroporation and the transformants were cultured on LB agar with zeocin and hygromycin (50 μg/mL). The resulting colonies were restreaked onto LB agar with either kanamycin or zeocin. The colonies that grew only on zeocin, and not on kanamycin plates, were used for miniprep. Region IV rescue (IV-R) BAC DNA was used for PCR verification along with WT and IV-Δ BACs (Figure 6). The IV-R BAC was also digested by EcoRI to check the integrity of the BAC DNA.

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