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Efficient conditional knockout targeting vector construction using co-selection BAC recombineering (CoSBR).

Newman RJ, Roose-Girma M, Warming S - Nucleic Acids Res. (2015)

Bottom Line: A simple and efficient strategy for Bacterial Artificial Chromosome (BAC) recombineering based on co-selection is described.We show that it is possible to efficiently modify two positions of a BAC simultaneously by co-transformation of a single-stranded DNA oligo and a double-stranded selection cassette.The use of co-selection BAC recombineering reduces the DNA manipulation needed to make a conditional knockout gene targeting vector to only two steps: a single round of BAC modification followed by a retrieval step.

View Article: PubMed Central - PubMed

Affiliation: Genentech, Inc., Department of Molecular Biology, 1 DNA Way, South San Francisco, CA 94080, USA.

No MeSH data available.


Strategy for generating a BAC targeting neo cassette by gene synthesis and recombineering. (A) A small sequence is synthesized and cloned into a standard pUC plasmid. The insert contains a 5′ mini homology arm flanking the future insertion site of the selection cassette, an FRT site, homology to the 5′ end of the mouse Pgk1 promoter, homology to the 3′ end of the bovine growth hormone (BGH) polyadenylation cassette, another FRT site, a loxP site and a 3′ homology arm flanking the future cassette insertion site. An EcoRI-BamHI fragment from PL452 containing the entire dual eukaryotic/prokaryotic neo cassette is introduced into the target plasmid by recombineering. Precise integration into the plasmid is achieved by recombination with the synthesized Pgk1 and BGH pA homology fragments. (B) Representative BamHI restriction digest showing the band sizes before and after insertion of the selection marker by recombineering. The bands in lane 1 result from a digest of the target plasmid with synthesized insert, and lane 2 contains a digest of the modified plasmid. This plasmid is a mixture of un-targeted and recombined plasmid. The 0.5 and 2.2 kb fragment represents synthesized fragment only and size-shifted cassette containing the selection cassette flanked by homology for insertion into the BAC, respectively. neo: neomycin; Pgk1: Phosphoglycerate kinase 1; em7: artificial prokaryotic promoter; BGH pA: bovine growth hormone polyadenylation signal; ori: plasmid origin of replication; Amp: cassette encoding β-lactamase for resistance to ampicillin/carbenicillin. Maps are not drawn to scale.
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Figure 1: Strategy for generating a BAC targeting neo cassette by gene synthesis and recombineering. (A) A small sequence is synthesized and cloned into a standard pUC plasmid. The insert contains a 5′ mini homology arm flanking the future insertion site of the selection cassette, an FRT site, homology to the 5′ end of the mouse Pgk1 promoter, homology to the 3′ end of the bovine growth hormone (BGH) polyadenylation cassette, another FRT site, a loxP site and a 3′ homology arm flanking the future cassette insertion site. An EcoRI-BamHI fragment from PL452 containing the entire dual eukaryotic/prokaryotic neo cassette is introduced into the target plasmid by recombineering. Precise integration into the plasmid is achieved by recombination with the synthesized Pgk1 and BGH pA homology fragments. (B) Representative BamHI restriction digest showing the band sizes before and after insertion of the selection marker by recombineering. The bands in lane 1 result from a digest of the target plasmid with synthesized insert, and lane 2 contains a digest of the modified plasmid. This plasmid is a mixture of un-targeted and recombined plasmid. The 0.5 and 2.2 kb fragment represents synthesized fragment only and size-shifted cassette containing the selection cassette flanked by homology for insertion into the BAC, respectively. neo: neomycin; Pgk1: Phosphoglycerate kinase 1; em7: artificial prokaryotic promoter; BGH pA: bovine growth hormone polyadenylation signal; ori: plasmid origin of replication; Amp: cassette encoding β-lactamase for resistance to ampicillin/carbenicillin. Maps are not drawn to scale.

Mentions: To generate a selection cassette for dual E. coli/ES cell selection, for each gene a fragment with the following components was synthesized and inserted into pUC (Figure 1A): A recognition site for a restriction enzyme (e.g. BamHI), 100 bp 5′ mini homology arm matching the sequence immediately 5′ of the cassette insertion site, a 34 bp FRT site, 71 bp homology to the 5′ end of the mouse Pgk1 promoter, 60 bp homology to the 3′ end of the bovine growth hormone polyA sequence, a 34 bp FRT site, an 18 bp spacer, a 34 bp loxP site, a 100 bp 3′ mini homology arm matching the sequence immediately 3′ of the cassette insertion site and a recognition site for a restriction enzyme (identical to, or different from, the 5′ site). To prepare the full-length selection cassette, a 1.9 kb EcoRI-BamHI fragment was isolated from PL452 (5) and 10 ng was co-transformed with the modified pUC plasmid into pre-made heat-shocked and recombineering-ready SW102 cells (prepared as described below) using electroporation and the following conditions: 2.5 kV, 25 μF, 200 Ω. After 1 h of outgrowth in 1 ml LB, 100 μl and 200 μl were plated on agar plates containing 50 μg/ml kanamycin and incubated overnight at 32°C. The following day the plasmid was isolated by miniprep (Qiagen). The plasmid prep contains a combination of parent and modified plasmid and the size-shifted targeting cassette was isolated from the backbone using the appropriate enzyme (e.g. BamHI) and gel-purified (GFX kit, GE Healthcare). In most cases three bands are visible on the gel (Figure 1B): the pUC vector backbone, the synthesized and un-modified insert, and the desired 2 kb size-shifted selection cassette resulting from recombination of the Pgk1-em7-neo-BGHpA fragment with the cassette homology arms. The cassette was eluted in 10 mM Tris-HCL pH 8.5 and 1 ng was used in the subsequent CoSBR experiment.


Efficient conditional knockout targeting vector construction using co-selection BAC recombineering (CoSBR).

Newman RJ, Roose-Girma M, Warming S - Nucleic Acids Res. (2015)

Strategy for generating a BAC targeting neo cassette by gene synthesis and recombineering. (A) A small sequence is synthesized and cloned into a standard pUC plasmid. The insert contains a 5′ mini homology arm flanking the future insertion site of the selection cassette, an FRT site, homology to the 5′ end of the mouse Pgk1 promoter, homology to the 3′ end of the bovine growth hormone (BGH) polyadenylation cassette, another FRT site, a loxP site and a 3′ homology arm flanking the future cassette insertion site. An EcoRI-BamHI fragment from PL452 containing the entire dual eukaryotic/prokaryotic neo cassette is introduced into the target plasmid by recombineering. Precise integration into the plasmid is achieved by recombination with the synthesized Pgk1 and BGH pA homology fragments. (B) Representative BamHI restriction digest showing the band sizes before and after insertion of the selection marker by recombineering. The bands in lane 1 result from a digest of the target plasmid with synthesized insert, and lane 2 contains a digest of the modified plasmid. This plasmid is a mixture of un-targeted and recombined plasmid. The 0.5 and 2.2 kb fragment represents synthesized fragment only and size-shifted cassette containing the selection cassette flanked by homology for insertion into the BAC, respectively. neo: neomycin; Pgk1: Phosphoglycerate kinase 1; em7: artificial prokaryotic promoter; BGH pA: bovine growth hormone polyadenylation signal; ori: plasmid origin of replication; Amp: cassette encoding β-lactamase for resistance to ampicillin/carbenicillin. Maps are not drawn to scale.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4627060&req=5

Figure 1: Strategy for generating a BAC targeting neo cassette by gene synthesis and recombineering. (A) A small sequence is synthesized and cloned into a standard pUC plasmid. The insert contains a 5′ mini homology arm flanking the future insertion site of the selection cassette, an FRT site, homology to the 5′ end of the mouse Pgk1 promoter, homology to the 3′ end of the bovine growth hormone (BGH) polyadenylation cassette, another FRT site, a loxP site and a 3′ homology arm flanking the future cassette insertion site. An EcoRI-BamHI fragment from PL452 containing the entire dual eukaryotic/prokaryotic neo cassette is introduced into the target plasmid by recombineering. Precise integration into the plasmid is achieved by recombination with the synthesized Pgk1 and BGH pA homology fragments. (B) Representative BamHI restriction digest showing the band sizes before and after insertion of the selection marker by recombineering. The bands in lane 1 result from a digest of the target plasmid with synthesized insert, and lane 2 contains a digest of the modified plasmid. This plasmid is a mixture of un-targeted and recombined plasmid. The 0.5 and 2.2 kb fragment represents synthesized fragment only and size-shifted cassette containing the selection cassette flanked by homology for insertion into the BAC, respectively. neo: neomycin; Pgk1: Phosphoglycerate kinase 1; em7: artificial prokaryotic promoter; BGH pA: bovine growth hormone polyadenylation signal; ori: plasmid origin of replication; Amp: cassette encoding β-lactamase for resistance to ampicillin/carbenicillin. Maps are not drawn to scale.
Mentions: To generate a selection cassette for dual E. coli/ES cell selection, for each gene a fragment with the following components was synthesized and inserted into pUC (Figure 1A): A recognition site for a restriction enzyme (e.g. BamHI), 100 bp 5′ mini homology arm matching the sequence immediately 5′ of the cassette insertion site, a 34 bp FRT site, 71 bp homology to the 5′ end of the mouse Pgk1 promoter, 60 bp homology to the 3′ end of the bovine growth hormone polyA sequence, a 34 bp FRT site, an 18 bp spacer, a 34 bp loxP site, a 100 bp 3′ mini homology arm matching the sequence immediately 3′ of the cassette insertion site and a recognition site for a restriction enzyme (identical to, or different from, the 5′ site). To prepare the full-length selection cassette, a 1.9 kb EcoRI-BamHI fragment was isolated from PL452 (5) and 10 ng was co-transformed with the modified pUC plasmid into pre-made heat-shocked and recombineering-ready SW102 cells (prepared as described below) using electroporation and the following conditions: 2.5 kV, 25 μF, 200 Ω. After 1 h of outgrowth in 1 ml LB, 100 μl and 200 μl were plated on agar plates containing 50 μg/ml kanamycin and incubated overnight at 32°C. The following day the plasmid was isolated by miniprep (Qiagen). The plasmid prep contains a combination of parent and modified plasmid and the size-shifted targeting cassette was isolated from the backbone using the appropriate enzyme (e.g. BamHI) and gel-purified (GFX kit, GE Healthcare). In most cases three bands are visible on the gel (Figure 1B): the pUC vector backbone, the synthesized and un-modified insert, and the desired 2 kb size-shifted selection cassette resulting from recombination of the Pgk1-em7-neo-BGHpA fragment with the cassette homology arms. The cassette was eluted in 10 mM Tris-HCL pH 8.5 and 1 ng was used in the subsequent CoSBR experiment.

Bottom Line: A simple and efficient strategy for Bacterial Artificial Chromosome (BAC) recombineering based on co-selection is described.We show that it is possible to efficiently modify two positions of a BAC simultaneously by co-transformation of a single-stranded DNA oligo and a double-stranded selection cassette.The use of co-selection BAC recombineering reduces the DNA manipulation needed to make a conditional knockout gene targeting vector to only two steps: a single round of BAC modification followed by a retrieval step.

View Article: PubMed Central - PubMed

Affiliation: Genentech, Inc., Department of Molecular Biology, 1 DNA Way, South San Francisco, CA 94080, USA.

No MeSH data available.