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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.


Related in: MedlinePlus

Representative analysis of a 96-well CoSBR experiment. (A) 1.5% agarose gel electrophoresis of PCR products generated using primers flanking the 5′ loxP insertion site in a Cdh11 BAC (RP23–60C23). Wild-type amplicon is 201 bp, and the loxP containing amplicon is 235 bp. White asterisks denote five wells chosen for subsequent retrieval. (B) Repeated PCR analysis of the five chosen wells from (A) to verify presence of loxP site. G12: this well had no bacterial growth. H12: no inoculation (neg: negative control). wt: wild-type, un-modified BAC. pos: plasmid positive control with loxP site (final CKO vector).
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Figure 5: Representative analysis of a 96-well CoSBR experiment. (A) 1.5% agarose gel electrophoresis of PCR products generated using primers flanking the 5′ loxP insertion site in a Cdh11 BAC (RP23–60C23). Wild-type amplicon is 201 bp, and the loxP containing amplicon is 235 bp. White asterisks denote five wells chosen for subsequent retrieval. (B) Repeated PCR analysis of the five chosen wells from (A) to verify presence of loxP site. G12: this well had no bacterial growth. H12: no inoculation (neg: negative control). wt: wild-type, un-modified BAC. pos: plasmid positive control with loxP site (final CKO vector).

Mentions: Based on the assumption of 1–4 kanR founder bacteria per well and data from our initial experiments, we developed and tested a more streamlined CoSBR protocol for generation of CKO vectors (outlined in Figure 4). Our 96-well PCR screening strategy uses a small amplicon with primers located outside of the loxP oligo homology arms so that the increase in amplicon size resulting from the extra 34 bp from the loxP site can be easily used to distinguish the two types of kanR BACs. After obtaining the synthesized fragments (starting plasmid for the targeting cassette, loxP oligo and retrieval vector), the CoSBR protocol can be completed in 7 days or less with minimal hands-on time. We applied CoSBR to generate two additional CKO vectors using 2–3 BACs per gene along with the corresponding lagging-strand oligo. The data for both genes is summarized in Table 2, and the result from screening a 96-well plate from one of these projects is shown in Figure 5A (Cdh11, RP23–60C23). The oligos used for CoSBR are 200 bp and a fraction of these oligos will most likely contain sequence errors. To make sure we are generating a CKO vector completely without any errors either in the loxP sequence or in the sequence flanking the loxP site and introduced by the oligo, for each experiment we combined five loxP-positive wells for subsequent retrieval/gap-repair. To verify that all five wells picked indeed contain bacteria with the loxP-modified BAC, the PCR was repeated (Figure 5B). By combining bacteria from five individual wells, the modified genomic fragment is retrieved from several independently targeted BACs, increasing the likelihood of identifying an error-free vector to be used for ES cell targeting. 36% of the wells from the RP23–60C23 experiment (Figure 5) were loxP positive (Table 2). After retrieval, 6/21 plasmids (29%) contained the loxP site and the remaining plasmids contained the neo cassette only. This suggested an average loxP:neo-only ratio of 1:2 in the five wells combined for retrieval. Of these six loxP positive plasmids, all contained an error-free loxP site. In the other Cdh11 experiment 2/95 wells were positive. Since identical neo cassettes were used in these two experiments and the only difference is the orientation of the oligo and the BAC genomic insert, it is possible that for this gene one orientation is favored over the other. Retrieval was not done from the second experiment. Data for the other gene, S100A8, is also summarized in Table 2. In one experiment 60/95 (63%) of the wells were loxP-positive, 6/17 (35%) of the retrievals contained the loxP site and 5/6 had an error-free loxP sequence. For the other S100A8 experiment two BACs with the same back-bone orientation were combined and 15/95 (54%) of the wells were loxP positive. Retrieval was not done from the second experiment.


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

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

Representative analysis of a 96-well CoSBR experiment. (A) 1.5% agarose gel electrophoresis of PCR products generated using primers flanking the 5′ loxP insertion site in a Cdh11 BAC (RP23–60C23). Wild-type amplicon is 201 bp, and the loxP containing amplicon is 235 bp. White asterisks denote five wells chosen for subsequent retrieval. (B) Repeated PCR analysis of the five chosen wells from (A) to verify presence of loxP site. G12: this well had no bacterial growth. H12: no inoculation (neg: negative control). wt: wild-type, un-modified BAC. pos: plasmid positive control with loxP site (final CKO vector).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Representative analysis of a 96-well CoSBR experiment. (A) 1.5% agarose gel electrophoresis of PCR products generated using primers flanking the 5′ loxP insertion site in a Cdh11 BAC (RP23–60C23). Wild-type amplicon is 201 bp, and the loxP containing amplicon is 235 bp. White asterisks denote five wells chosen for subsequent retrieval. (B) Repeated PCR analysis of the five chosen wells from (A) to verify presence of loxP site. G12: this well had no bacterial growth. H12: no inoculation (neg: negative control). wt: wild-type, un-modified BAC. pos: plasmid positive control with loxP site (final CKO vector).
Mentions: Based on the assumption of 1–4 kanR founder bacteria per well and data from our initial experiments, we developed and tested a more streamlined CoSBR protocol for generation of CKO vectors (outlined in Figure 4). Our 96-well PCR screening strategy uses a small amplicon with primers located outside of the loxP oligo homology arms so that the increase in amplicon size resulting from the extra 34 bp from the loxP site can be easily used to distinguish the two types of kanR BACs. After obtaining the synthesized fragments (starting plasmid for the targeting cassette, loxP oligo and retrieval vector), the CoSBR protocol can be completed in 7 days or less with minimal hands-on time. We applied CoSBR to generate two additional CKO vectors using 2–3 BACs per gene along with the corresponding lagging-strand oligo. The data for both genes is summarized in Table 2, and the result from screening a 96-well plate from one of these projects is shown in Figure 5A (Cdh11, RP23–60C23). The oligos used for CoSBR are 200 bp and a fraction of these oligos will most likely contain sequence errors. To make sure we are generating a CKO vector completely without any errors either in the loxP sequence or in the sequence flanking the loxP site and introduced by the oligo, for each experiment we combined five loxP-positive wells for subsequent retrieval/gap-repair. To verify that all five wells picked indeed contain bacteria with the loxP-modified BAC, the PCR was repeated (Figure 5B). By combining bacteria from five individual wells, the modified genomic fragment is retrieved from several independently targeted BACs, increasing the likelihood of identifying an error-free vector to be used for ES cell targeting. 36% of the wells from the RP23–60C23 experiment (Figure 5) were loxP positive (Table 2). After retrieval, 6/21 plasmids (29%) contained the loxP site and the remaining plasmids contained the neo cassette only. This suggested an average loxP:neo-only ratio of 1:2 in the five wells combined for retrieval. Of these six loxP positive plasmids, all contained an error-free loxP site. In the other Cdh11 experiment 2/95 wells were positive. Since identical neo cassettes were used in these two experiments and the only difference is the orientation of the oligo and the BAC genomic insert, it is possible that for this gene one orientation is favored over the other. Retrieval was not done from the second experiment. Data for the other gene, S100A8, is also summarized in Table 2. In one experiment 60/95 (63%) of the wells were loxP-positive, 6/17 (35%) of the retrievals contained the loxP site and 5/6 had an error-free loxP sequence. For the other S100A8 experiment two BACs with the same back-bone orientation were combined and 15/95 (54%) of the wells were loxP positive. Retrieval was not done from the second 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.


Related in: MedlinePlus