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- W2022302103 abstract "Application of the founder principle from population genetics to variant selection after recombineering allows the isolation of rare unselected recombinants. Current methods to isolate rare (1:10,000–1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle–driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof of principle, we isolated 1:100,000 seamless fluorescent protein–modified Nodal BACs and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1 phage–derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated with <40 PCRs, and we developed a web-based calculator to optimize FPE." @default.
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- W2022302103 date "2014-07-13" @default.
- W2022302103 modified "2023-09-29" @default.
- W2022302103 title "Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis" @default.
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- W2022302103 doi "https://doi.org/10.1038/nmeth.3030" @default.
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