Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 25028895.
- Also identified by DOI 10.1038/nmeth.3030 and PMC identifier 4149595.
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Abstract
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.
Medical subject headings
- Chromosomes, Artificial, Bacterial
- Mutagenesis, Site-Directed
- Protein Engineering
- Recombinant Proteins