Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis.

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 PCR

SUBMITTER: Lyozin GT 

PROVIDER: S-EPMC4149595 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature