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

Nat Methods.. 2014-07; 
Lyozin GT, Bressloff PC, Kumar A, Kosaka Y, Demarest BL, Yost HJ, Kuehn MR, Brunelli L. Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah, USA.
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摘要

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. Statis... More

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