Isolation of rare recombinants without using selectable markers for one-step seamless BACmutagenesis

George T. Lyozin, Paul C. Bressloff, Amit Kumar, Yasuhiro Kosaka, Bradley L. Demarest, H. Joseph Yost, Michael R. Kuehn, Luca Brunelli

Research output: Contribution to journalArticle

5 Scopus citations

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 s, and we developed a web-based calculator to optimize FPE.

Original languageEnglish (US)
Pages (from-to)966-970
Number of pages5
JournalNature Methods
Volume11
Issue number9
DOIs
StatePublished - Jan 1 2014

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ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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