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

6 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 - 2014

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Isolation of rare recombinants without using selectable markers for one-step seamless BACmutagenesis'. Together they form a unique fingerprint.

  • Cite this

    Lyozin, G. T., Bressloff, P. C., Kumar, A., Kosaka, Y., Demarest, B. L., Yost, H. J., Kuehn, M. R., & Brunelli, L. (2014). Isolation of rare recombinants without using selectable markers for one-step seamless BACmutagenesis. Nature Methods, 11(9), 966-970. https://doi.org/10.1038/nmeth.3030