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

Nat Methods. 2014 Sep;11(9):966-970. doi: 10.1038/nmeth.3030. Epub 2014 Jul 13.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromosomes, Artificial, Bacterial / genetics*
  • Genetic Markers / genetics
  • Mice
  • Mutagenesis, Site-Directed / methods*
  • Protein Engineering / methods*
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / isolation & purification*


  • Genetic Markers
  • Recombinant Proteins