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

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

Research output: Contribution to journalArticle

3 Citations (Scopus)

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

Fingerprint

Bacterial Artificial Chromosomes
Chromosomes
Mutagenesis
Nodal Protein
P1 Bacteriophage Artificial Chromosomes
Bacteriophage P1
Bacteriophages
Statistical methods
Polymerase Chain Reaction
Proteins

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Isolation of rare recombinants without using selectable markers for one-step seamless BACmutagenesis. / Lezin, George T; Bressloff, Paul C.; Kumar, Amit; Kosaka, Yasuhiro; Demarest, Bradley L.; Yost, H. Joseph; Kuehn, Michael R.; Brunelli, Luca.

In: Nature Methods, Vol. 11, No. 9, 01.01.2014, p. 966-970.

Research output: Contribution to journalArticle

Lezin, George T ; Bressloff, Paul C. ; Kumar, Amit ; Kosaka, Yasuhiro ; Demarest, Bradley L. ; Yost, H. Joseph ; Kuehn, Michael R. ; Brunelli, Luca. / Isolation of rare recombinants without using selectable markers for one-step seamless BACmutagenesis. In: Nature Methods. 2014 ; Vol. 11, No. 9. pp. 966-970.
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