title: Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis.
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 PCRs, and we developed a web-based calculator to optimize FPE.
authors.affiliation: Department of Pediatrics (Neonatology), The University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: George T
authors.initials: GT
authors.lastname: Lyozin
authors.affiliation: Department of Mathematics, The University of Utah, Salt Lake City, Utah, USA.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Bressloff
authors.affiliation: Laboratory of Protein Dynamics and Signaling, National Cancer Institute, Frederick, Maryland, USA.
authors.forename: Amit
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Pediatrics (Neonatology), The University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kosaka
authors.affiliation: Department of Neurobiology and Anatomy, and.
authors.forename: Bradley L
authors.initials: BL
authors.lastname: Demarest
authors.affiliation: Department of Neurobiology and Anatomy, and.|Department of Pediatrics, The University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: H Joseph
authors.initials: HJ
authors.lastname: Yost
authors.affiliation: Laboratory of Protein Dynamics and Signaling, National Cancer Institute, Frederick, Maryland, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kuehn
authors.affiliation: Department of Pediatrics (Neonatology), The University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: Luca
authors.initials: L
authors.lastname: Brunelli
mesh_terms: D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D005819:Genetic Markers; D011994:Recombinant Proteins