High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes
- PMID: 22138824
- DOI: 10.1038/nmeth.1803
High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes
Abstract
Whereas bacterial artificial chromosomes (BACs) offer many advantages in studies of gene and protein function, generation of seamless, precisely mutated BACs has been difficult. Here we describe a counterselection-based recombineering method and its accompanying reagents. After identifying intramolecular recombination as the major problem in counterselection, we built a strategy to reduce these unwanted events by expressing Redβ alone at the crucial step. We enhanced this method by using phosphothioated oligonucleotides, using a sequence-altered rpsL counterselection gene and developing online software for oligonucleotide design. We illustrated this method by generating transgenic mammalian cell lines carrying small interfering RNA-resistant and point-mutated BAC transgenes. Using this approach, we generated mutated TACC3 transgenes to identify phosphorylation-specific spindle defects after knockdown of endogenous TACC3 expression. Our results highlight the complementary use of precisely mutated BAC transgenes and RNA interference in the study of cell biology at physiological expression levels and regulation.
Similar articles
-
Recombineering-based procedure for creating BAC transgene constructs for animals and cell lines.Curr Protoc Mol Biol. 2011 Jul;Chapter 23:Unit 23.14. doi: 10.1002/0471142727.mb2314s95. Curr Protoc Mol Biol. 2011. PMID: 21732318 Free PMC article.
-
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. Nat Methods. 2014. PMID: 25028895 Free PMC article.
-
Improved seamless mutagenesis by recombineering using ccdB for counterselection.Nucleic Acids Res. 2014 Mar;42(5):e37. doi: 10.1093/nar/gkt1339. Epub 2013 Dec 24. Nucleic Acids Res. 2014. PMID: 24369425 Free PMC article.
-
An overview on the generation of BAC transgenic mice for neuroscience research.Curr Protoc Neurosci. 2005 May;Chapter 5:Unit 5.20. doi: 10.1002/0471142301.ns0520s31. Curr Protoc Neurosci. 2005. PMID: 18428622 Review.
-
Recombineering BAC transgenes for protein tagging.Methods. 2011 Feb;53(2):113-9. doi: 10.1016/j.ymeth.2010.09.003. Epub 2010 Sep 22. Methods. 2011. PMID: 20868752 Review.
Cited by
-
Proof of ssDNA degraded from dsDNA for ET recombination.Biochem Biophys Rep. 2024 Jun 18;39:101750. doi: 10.1016/j.bbrep.2024.101750. eCollection 2024 Sep. Biochem Biophys Rep. 2024. PMID: 39035021 Free PMC article.
-
Recombineering: Genetic Engineering in Escherichia coli Using Homologous Recombination.Curr Protoc. 2023 Feb;3(2):e656. doi: 10.1002/cpz1.656. Curr Protoc. 2023. PMID: 36779782 Free PMC article.
-
Direct cloning of a herpesvirus genome for rapid generation of infectious BAC clones.J Adv Res. 2023 Jan;43:97-107. doi: 10.1016/j.jare.2022.02.012. Epub 2022 Feb 23. J Adv Res. 2023. PMID: 36585118 Free PMC article.
-
Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.Nucleic Acids Res. 2022 Aug 26;50(15):8947-8960. doi: 10.1093/nar/gkac649. Nucleic Acids Res. 2022. PMID: 35920321 Free PMC article.
-
BAC Recombineering and Transgenesis to Study Cell Polarity and Polarized Tissue Morphogenesis in Mice.Methods Mol Biol. 2022;2438:197-216. doi: 10.1007/978-1-0716-2035-9_13. Methods Mol Biol. 2022. PMID: 35147944 Free PMC article.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources