ArticleNature methods2011
High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes.
Article in Nature methods, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 36 papers.
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Who cites it
36 citing papers in PubMed.
- Proof of ssDNA degraded from dsDNA for ET recombination.Biochemistry and biophysics reports · 2024Article
- Accelerating Genetic Sensor Development, Scale-up, and Deployment Using Synthetic Biology.Biodesign research · 2024Article
- Recombineering: Genetic Engineering in Escherichia coli Using Homologous Recombination.Current protocols · 2023Article
- Direct cloning of a herpesvirus genome for rapid generation of infectious BAC clones.Journal of advanced research · 2023Article
- Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.Nucleic acids research · 2022Article
- Novel Group C Oncolytic Adenoviruses Carrying a miRNA Inhibitor Demonstrate Enhanced Oncolytic Activity In Vitro and In Vivo.Molecular cancer therapeutics · 2022Article
- BAC Recombineering and Transgenesis to Study Cell Polarity and Polarized Tissue Morphogenesis in Mice.Methods in molecular biology (Clifton, N.J.) · 2022Article
- ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes.Life science alliance · 2021Article
- Generation and characterization of a Meflin-CreERT2 transgenic line for lineage tracing in white adipose tissue.PloS one · 2021Article
- Improved bacterial recombineering by parallelized protein discovery.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- Clathrin's adaptor interaction sites are repurposed to stabilize microtubules during mitosis.The Journal of cell biology · 2020Article
- Genetic Engineering by DNA Recombineering.Current protocols in chemical biology · 2019Article
- Escherichia coli vectors having stringently repressible replication origins allow a streamlining of Crispr/Cas9 gene editing.Plasmid · 2019Article
- Plasmid-based gap-repair recombineered transgenes reveal a central role for introns in mutually exclusive alternative splicing in Down Syndrome Cell Adhesion Molecule exon 4.Nucleic acids research · 2019Article
- A dual role of the extracellular domain ofBiology open · 2018Article
- Article
- GTSE1 tunes microtubule stability for chromosome alignment and segregation by inhibiting the microtubule depolymerase MCAK.The Journal of cell biology · 2016Article
- High production of fatty alcohols in Escherichia coli with fatty acid starvation.Microbial cell factories · 2016Article
- λ Recombination and Recombineering.EcoSal Plus · 2016Review
- Interrogation ofSynthetic and systems biotechnology · 2016Article
Corrections and comments
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Authors and funding
8 authors.
Funding
No grant is acknowledged in the PubMed record.
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.
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