title: Recombineering: using drug cassettes to knock out genes in vivo.
abstract: A 'gene knockout' or 'knockout' is a mutation that inactivates a gene function. These mutations are very useful for classical genetic studies as well as for modern techniques including functional genomics. In the past, knockouts of bacterial genes were often made by transposon mutagenesis. In this case, laborious screens are required to find a knockout in the gene of interest. Knockouts of other organisms have traditionally been made by first using in vitro genetic engineering to modify genes contained on plasmids or bacterial artificial chromosomes (BACs) and later moving these modified constructs to the organism of interest by cell culture techniques. Other methods utilizing a combination of genetic engineering and in vivo homologous recombination were inefficient at best. Recombineering provides a new way to generate knockout mutations directly on the bacterial chromosome or to modify any plasmid or BAC in vivo as a prelude to making knockouts in other organisms. The constructs are designed to the base pair and are not dependent on suitable restriction sites. A drug cassette can be placed anywhere within a gene or the open reading frame of the gene can be replaced with the drug cassette. Either way, the desired construct is selected for.
authors.affiliation: Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA. Electronic address: sawitzkj@mail.nih.gov.
authors.forename: James A
authors.initials: JA
authors.lastname: Sawitzke
authors.forename: Lynn C
authors.initials: LC
authors.lastname: Thomason
authors.forename: Mikhail
authors.initials: M
authors.lastname: Bubunenko
authors.forename: Xintian
authors.initials: X
authors.lastname: Li
authors.forename: Nina
authors.initials: N
authors.lastname: Costantino
authors.forename: Donald L
authors.initials: DL
authors.lastname: Court
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D010582:Bacteriophage lambda / Q000187:drug effects / Q000235:genetics; D002876:Chromosomes, Bacterial; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000295:instrumentation / Q000379:methods*; D005818:Genetic Engineering / Q000295:instrumentation / Q000379:methods*; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D014764:Viral Proteins / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C065410:gam protein, Coliphage
keywords: Bacterial artificial chromosomes (BACs); Confirming knockout mutations; Design construct and order primers; Genes knock out; Linear substrate by polymerase chain reaction; Recombineering- ready cells; Selecting for knockout mutations