title: Reliable handling of highly A/T-rich genomic DNA for efficient generation of knockin strains of Dictyostelium discoideum.
abstract: BACKGROUND
  Social amoeba, Dictyostelium discoideum, is a well-established model organism for studying cellular physiology and developmental pattern formation. Its haploid genome facilitates functional analysis of genes by a single round of mutagenesis including targeted disruption. Although the efficient generation of knockout strains based on an intrinsically high homologous recombination rate has been demonstrated, successful reports for knockin strains have been limited. As social amoeba has an exceptionally high adenine and thymine (A/T)-content, conventional plasmid-based vector construction has been constrained due to deleterious deletion in E. coli.
  
  
  RESULTS
  We describe here a simple and efficient strategy to construct GFP-knockin cassettes by using a linear DNA cloning vector derived from N15 bacteriophage. This allows reliable handling of DNA fragments whose A/T-content may be as high as 85 %, and which cannot be cloned into a circular plasmid. By optimizing the length of recombination arms, we successfully generate GFP-knockin strains for five genes involved in cAMP signalling, including a triple-colour knockin strain.
  
  
  CONCLUSIONS
  This robust strategy would be useful in handling DNA fragments with biased A/T-contents such as the genome of lower organisms and the promoter/terminator regions of higher organisms.
authors.affiliation: Division of Bioimaging, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima City, Tokushima, 770-8503, Japan.
authors.forename: Asuka
authors.initials: A
authors.lastname: Mukai
authors.affiliation: Division of Bioimaging, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima City, Tokushima, 770-8503, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Ichiraku
authors.affiliation: Division of Bioimaging, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima City, Tokushima, 770-8503, Japan. horikawa.kazuki@tokushima-u.ac.jp.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Horikawa
mesh_terms: D020856:AT Rich Sequence / Q000235:genetics*; D000225:Adenine; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics*; D055879:Gene Knock-In Techniques / Q000379:methods*; D016678:Genome / Q000235:genetics*; D013941:Thymine
chemical_list: D016054:DNA, Protozoan; D000225:Adenine; D013941:Thymine
keywords: A/T-rich genome; Dictyostelium discoideum; Knockin; Linear DNA cloning