Abstract
BackgroundSocial 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.ResultsWe 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0267-8) contains supplementary material, which is available to authorized users.
Highlights
Social amoeba, Dictyostelium discoideum, is a well-established model organism for studying cellular physiology and developmental pattern formation
Robustness of our strategy was demonstrated by multiple labelling of a gene with three different fluorescent proteins. These results suggest that our simple strategy would facilitate genomic manipulations which had previously been hampered by the inability to clone DNA fragments with biased A/T-contents for a variety of model organisms
A universal knockin module was independently prepared as pUniv_CKI_mEGFP (Fig. 1a and Additional file 2: Figure S1). It contains cDNA encoding mEGFP followed by a generic terminator from myosin heavy chain A (MHC), which allows the expression of a green fluorescent protein (GFP)-fusion protein just after the knockin event
Summary
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. 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. The eukaryote Dictyostelium discoideum (D. discoideum), called social amoeba, is an excellent model to study the principles of unicellular physiology and multicellular development. Homologous recombination has been effective for introducing site-specific mutations, or for the targeted disruption of genes of interest (GOI). As with other model organisms, gene targeting has been performed by introducing a DNA cassette consisting of a selection. In spite of potential applicability, successful generation of knockin strains has been limited to a few loci, while knockout strains have been routinely generated [6,7,8,9,10,11,12]
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