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ID 114375
Author
Mukai, Asuka Tokushima University
Ichiraku, Aya Tokushima University
Keywords
A/T-rich genome
Linear DNA cloning
Knockin
Dictyostelium discoideum
Content Type
Journal Article
Description
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.
Journal Title
BMC Biotechnology
ISSN
14726750
NCID
AA12034730
Publisher
BioMed Central|Springer Nature
Volume
16
Start Page
37
Published Date
2016-04-14
Rights
© 2016 Mukai et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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language
eng
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departments
Medical Sciences