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Reliable handling of highly A/T-rich genomic DNA for efficient generation of knockin strains of Dictyostelium discoideum

Overview of attention for article published in BMC Biotechnology, April 2016
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Title
Reliable handling of highly A/T-rich genomic DNA for efficient generation of knockin strains of Dictyostelium discoideum
Published in
BMC Biotechnology, April 2016
DOI 10.1186/s12896-016-0267-8
Pubmed ID
Authors

Asuka Mukai, Aya Ichiraku, Kazuki Horikawa

Abstract

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. 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. 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.

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Mendeley readers

Mendeley readers

The data shown below were compiled from readership statistics for 29 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
United States 1 3%
Portugal 1 3%
Italy 1 3%
Unknown 26 90%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 8 28%
Student > Master 5 17%
Researcher 5 17%
Student > Doctoral Student 2 7%
Student > Bachelor 1 3%
Other 2 7%
Unknown 6 21%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 11 38%
Agricultural and Biological Sciences 8 28%
Unspecified 1 3%
Immunology and Microbiology 1 3%
Unknown 8 28%
Attention Score in Context

Attention Score in Context

This research output has an Altmetric Attention Score of 2. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 23 November 2016.
All research outputs
#14,257,527
of 22,862,742 outputs
Outputs from BMC Biotechnology
#626
of 935 outputs
Outputs of similar age
#160,523
of 300,620 outputs
Outputs of similar age from BMC Biotechnology
#8
of 22 outputs
Altmetric has tracked 22,862,742 research outputs across all sources so far. This one is in the 35th percentile – i.e., 35% of other outputs scored the same or lower than it.
So far Altmetric has tracked 935 research outputs from this source. They typically receive more attention than average, with a mean Attention Score of 7.8. This one is in the 31st percentile – i.e., 31% of its peers scored the same or lower than it.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 300,620 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 43rd percentile – i.e., 43% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 22 others from the same source and published within six weeks on either side of this one. This one has gotten more attention than average, scoring higher than 54% of its contemporaries.