Dataset Information

Rapid and reliable identification of insulin 2 gene mutation in Akita diabetic mice by a tetra-primer-ARMS-PCR method.

ABSTRACT: The Akita mouse, one of the most frequently used animal models for the study of diabetes mellitus and its complications, carries a heterozygous missense mutation (C96Y) in the insulin 2 (Ins2) gene that results in proinsulin misfolding in the endoplasmic reticulum (ER), ER stress, pancreatic beta cell death and ultimately diabetes. Maintenance of Akita mice entails genotyping for the identification of the heterozygous Akita mutation. Current genotyping methods for the Akita mouse strain are time consuming, expensive, or needing special device. Here, we develop a simple, fast, cost-effective, and reliable genotyping methodology for the Akita mice. Utilizing the tetra-primer amplification-refractory mutation system polymerase chain reaction (ARMS-PCR) with primers that are specific for normal alleles or Akita mutant alleles, we obtained amplified PCR products that allowed us to distinguish between the wild-type (+/+), heterozygous (Ins2 ^Akita /+), and homozygous (Ins2 ^Akita /Ins2 ^Akita ) mice within 3 hours. These results present the ARMS-PCR analysis as highly desirable and suitable for the identification of the Akita mutation, which is expected to significantly facilitate and promote the Akita mouse-related studies.

SUBMITTER: Undi R

PROVIDER: S-EPMC6329700 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Rapid and reliable identification of insulin 2 gene mutation in Akita diabetic mice by a tetra-primer-ARMS-PCR method.

Undi Rambabu R Lim Hui-Ying HY Wang Weidong W

Heliyon 20190111 1

The Akita mouse, one of the most frequently used animal models for the study of diabetes mellitus and its complications, carries a heterozygous missense mutation (C96Y) in the insulin 2 (Ins2) gene that results in proinsulin misfolding in the endoplasmic reticulum (ER), ER stress, pancreatic beta cell death and ultimately diabetes. Maintenance of Akita mice entails genotyping for the identification of the heterozygous Akita mutation. Current genotyping methods for the Akita mouse strain are time ...[more]

PMID: 30656224

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Project description:BackgroundWidely used restriction-dependent cloning methods are labour-intensive and time-consuming, while several types of ligase-independent cloning approaches have inherent limitations. A rapid and reliable method of cloning native DNA sequences into desired plasmids are highly desired.ResultsThis paper introduces ABI-REC, a novel strategy combining asymmetric bridge PCR with intramolecular homologous recombination in bacteria for native DNA cloning. ABI-REC was developed to precisely clone inserts into defined location in a directional manner within recipient plasmids. It featured an asymmetric 3-primer PCR performed in a single tube that could robustly amplify a chimeric insert-plasmid DNA sequence with homologous arms at both ends. Intramolecular homologous recombination occurred to the chimera when it was transformed into E.coli and produced the desired recombinant plasmids with high efficiency and fidelity. It is rapid, and does not involve any operational nucleotides. We proved the reliability of ABI-REC using a double-resistance reporter assay, and investigated the effects of homology and insert length upon its efficiency. We found that 15 bp homology was sufficient to initiate recombination, while 25 bp homology had the highest cloning efficiency. Inserts up to 4 kb in size could be cloned by this method. The utility and advantages of ABI-REC were demonstrated through a series of pig myostatin (MSTN) promoter and terminator reporter plasmids, whose transcriptional activity was assessed in mammalian cells. We finally used ABI-REC to construct a pig MSTN promoter-terminator cassette reporter and showed that it could work coordinately to express EGFP.ConclusionsABI-REC has the following advantages: (i) rapid and highly efficient; (ii) native DNA cloning without introduction of extra bases; (iii) restriction-free; (iv) easy positioning of directional and site-specific recombination owing to formulated primer design. ABI-REC is a novel approach to DNA engineering and gene functional analysis.

Dataset Information

Rapid and reliable identification of insulin 2 gene mutation in Akita diabetic mice by a tetra-primer-ARMS-PCR method.

Publications

Rapid and reliable identification of insulin 2 gene mutation in Akita diabetic mice by a tetra-primer-ARMS-PCR method.

Similar Datasets

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