Simultaneous Detection of Multiple Transgenes for Genetically-Modified Mouse Strains

Nakata, Hatsumi; Hashimoto, Tomomi; Seki, Yukiko; Mekada, Kazuyuki; Obata, Yuichi; Yoshiki, Atsushi

doi:10.1538/expanim.58.437

Cited by 5 publications

(7 citation statements)

References 28 publications

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“…PCR tests using the developed universal primers can be widely applied to the various conditional genetic models containing loxP s and FRT s currently available at the core facility of the National BioResource Project by the Japanese Ministry of Education, Culture, Sports, Science and Technology and the Agency for Medical Research and Development (Yoshiki et al., 2009). To establish a comprehensive genetic QC program, it is recommended that the loxP ‐ and FRT ‐PCR tests be conducted along with other genetic QC tests, such as marker gene detection (Nakata et al., 2009) and strain‐specific genotyping and sequencing. The developed universal primers should also play an important role in the quick validation of conditional mice with cis‐ versus trans‐ integrated loxP s via genome editing knock‐in technology (Gurumurthy et al., 2019).…”

Section: Resultsmentioning

confidence: 99%

“…Mouse genomic DNA was extracted from tail tips as previously described using automated DNA isolator (GENE PREP STAR PI‐80X, Kurabo; Nakata et al., 2009; Figure S2). Primer sets with 5' end containing restriction sites for loxP and FRT as shown in Figure 1 and listed in Table 1 were used.…”

Section: Methodsmentioning

confidence: 99%

“…Recent advancements in genome editing together with DNA recombination technologies have resulted in the creation of thousands of conditional mouse strains carrying increasingly complex and sophisticated floxed and/or Flrted alleles for in vivo studies of gene functions and disease processes (Gurumurthy et al., 2019; Skarnes et al., 2011). To obtain reproducible and reliable experimental results using the floxed and/or Flrted mice, the genetic quality of the floxed and Flrted alleles, including their complete structure as originally designed using functional components, should be validated prior to experiments (Nakata et al., 2009). In large‐scale knockout mouse production facilities, the validation of conditional alleles is conducted by long‐range PCR with primers designed based on the information of gene cassette and genome sequences adjacent to the loxP followed by sequencing (Skarnes et al., 2011).…”

Section: Introductionmentioning

confidence: 99%

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Quick validation of genetic quality for conditional alleles in mice

2021

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Site-specific conditional inactivation technologies using Cre-loxP or Flp-FRT systems are becoming increasingly important for the elucidation of gene function and disease mechanism in vivo. A large number of gene knockout mouse models carrying complex conditional alleles have been generated by global community efforts and made available for biomedical researchers. The structures of conditional alleles in these mice are becoming increasingly complex and sophisticated, and so the validation of the genetic quality of these alleles is likewise becoming a laborious task for individual researchers. To ensure the reproducibility of conditional experiments, the researcher should confirm that loxP or FRT is integrated at the correct positions in the genome prior to start of the experiments. We report the successful design of universal PCR primers specific to loxP and FRT for the quick validation of conditional floxed and Flrted alleles. The primer set consists of forward and reverse primers complimentary to the loxP or FRT sequences with partial modifications at the 5ʹ end containing 6-base restriction endonuclease recognition sites. The universal primer set was tested to detect genomic intervals between a pair of cis-integrated loxP or FRT and was useful for quickly validating various floxed or Flrted alleles in conditional mice.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Quick validation of genetic quality for conditional alleles in mice

2021

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“…If there is any discrepancy between the depositor's information and our test results, we communicate the results to the depositor and ask for a correction of the supplied information or resending correct mice. We use different genetic QC tests (Nakata et al 2009(Nakata et al , 2021, particularly PCR, depending on various genetic mutations (Table 3). The test results and protocols for each strain are posted in the strain data sheet in the web catalog when tested.…”

Section: Genetic Quality Controlmentioning

confidence: 99%

Mouse resources at the RIKEN BioResource Research Center and the National BioResource Project core facility in Japan

et al. 2021

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The RIKEN BioResource Research Center (BRC) was established in 2001 as a comprehensive biological resource center in Japan. The Experimental Animal Division, one of the BRC infrastructure divisions, has been designated as the core facility for mouse resources within the National BioResource Project (NBRP) by the Japanese government since FY2002. Our activities regarding the collection, preservation, quality control, and distribution of mouse resources have been supported by the research community, including evaluations and guidance on advancing social and research needs, as well as the operations and future direction of the BRC. Expenditure for collection, preservation, and quality-control operations of the BRC, as a national core facility, has been funded by the government, while distribution has been separately funded by users’ reimbursement fees. We have collected over 9000 strains created mainly by Japanese scientists including Nobel laureates and researchers in cutting-edge fields and distributed mice to 7000 scientists with 1500 organizations in Japan and globally. Our users have published 1000 outstanding papers and a few dozen patents. The collected mouse resources are accessible via the RIKEN BRC website, with a revised version of the searchable online catalog. In addition, to enhance the visibility of useful strains, we have launched web corners designated as the “Mouse of the Month” and “Today’s Tool and Model.” Only high-demand strains are maintained in live colonies, while other strains are cryopreserved as embryos or sperm to achieve cost-effective management. Since 2007, the RIKEN BRC has built up a back-up facility in the RIKEN Harima branch to protect the deposited strains from disasters. Our mice have been distributed with high quality through the application of strict microbial and genetic quality control programs that cover a globally accepted pathogens list and mutated alleles generated by various methods. Added value features, such as information about users’ publications, standardized phenotyping data, and genome sequences of the collected strains, are important to facilitate the use of our resources. We have added and disseminated such information in collaboration with the NBRP Information Center and the NBRP Genome Information Upgrading Program. The RIKEN BRC has participated in international mouse resource networks such as the International Mouse Strain Resource, International Mouse Phenotyping Consortium, and Asian Mouse Mutagenesis and Resource Association to facilitate the worldwide use of high-quality mouse resources, and as a consequence it contributes to reproducible life science studies and innovation around the globe.

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“…Since animal experiments increasingly use multiple genetically modified mouse strains and their crosses, it is necessary to thoroughly maintain those genetic modifications to meet scientific and legal requirements. For this purpose, we established a simultaneous PCR test to detect the presence of multiple transgenes 25 . This PCR test, the "KO-survey," provides users with mouse strains of the highest genetic quality and accurate information on their genetic modifications.…”

Section: Genetic Qualitymentioning

confidence: 99%

Simultaneous Detection of Multiple Transgenes for Genetically-Modified Mouse Strains

Cited by 5 publications

References 28 publications

Quick validation of genetic quality for conditional alleles in mice

Quick validation of genetic quality for conditional alleles in mice

Mouse resources at the RIKEN BioResource Research Center and the National BioResource Project core facility in Japan

Dissemination of Advanced Mouse Resources and Technologies at RIKEN BioResource Center

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