著者

Masaharu YOSHIHARA Teppei NISHINO Naoto SAMBE Takahiro NAYAKAMA Freddy RADTKE Seiya MIZUNO Satoru TAKAHASHI

出版者

Japanese Association for Laboratory Animal Science

雑誌

Experimental Animals (ISSN:13411357)

巻号頁・発行日

pp.21-0202, (Released:2022-04-21)

被引用文献数

4

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Cell labeling technologies, including the Cre/loxP system, are powerful tools in developmental biology. Although the conventional Cre/loxP system has been extensively used to label the expression of specific genes, it is less frequently used for labeling protein-protein interactions owing to technical difficulties. In the present study, we generated a new Gal4-dependent transgenic reporter mouse line that expressed Cre recombinase and a near-infrared fluorescent protein, miRFP670. To examine whether this newly generated transgenic mouse line is applicable in labeling of protein-protein interaction, we used a previously reported transgenic mouse lines that express Notch1 receptor with its intracellular domain replaced with a yeast transcription factor, Gal4. Upon the binding of this artificial Notch1 receptor and endogenous Notch1 ligands, Gal4 would be cleaved from the cell membrane to induce expression of Cre recombinase and miRFP670. Indeed, we observed miRFP670 signal in the mouse embryos (embryonic day 14.5). In addition, we examined whether our Cre recombinase was functional by using another transgenic mouse line that express dsRed after Cre-mediated recombination. We observed dsRed signal in small intestine epithelial cells where Notch1 signal was suggested to be involved in the crypt stem cell maintenance, suggesting that our Cre recombinase was functional. As our newly generated mouse line required only the functioning of Gal4, it could be useful for labeling several types of molecular activities in vivo.

著者

Seiya MIZUNO Saori IIJIMA Tomoko OKANO Noriko KAJIWARA Satoshi KUNITA Fumihiro SUGIYAMA Ken-ichi YAGAMI

出版者

公益社団法人 日本実験動物学会

雑誌

Experimental Animals (ISSN:13411357)

巻号頁・発行日

vol.60, no.2, pp.161-167, 2011 (Released:2011-04-21)

参考文献数

14

被引用文献数

5 18

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We found 6 spontaneous mutant mice with long pelage hair in our ICR breeding colony. The abnormal trait was restricted to long hair in these mice, which we named moja. They were fertile and showed the same growth and behavior as wild-type mice. To investigate the manner of the genetic inheritance of the moja allele, offspring were bred by mating the moja mice; all offspring had long pelage hair. Furthermore, we performed a reciprocal cross between moja mice and wild-type ICR mice with normal hair. All offspring exhibited normal hair suggesting an autosomal recessive inheritance of the trait. The moja/moja hair phenotype was maintained in skin grafted onto nude mice, suggesting that circulating or diffusible humoral factors regulating the hair cycle are not involved in the abnormal trait. The phenotype of moja/moja mice is similar to that of Fgf5-deficient mice. Therefore, we examined the expression of Fgf5 by RT-PCR in moja/moja mice. As expected, no Fgf5 expression was found in moja/moja mouse skin. PCR and DNA sequence analyses were performed to investigate the structure of the Fgf5 gene. We found a deletion of a 9.3-kb region in the Fgf5 gene including exon 3 and its 5’ and 3’ flanking sequences. Interestingly, the genomic deletion site showed insertion of a 498-bp early transposon element long terminal repeat. Taken together, these results suggest that the long hair mutation of moja/moja mice is caused by disruption of Fgf5 mediated by insertion of a retrotransposon.

著者

杉山 文博 高橋 智 水野 聖哉 Channabasavaiah B. Gurumurthy Aidan R. O'Brien Rolen M. Quadros John Adams Pilar Alcaide Shinya Ayabe Johnathan Ballard Surinder K. Batra Marie-Claude Beauchamp Kathleen A. Becker Guillaume Bernas David Brough Francisco Carrillo-Salinas Wesley Chan Hanying Chen Ruby Dawson Victoria DeMambro Jinke D'Hont Katharine M. Dibb James D. Eudy Lin Gan Jing Gao Amy Gonzales Anyonya R. Guntur Huiping Guo Donald W. Harms Anne Harrington Kathryn E. Hentges Neil Humphreys Shiho Imai Hideshi Ishii Mizuho Iwama Eric Jonasch Michelle Karolak Bernard Keavney Nay-Chi Khin Masamitsu Konno Yuko Kotani Yayoi Kunihiro Imayavaramban Lakshmanan Catherine Larochelle Catherine B. Lawrence Lin Li Volkhard Lindner Xian-De Liu Gloria Lopez-Castejon Andrew Loudon Jenna Lowe Loydie A. Jerome-Majewska Taiji Matsusaka Hiromi Miura Yoshiki Miyasaka Benjamin Morpurgo Katherine Motyl Yo-ichi Nabeshima Koji Nakade Toshiaki Nakashiba Kenichi Nakashima Yuichi Obata Sanae Ogiwara Mariette Ouellet Leif Oxburgh Sandra Piltz Ilka Pinz Moorthy P. Ponnusamy David Ray Ronald J. Redder Clifford J. Rosen Nikki Ross Mark T. Ruhe Larisa Ryzhova Ane M. Salvador Sabrina Shameen Alam Radislav Sedlacek Karan Sharma Chad Smith Katrien Staes Lora Starrs Fumihiro SUGIYAMA Satoru TAKAHASHI Tomohiro Tanaka Andrew W. Trafford Yoshihiro Uno Leen Vanhoutte Frederique Vanrockeghem Brandon J. Willis Christian S. Wright Yuko Yamauchi Xin Yi Kazuto Yoshimi Xuesong Zhang Yu Zhang Masato Ohtsuka Satyabrata Das Daniel J. Garry Tino Hochepied Paul Thomas Jan Parker-Thornburg Antony D. Adamson Atsushi Yoshiki Jean-Francois Schmouth Andrei Golovko William R. Thompson K. C. Kent Lloyd Joshua A. Wood Mitra Cowan Tomoji Mashimo Seiya MIZUNO Hao Zhu Petr Kasparek Lucy Liaw Joseph M. Miano Gaetan Burgio

出版者

BMC

雑誌

Genome Biology (ISSN:1474760X)

巻号頁・発行日

vol.20, no.1, 2019-08

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BackgroundCRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method).ResultsWe re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach.ConclusionWe propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

著者

Yu-Hsin CHANG Megumi C. KATOH Ahmed M. ABDELLATIF Guli XIAFUKAITI Abdelaziz ELZEFTAWY Masami OJIMA Seiya MIZUNO Akihiro KUNO Satoru TAKAHASHI

出版者

Japanese Association for Laboratory Animal Science

雑誌

Experimental Animals (ISSN:13411357)

巻号頁・発行日

pp.19-0105, (Released:2019-12-02)

被引用文献数

1

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Cre/loxP is a site-specific recombination system extensively used to enable the conditional deletion or activation of target genes in a spatial- and/or temporal-specific manner. A number of pancreatic-specific Cre driver mouse lines have been broadly established for studying the development, function and pathology of pancreatic cells. However, only a few models are currently available for glucagon-producing α-cells. Disagreement exists over the role of the MAFB transcription factor in glucagon expression during postnatal life, which might be due to the lack of α-cell-specific Cre driver mice. In the present study, we established a novel Gcg-Cre knock-in mouse line with the Cre transgene expressed under the control of the preproglucagon (Gcg) promoter without disrupting the endogenous Gcg gene expression. Then, we applied this newly developed Gcg-Cre mouse line to generate a new α-cell-specific Mafb conditional knockout mouse model (MafbΔGcg). Not only α-cell number but also glucagon production were significantly decreased in MafbΔGcg mice compared to control littermates, suggesting an indispensable role of MAFB in both α-cell development and function. Taken together, our newly developed Gcg-Cre mouse line, which was successfully utilized to uncover the role of MAFB in α-cells, is a useful tool for genetic manipulation in pancreatic α-cells, providing a new platform for future studies in this field.