TY - JOUR
T1 - A novel positive detection system of in vivo mutations in rpsL (strA) transgenic mice
AU - Gondo, Yoichi
AU - Shioyama, Yoshiyuki
AU - Nakao, Kazuki
AU - Katsuki, Motoya
N1 - Funding Information:
We appreciate the excellent technical contributions by Yumiko Ikeda, Yoshiko Motegi, Aya Takeshita, Michie Takahashi and Toshiko Yokoyama. We also thank Dr. Minesuke Yokoyama and Takanori Hasegawa for their help in constructing the transgenic mice; Kuniko Katsuki and Yusuke Sotomaru for maintaining the mice; Dr. Brian T. Quinn for help in the preparation of this manuscript; and especially to Drs. Hisaji Maki and Mutsuo Sekiguchi for their advice and for generously providing us pML4, NM554 and MY1. This work was supported by Grants-in-Aids for Scientific Research Priority Areas, for Cancer Research, for Scientific Research and for Developmental Scientific Research from the Ministry of Education, Science and Culture, Japan, grants from the Ministry of Health and Welfare, Japan, and to M.K. from the Science and Technology Agency, Japan. M.K. and Y.G. are recipients of Research Grants of the Princess Takamatsu Cancer Research Fund. Y.G. is also a recipient of funds from the Kato Memorial Bioresearch Foundation and the Fukuoka Cancer Society.
PY - 1996
Y1 - 1996
N2 - To positively detect the in vivo mutations accumulated in different mouse organs, we have developed a transgenic mouse system. This transgenic mouse carried an Escherichia coli (E. coli) plasmid pML4 as a shuttle vector that consisted of a replication origin (ori), the kanamycin-resistant gene (Kan(R)) and the rpsL+ gene (strA(S)) derived from E. coli. These E. coli elements were expected to be inert in the transgenic mouse system; thus, neutral mutations would be accumulated on the shuttle plasmid in the transgenic mice. The shuttle plasmid vector was recovered from the mouse genomic DNA and introduced into kanamycin-sensitive (Km(S)) and streptomycin-resistant (Sm(R)) E. coli cells by using electroporation. The original pML4 shuttle plasmid transformed the host E. coli to Km(R) and Sm(S), since both the Kan(R) and rpsL genes exhibited dominant traits of Km(R) and Sm(S) respectively. On the other hand, when the retrieved pML4 shuttle plasmid carried a mutated rpsL gene, it could be positively detected as both Km(R) and Sm(R). Based on this principle, we were able to positively detect the in vivo mutations accumulated in the rpsL transgene of the shuttle vector pML4 integrated into the mouse genome. The total number of rescued shuttle plasmids were counted on the plates containing Km alone, while only mutants were detected on the plates containing both Km and Sm. We have so far established 22 independent transgenic mouse lines that carried up to approx. 750 copies of the shuttle plasmid pML4 in a haploid genome. By using high-copy-number transgenic mouse lines which carried 350 copies or more of the shuttle vector, we also developed a simple and proficient method for retrieving the shuttle plasmid from various tissues of the transgenic mice. The background mutant frequency was approx. 5 x 10-5. In order to validate the applicability of the positive-detection transgenic system for the induced mutagenicity assay, methylnitrosourea (MNU) was administered to the transgenic mice, and an increase in the number of mutant frequencies was seen in all tested organs including spleen, liver and brain. The rpsL transgenic mouse system was therefore considered to provide a quick-and-easy risk assessment test for in vivo tissue-specific mutagenicity, using positive detection by streptomycin.
AB - To positively detect the in vivo mutations accumulated in different mouse organs, we have developed a transgenic mouse system. This transgenic mouse carried an Escherichia coli (E. coli) plasmid pML4 as a shuttle vector that consisted of a replication origin (ori), the kanamycin-resistant gene (Kan(R)) and the rpsL+ gene (strA(S)) derived from E. coli. These E. coli elements were expected to be inert in the transgenic mouse system; thus, neutral mutations would be accumulated on the shuttle plasmid in the transgenic mice. The shuttle plasmid vector was recovered from the mouse genomic DNA and introduced into kanamycin-sensitive (Km(S)) and streptomycin-resistant (Sm(R)) E. coli cells by using electroporation. The original pML4 shuttle plasmid transformed the host E. coli to Km(R) and Sm(S), since both the Kan(R) and rpsL genes exhibited dominant traits of Km(R) and Sm(S) respectively. On the other hand, when the retrieved pML4 shuttle plasmid carried a mutated rpsL gene, it could be positively detected as both Km(R) and Sm(R). Based on this principle, we were able to positively detect the in vivo mutations accumulated in the rpsL transgene of the shuttle vector pML4 integrated into the mouse genome. The total number of rescued shuttle plasmids were counted on the plates containing Km alone, while only mutants were detected on the plates containing both Km and Sm. We have so far established 22 independent transgenic mouse lines that carried up to approx. 750 copies of the shuttle plasmid pML4 in a haploid genome. By using high-copy-number transgenic mouse lines which carried 350 copies or more of the shuttle vector, we also developed a simple and proficient method for retrieving the shuttle plasmid from various tissues of the transgenic mice. The background mutant frequency was approx. 5 x 10-5. In order to validate the applicability of the positive-detection transgenic system for the induced mutagenicity assay, methylnitrosourea (MNU) was administered to the transgenic mice, and an increase in the number of mutant frequencies was seen in all tested organs including spleen, liver and brain. The rpsL transgenic mouse system was therefore considered to provide a quick-and-easy risk assessment test for in vivo tissue-specific mutagenicity, using positive detection by streptomycin.
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U2 - 10.1016/S0165-1161(96)90231-9
DO - 10.1016/S0165-1161(96)90231-9
M3 - Article
C2 - 8657204
AN - SCOPUS:0029947527
VL - 360
SP - 1
EP - 14
JO - Mutation Research - Environmental Mutagenesis and Related Subjects Including Methodology
JF - Mutation Research - Environmental Mutagenesis and Related Subjects Including Methodology
SN - 0165-1161
IS - 1
ER -