RERF Report No. 10-14

Paper published in PLOS ONE §“Creation of mice bearing a partial duplication of HPRT gene marked with a GFP
gene and detection of revertant cells in situ as GFP-positive somatic cells”
Asao Noda, Hirofumi Suemori, Yuko Hirai, Kanya Hamasaki, Yoshiaki Kodama, Hiroshi
Mitani, Reid D. Landes, Nori Nakamura
PLOS ONE (August 21, 2015); 10(8): e0136041.
(doi: 10.1371/journal.pone.0136041)

Study Findings
We created transgenic mice that allow in situ (i.e., in tissue) visualization of mutations in
any cell throughout the body of the animal. The mice are technically called HPRT-dup-GFP
knock-in mice. We used this knock-in mouse model to detect mutant cells arising in organs,including the liver, pancreas, small intestine, large intestine, lungs, thyroid, and testes. Large
differences among individuals in terms of spontaneous mutation frequencies in were observed insomatic cells. Such high spontaneous frequencies impeded detection of mutations induced byradiation. Lowering the background mutation frequency is the next challenge for better use ofthis system in radiation research.
1. Objectives
One objective was to create mice to assist in the detection of radiation-induced mutations
in somatic and germ-line cells. Cells developing a mutation in this mouse model survive andfluoresce green (because they contain green fluorescent protein, or GFP). Another objective wasto use this mouse model to observe where radiation-induced mutant cells occur and grow in thebody. The final objective was to establish a system for monitoring the genetic effects ofradiation in individual organisms.
2. Methods
Embryological engineering techniques were used for manipulating the mouse HPRT gene(a structural gene involved in nucleic acid synthesis) to be partially duplicated in conjunction
withthe GFP gene on

the Xchromosome,therebycreating

transgenicmice.

Recombination-mediated reversion from the partial duplications of the HPRT (i.e., reactivationof gene activity) in the cells of these mice led to expression of the HPRT-GFP fusion protein andcaused the affected cells to fluoresce green.
Radiation exposure effects were evaluated using fluorescence to determine mutation
frequency in the mouse cells after the mice were exposed to radiation.
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3. Results(1) We successfully created transgenic mice (HPRT-dup-GFP mice) by double gene knock-in(the insertion of a gene in a specific location in a chromosome) in mouse embryonic stem cells(cells from the early embryo that can differentiate into any cell type). On the HPRT-dup-GFP
locus of the mice, in vivo arising, somatic mutant cells, which are revertants from the partial
duplicated allele of the HPRT gene, were confirmed as originating from gene deletion vianormal homologous recombination repair. Mutant cells in any of the tissues except the brain
fluoresced green, but the frequencies of spontaneous mutations differed among tissues.
(2) Large individual differences were seen in the frequency of somatic cell mutations, even in
the mice not exposed to radiation. Around 1% of the mice developed mutations too numerous
to be countable. We used the term “extreme jackpot mutations” to describe such cases. The
large differences in mutation frequency among individual mice may depend on when during
development and growth cycles mutant cells arise. The investigation thus can be conceived asa fluctuation test of mutations at the level of the individual.
(3) Somatic cell mutation frequencies were measured in different tissues of mice 3 months after
exposure to a dose of 3 Gy as a model of radiation exposure. Mutation frequency was observed
to be little changed in certain tissues (pancreas and lymphocytes) but two-fold higher in others(e.g., liver, small intestine).
Study SignificanceWe successfully created mice whose living cells fluoresce when developing a mutation.
Although the model is useful, background noise from spontaneous mutations will have to be
reduced for the model to be a reliable indicator of the effects of radiation exposure. Our findings
strongly suggest that many of the somatic cells making up our bodies already contain mutations
that give us a genetically mosaic appearance.
The Radiation Effects Research Foundation has studied A-bomb survivors and their offspring in
Hiroshima and Nagasaki for more than 60 years. RERF’s research achievements are considered theprincipal scientific basis for radiation risk assessment by the United Nations Scientific Committee on theEffects of Atomic Radiation (UNSCEAR) and for recommendations regarding radiation protection
standards by the International Commission on Radiological Protection (ICRP). RERF expresses its
profound gratitude to the A-bomb survivors and survivors’ offspring for their cooperation in our studies.

§PLOS ONE is an online, open access, and internationally peer-reviewed journal published by the Public

Library of Science, a nonprofit corporation. The journal features original articles in all fields of scienceand medicine. (Impact factor in 2014: 3.53)

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