【Congress Award Lecture】
Establishment of genetically modified models and development of the usefulness as the model using Common marmoset ○ Erika Sasaki Central Institute for Experimental Animals, Keio Advanced Research Center, Kio University
Experimental animals including non-human primates （NHP） bridge the gap between basic researches and clinics in biomedical science. In Japan, history of development of NHP experimental animals was started in 1975 by Dr. Tastuji Nomura and his colleagues. They compared 12 kinds of NHP species and selected Common marmoset（Callithrix jacchus, marmoset） to develop as the NHP experimental animals because marmoset has several valuable biological characteristics such as similarities of human physiologic characteristics, fecundity and small body size. Using this marmoset, several disease models have been developed by drug induce or surgical methods. However, to understand molecular mechanisms of onset and progression of disease for developing new therapy, genetically modified marmoset models were required. Therefore, to produce transgenic marmosets, we established developmental engineering technics in the marmoset. For this purpose, our research team started from controlling marmoset ovarian cycles and collecting marmoset naturally mated embryos from
uterus. Using these results, we established marmoset ES cell（ESCs）lines. Furthermore, based on these technics, transgenic marmosets using lentiviral vectors were produced. On the otherhand, it was difficult to produce targeted gene knock-out（KO）marmosets, because NHPs ESCs lack the ability to contribute to germ cells. Recent development of innovative genome-editing technologies makes it possible to KO marmosets. As this, we developed screening system of the genome editing tools and results in we produced immunodeficient marmosets. Furthermore, collaborating with many researchers, we also developed research tools, which are genome sequences, BAC library, cDNA library, antibodies and brain atlas to analyze physiological status of the marmosets. I am delighted to be chosen for this award and I would like to continue this work to realize practical application of the genetically modified marmosets in biomedical science.
Development of cre-reporter and cre-driver mice L-2
○ Yoshikazu Hasegawa Laboratory Animal Resource Center, University of Tsukuba
The cre-loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. In this study, I developed cre-reporter and cre-driver mice to facilitate in vivo gene modification, In most reporter mouse strains, it has remained unclear whether a lack of reporter signal indicates either no cre recombinase expression or insufficient reporter gene promoter activity. I produced a novel cre-reporter mice exhibiting green emission before and red after cre-mediated recombination（R26GRR）. Ubiquitous and local cre-loxP recombination was demonstrated by observing fluorescence in tissues of R26GRR x Ayu1-cre and R26GRR x Tei2-cre progeny, respectively. Next, I produced transgenic mice carrying cre driven by mouse Ins1 using a bacterial artificial chromosome （BAC）construct. A founder strain, BAC Ins1-cre25,
was crossed with two different cre-reporter strains. Reporter signals after cre-loxP recombination were detected in pancreatic beta cells of both F 1 mice, but not in other cells. Finally, I generated CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice（C57BL/6J-Ins1em1（cre） Utr ）. R26GRR x C57BL/6J-Ins1em1（cre）Utr F1 mice were histologically characterized and cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed red fluorescence. Furthermore, there were no differences in results of glucose tolerance test among genotypes. Taken together, these mouse strains could be useful and novel cre-reporter and cre-driver mouse strains. In future, I would like to progress the development of cre-driver mice and the cre-loxP evaluation system.
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Analysis of mouse models of male infertility using gene manipulation L-3
○ Yoshitaka Fujihara Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University
We have many types of differentiated cells in our body, but they can be divided into two major groups: somatic cells and germ cells. Germ cells are defined as the only cells that can transmit genetic information to the next generation. Haploid gametes, eggs and sperm, are terminally differentiated from germ cells. Fertilization consists of a complicated multi-step event: development and maturation of the eggs and sperm, sperm migration into the oviduct, sperm-egg interaction in the oviduct, and then sperm-egg fusion. The classical analyses of fertilization were based on in vitro experiments and many factors emerged to be required for fertilization. However, almost all of these factors showed no or minor phenotypes related to fertility when analyzed using gene knockout（KO） mice. Therefore, in vivo analyses using KO mice have reconstructed the mechanisms behind fertilization.
TEX101 is a germ cell-specific glycosylphosphatidylinositol （GPI）-anchored protein. Tex101 KO mice cause sterility because of an ADAM3 deficiency on the sperm plasma membrane. Angiotensin-converting enzyme （ACE） has the ability to regulate blood pressure and GPI-anchored protein-releasing activity. The removal of TEX101 by ACE is essential to produce fertile spermatozoa. Currently, I am focusing on sperm membrane proteins that are conserved in mice and humans for the sperm migrating ability into the oviduct. Furthermore, we have improved the method for generating KO mice by CRISPR/Cas9. By this method, I have found some essential factors for spermatogenesis and fertilization, and plan to continue my research to elucidate the molecular mechanisms of mammalian fertilization.
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