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Technology Licensing Opportunity Medaka Fish Haploid Embryonic Stem Cells Key Features • First haploid ES cell lines in the world • Pluripotent • Genetically stable • Mendelian germline transmission • First proof-ofconcept of semicloning technique Potential Applications • Reproductive medicine • Genetic diagnostics • Invitro drug screening • Disease models Abstract Medaka fish is a small laboratory fish that is widely used as a model organism in biomedical research. Repeated attempts in Drosophila, frog, mice and even humans have failed to derive homologous haploid cell populations. For the first time, we have successfully established the culture conditions for medaka haploid ES cell, and obtained three stable haploid ES cell lines, HX1, HX2 and HX3, and gave birth to Holly the fish, the world’s first semi-cloned animal. The ability to derive haploid ES (hES) cells combining the advantages of both haploidy and pluripotency has enormous potential in biomedicine, because they would provide unique opportunities for genetic analyses of recessive and disease phenotypes in various cell lineages in vitro. Technology Summary Haploid embryonic stem (ES) cells combine haploidy and pluripotency, enabling direct genetic analyses of recessive phenotypes in vertebrate cells. Haploid cells have been elusive for culture, due to their inferior growth and genomic instability. We have generated gynogenetic medaka embryos and obtained three haploid ES cell lines that retained pluripotency and competitive growth. Upon nuclear transfer into unfertilized oocytes, the haploid ES cells, even after genetic engineering, generated viable offspring capable of germline transmission. Our haploid medaka ES cells stably maintain normal growth, pluripotency, and genomic integrity. Fig 1. (A) Expression of pluripotency genes by reverse transcription polymerase chain reaction. (B) Expression of pluripotency genes and differentiation genes in haploid ES cells after 10 days of EB formation. Fig 2. (C to G) Immunostaining (green) and propidium iodide (red) nuclear staining of replated cells from 10-day-old EBs. (C) NF200-positive neurons. (D) Map2- positive neuron. (E) Glial fibrillary acidic protein –positive astrocyte. (F) PanCK positive squamous epithelium. (G) Actinin2-positive matured muscle with striations (arrows). Disclaimer Although due care and attention have been taken to ensure that the preparation of this material is as accurate as possible, the contents of this brochure are provided for information purposes only. Neither the Agency for Science, Technology and Research, Exploit Technologies Pte Ltd nor the inventors offer any warranty, written express or implied, as to the accuracy of the said contents. Applicants are advised to undertake independent evaluation of the technology. Technology Licensing Opportunity Figure 3. Stable growth and ploidy. (A and B) Growth curves. Similar doubling time (Td) is seen between haploid clone HX1a (A) and diploid MES1(B). Figure 4. Generation of fertile nuclear transplant and germline transmission. (A and B) Nuclear transplant founder NT1 embryo at day 7 stably labelled with nuclear GFP. Wild-type pigmentation is seen in the eye and body surface [arrows in (A)], and nuclear GFP is throughout the embryo (B). (C) Fry NT1 showing GFPexpression and eye pigmentation. (D) Holly, NT1 fertile female laying eggs. Patents One patent application has been filed for this technology Publications Yi, M. et al. Generation of Medaka Fish Haploid Embryonic Stem Cells; Science 326, 430 (2009) Commercialisation Opportunities Technology is available for licensing from Exploit Technologies Pte Ltd. Contact Us Exploit Technologies Private Limited (A member of A*STAR) 30 Biopolis Street, #09-02 Matrix Singapore 138671 Email: [email protected] Disclaimer Although due care and attention have been taken to ensure that the preparation of this material is as accurate as possible, the contents of this brochure are provided for information purposes only. Neither the Agency for Science, Technology and Research, Exploit Technologies Pte Ltd nor the inventors offer any warranty, written express or implied, as to the accuracy of the said contents. Applicants are advised to undertake independent evaluation of the technology.
