Abstract
Totipotent embryonic stem (ES) cells represent a bridge that links in vitro and in vivo manipulations of animal genomes and have enormous potential for genetic engineering of livestock. We have recently established feeder cell-free conditions for culturing cells of midblastula embryos (MBE) of the medaka (Oryzias latipes) and obtained several stable cell lines that show all features of mouse ES cells in vitro. One of these lines, MES1, has been demonstrated to retain a diploid karyotype and can be induced to differentiate into various cell types in vitro. Upon microinjection into albino host blastulae, MES1 cells are able to form pigmented chimeras. Genotype-specific PCR analysis revealed that 90% of host blastulae transplanted with MES1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. Transplantation of genetically labelled MES1 cells revealed a wide contribution to numerous organs derived from all three germ layers and differentiation into various types of functional cells. These ES properties of MES1 line was not abolished by stable gene transfer and long-term selection. Thus MES1 cells may represent a first promising cellular vehicle for the production of genetically modified fish. The genetic background has been found to have a profound effect on the efficacy of ES cell derivation and of chimera formation.
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