Production of medakafish chimeras from a stable embryonic stem cell line

Abstract

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for in vitro selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (Oryzias latipes) that shows all characteristics of mouse ES cells in vitro. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 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. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology.

Accessibility of host cell lineages to medaka stem cells depends on genetic background and irradiation of recipient embryos

“…However, both strains have iridophores that produce yellow (positive for both green and red fluorescence) autofluorescence at 3 days post-fertilization (dpf), heavily interfering with the observation of fluorescence-labeled donor cells in advanced embryos [10]. By combining three recessive genetic loci including i 3 , Wakamatsu et al [35] developed an autofluorescencefree strain called see-through (ST2), which lacks melanophores and iridophores.…”

“…Irradiated and non-irradiated embryos were dechorionated and transplanted at the midblastula stage with approximately 100 MES1 cells [10,21] or non-cultivated blastomeres [16,21,32]. In some experiments, embryos transplanted with blastula cells were reared in the presence of 1 mM of phenylthiourea to prevent pigmentation for easier fluorescence observations.…”

“…Several ES cell lines comparable to mouse ES cells are available in this organism [9,10]. One of these lines, MES1, has been characterized as being pluripotent in vitro and in vivo.…”

“…MES1 has a high efficiency for transient and stable gene transfer by physical/chemical procedures [13] and baculovirus transduction [14], and retains its developmental pluripotency even after long-term drug selection and stable gene transfer [13]. In vivo, MES1 after transplantation into host embryos can proliferate and differentiate during chimeric embryogenesis into many functional cell types that contribute to various organs [10]. The availability of ES cells and the possibility for robust genetic manipulations render medaka a promising model organism for cell culture-mediated germline transmission of defined genetic manipulations such as gene targeting.…”

“…In medaka, MBE-derived ES cell cultures including MES1 give rise to a high (up to 100%) frequency but a low (5% at best) degree of chimerism for somatic chimera formation [10]. This feature is common to ES cells after extended growth beyond 50 days of culture [21].…”