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. 1998 Mar 31;95(7):3679-84.
doi: 10.1073/pnas.95.7.3679.

Production of medakafish chimeras from a stable embryonic stem cell line

Y Hong  1 , C Winkler, M Schartl
Affiliations

Production of medakafish chimeras from a stable embryonic stem cell line

Y Hong et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Figure 1
Figure 1
Pigmented chimeras obtained from transplanted Mes1 cells. (A) Embryos (at day 7) of the donor (Left) and host (Right) strains. The donor, but not host, strain, shows dark pigmentation in the eye, head, trunk, and yolk sac. (B–E) Pigmented chimeras from Mes1 cells transplanted at different passages. Transplantation of cells at passage 31 (234 days of culture) (B), at passage 40 (320 days) (C and D), and at passage 36 (252 days) (E). Arrows indicate Mes1 cell-derived wild-type melanocytes in the chimeras. (B) A chimera showing a melanocyte on the head. (C and D) A chimera at different developmental stages. At day 4 (C), only a single small pigmented area is evident in the retina. By day 10 (D), expansion of this pigmented area to approximately one-third of the whole retina is paralleled by the appearance of two other pigmented areas, indicating proliferation and differentiation of Mes1 cells. (E) Pigmented chimeric fry showing melanocytes inside the head in the opercular region. (Bars = 200 μm.)
Figure 2
Figure 2
PCR detection of Mes1-derived chimeras. (A) Schematic structure of the tyrosinase gene in the wild-type Mes1 donor (HB32C) and albino host strains (i1). Only the first exon (black box) of the gene and the 1.9-kb insert (open box) interrupting the exon are shown. PCR primers are represented by arrows. TyrA and TyrC define a fragment of 369 bp specific to the donor strain, whereas TyrB and TyrC give rise to a 257-bp fragment unique to the host strain. (B) Sensitivity of the PCR assay. Lane M, 1-kb marker (GIBCO), with sizes shown in base pairs. Numbers in percentages indicate proportions of donor strain-derived DNA diluted with that of the host strain. The 369-bp, donor-specific band is detectable if the donor DNA represents at least 1% of the input DNA. (C) Screening of Mes1-injected embryos from the third transplantation experiment (Table 1). The 369-bp, donor-specific band is evident in 18 of 20 hatchlings and fry from Mes1-transplanted embryos (lanes 1–20) and in the donor (lane D) but not the host (lane H) strain. All host embryos display the 257-bp band. Lanes 1–20, pigmented (lanes 1 and 2) and nonpigmented (lanes 3–20) fry (lanes 1–11) and hatchlings (lanes 12–20). The second pigmented chimeric fry (lane 2) is shown in Fig. 1E. (D) Graphs of intensities of the 369-bp, donor-specific band in the samples shown in B and C. The intensity of the 369-bp band in the 1% lane (B) is equivalent to 1 arbitrary unit.
Figure 3
Figure 3
GFP-expressing Mes1 cells in 10-day-old chimeras. (A) Number of GFP-expressing Mes1 cells per chimera. (B) Number of distribution sites of GFP-expressing Mes1 cells per chimera. (C) Sites of GFP-expressing Mes1 cells in chimeras. “Head” includes the brain, gill operculum, jaws, otic vesicle; “trunk” includes the tail, spinal cord, notocord, and somites; “eye” means the retina and lens; and “heart” includes the atrium, ventricle, ventral body wall, aorta, and conus arteriosus.
Figure 4
Figure 4
Chimeric fry from Mes1 cells transiently expressing GFP. (A, C, E, and G) Bright-field micrographs. (B, D, F, H, and I) Dark-field fluorescent micrographs. (A and B) GFP-expressing Mes1 cells in the myotome (mt; large arrows) and the ventral fin (vt) as clustered (small arrow) and single (hollow arrows) cells. (C and D) GFP-expressing Mes1 cells in the ventral fin (vf; arrows) and the gut (gt; hollow arrows). Note the distinct epithelial phenotype of GFP-expressing cells that are morphologically indistinguishable from surrounding recipient cells. The gut is recognizable by its background autofluorescence. (E and F) GFP-expressing Mes1 cells in the trunk (arrows). GFP-positive cells show green fluorescence and can be distinguished from large, yellow autofluorescent recipient pigment cells. (G and H) GFP-expressing Mes1 cells in the ventral body wall surrounding the pericard (pc; hollow arrows), the ventricle (vt; large arrows), and conus arteriosus (ca; small arrow) of the heart. (I) GFP-expressing Mes1 cells in the atrium (solid arrow) and ventricle (hollow arrow) of the heart. The anterior is to the right, the dorsal side is up. [Bar = 50 μm (A–F and I) and 10 μm (G and H).]
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