Unpigmented, chimeric zebrafish (top) and its pigmented transgenic offpsring. Courtesy of Paul Collodi, Purdue University, Indiana, USA.

One gadget noticeably absent from the zebrafish geneticist's toolkit is an embryonic cell line to use for gene-targeting experiments. But the wait for this vital tool might now be over, with the publication of a technique that maintains undifferentiated, zebrafish embryonic cells in culture, which — when transplanted into embryos — can contribute to the zebrafish germ line.

The secret to the authors' success was their use of a rainbow trout spleen cell line (called RTS34st) to create a monolayer of feeder cells on which they cultured cells derived from gastrula-stage zebrafish embryos. When these embryo cells are cultured without feeder cells, they cease to express the primordial germ-cell marker vasa and begin to differentiate after five days. By contrast, when cultured on this feeder layer, embryo cells remain undifferentiated and express vasa for at least 25 days. Embryo cells cultured without feeder cells but with RTS34st-conditioned medium also express vasa but show earlier signs of differentiation.

But can these cultured cells contribute to the zebrafish germ line — something previous embryonic cell lines have failed to do? To test this, Ma et al. injected cultured embryo cells derived from a pigmented, neo-expressing transgenic zebrafish strain into unpigmented (GASSI) zebrafish embryos. Of the embryos that survived injection, four produced pigmented, neo-positive offspring when crossed to GASSI mates (see picture), indicating that they were germline chimeras. Although the frequency and the degree of germline chimerism are low among the fish that survived injection, the authors reason that these potential barriers to success are offset by the large numbers of offspring that fish produce — this provides many embryos for injection and means that fish with only low rates of chimerism can still produce transgenic offspring.

The next step for Ma et al. is to establish longer-term cultures to allow time for gene targeting and selection. This might require further investigation into what the feeder cells provide to embryo cells to keep them undifferentiated.