Reverse transcription–PCR analysis of vasa mRNA in
zebrafish embryo cell cultures. cDNA was synthesized from total RNA
obtained from embryo cell cultures. PCR amplification was performed
with vasa-specific primers designed to generate a 505-bp
product. Product identity was confirmed by sequencing. Lane a, MW
markers; lanes b, c, and d, embryo cells maintained for 5, 15, and 25
days, respectively, in RTS34st cell-conditioned medium; lanes e, f, and
g, embryo cells maintained for 5, 15, and 25 days, respectively, on an
RTS34st feeder layer; lane h, embryo cells maintained at first for 24
days in RTS34st cell-conditioned medium, and then (after passaging) for
8 days on an RTS34st feeder layer; lanes i, j, and k, embryo cell
cultures maintained for 1, 3, and 5 days, respectively, in the absence
of RTS34st feeder cells or cell-conditioned medium; lane l, negative
control (no template); lane m, RTS34st cells cultured in the absence of
zebrafish embryo cells. Primers that amplify fibronectin cDNA were used
to control for equal amounts of sample in each lane.