Sp6 or t7 rna polymerase

Embryos were collected and fixed in 4% PFA overnight at 4 °C before performing WISH, as previously described [35 (link)]. Probes for fer, fli1, gata1, scl, c-myb, hbbe2, and runx1 were generated from linearized plasmid containing the cDNA of interest, using either T7 or SP6 RNA polymerase (Ambion) and digoxygenin-UTP labeling mix (Roche). The embryos were washed in a series of glycerol/PBS solutions, where the percent glycerol was increased from 30 to 70 percent, prior to mounting on slides and imaging.

RNA for in vitro experiments was transcribed with T7 or SP6 RNA polymerase (Ambion) from PCR-amplified templates with T7 forward 5′-TAATACGACTCACTATAG-3′ and SP6 Reverse 5′-ATTTAGGTGACACTATAG-3′ primers. According to the manufacturer’s instructions, the RNA fragments, including 5’UTR, CDS, 3’UTR, 3’UTR-mut, and 3’UTR-AS, were purified and biotin-labeled using the RNA 3’-End Biotinylation Kit (Thermo Fisher Scientific). The in vitro binding assays of biotin-labeled VEGF-C mRNA fragments and ZFP36 protein were performed. Briefly, cells (1 × 10⁷) were lysed with 1 ml of binding buffer (50 mM Tris-HCl pH 7.9, 10% glycerol, 100 mM KCl, 5 mM MgCl₂, 10 mM β-ME, 0.1% NP-40, 1 mM PMSF, 1× Superase-in, and 1× protease inhibitor cocktail). Labeled RNA (50 pmol) was incubated with cell lysates for 1 h at RT. Then 50 μl of washed streptavidin-conjugated magnetic beads (Thermo Fisher Scientific) were added to each reaction and incubated at RT for 30 min. Beads were washed five times, and the retrieved protein was subjected to western blot analysis.