On the use of α‐amanitin as a transcriptional blocking agent in mouse embryos: A cautionary note

Abstract

We have tested the effect of α‐amanitin at 10, 50, and 100 μg/ ml, on precursor uptake and incorporation into poly(A)⁺ RNA and poly(A)⁻ RNA of mouse embryos on days 2, 3 and 4 of gestation. Embryos were pretreated with the inhibitor for 2 hr, then labeled for 2 hr in its continued presence. RNA fractions were separated by affinity chromatography on oligo(dT)‐cellulose. α‐Amanitin did not suppress uptake of RNA precursors at any of the concentrations tested in any stage. At 10 μg/ml, we could not detect any effect on incorporation into either RNA fraction in any stage. Only the highest concentration tested, 100 μg/ml, was effective in all stages in substantially suppressing incorporation into poly(A)⁺ RNA within 2 hr. Longer treatments increased the level of suppression to a maximum of about 80%. Incorporation into poly(A)⁻ RNA was suppressed to roughly the same extent. Despite previously reported data, it cannot be assumed that α‐amanitin at concentrations less than 100 μg/ml brings about a quick interruption of mRNA synthesis in preimplantation mouse embryos.