On the use of α-amanitin as a transcriptional blocking agent in mouse embryos: A cautionary note
✍ Scribed by Kidder, Gerald M. ;Green, A. Fred ;McLachlin, Jeanne R.
- Publisher
- John Wiley and Sons
- Year
- 1985
- Tongue
- English
- Weight
- 385 KB
- Volume
- 233
- Category
- Article
- ISSN
- 0022-104X
No coin nor oath required. For personal study only.
✦ Synopsis
We have tested the effect of alpha-amanitin at 10, 50 and 100 micrograms/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. alpha-Amanitin did not suppress uptake of RNA precursors at any of the concentrations tested in any stage. At 10 micrograms/ml, we could not detect any effect on incorporation into either RNA fraction in any stage. Only the highest concentration tested, 100 micrograms/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 alpha-amanitin at concentrations less than 100 micrograms/ml brings about a quick interruption of mRNA synthesis in preimplantation mouse embryos.