On the use of alpha-amanitin as a transcriptional blocking agent in mouse embryos: a cautionary note.
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.
Kidder
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Green
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McLachlin
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D000241:Adenosine / Q000378:metabolism; D000546:Amanitins / Q000494:pharmacology*; D000818:Animals; D001755:Blastocyst / Q000187:drug effects* / Q000378:metabolism; D002846:Chromatography, Affinity; D003470:Culture Media; D066298:In Vitro Techniques; D051379:Mice; D008815:Mice, Inbred Strains; D011061:Poly A / Q000096:biosynthesis*; D012313:RNA / Q000096:biosynthesis*; D012333:RNA, Messenger; D012334:RNA, Neoplasm / Q000096:biosynthesis*; D014158:Transcription, Genetic / Q000187:drug effects*; D014316:Tritium; D014529:Uridine / Q000378:metabolism
D000546:Amanitins; D003470:Culture Media; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014316:Tritium; D011061:Poly A; D012313:RNA; D000241:Adenosine; D014529:Uridine