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David Natale1 , Mark Westhusin2 *, Andrew Watson1 *
Depts of Ob/Gyn & Physiology, The University of Western Ontario, London, Canada 1
Depts. Vet. Physiology & Pharmacology, Texas A & M University, College Station, Texas 2
This study utilized Differential Display-Reverse Transcription-Polymerase Chain Reaction to characterize patterns of -amanitin sensitive gene expression during bovine early development. Total RNA was isolated from pools (20-50/pool)of in vitro matured oocytes, 2-5 cell, 6-8 cell, 8-16 cell, morula and blastocyst stage embryos, cultured in synthetic oviduct fluid medium + citrate and amino acids (cSOFMaa) with and without -amanitin (100g/mL) for 4 and 12 hr. Total RNA was reverse transcribed using a T11MC 3'anchored oligo-dT primer. mRNA profiles were displayed by PCR using the T11MC 3'primer in combination with two 5'-10 mer oligonucleotides (A and N) at an annealing temperature of 42oC for 40 cycles. Triplicate reactions from each sample were displayed and only consistent banding variations were recorded. Results employing primer A applied to zygotes treated with a-amanitin for 4 hr revealed total band counts of 114, 90, 95, 95, 117 and 139 for oocytes, 2-5 cell, 6-8 cell, 8-16 cell, morulae and blastocysts, respectively. For 12 hr treatments total band counts were 109, 87, 50, 86, 130, and 165 respectively. The frequency of a-amanitin sensitive bands for the 4 hr treatments were 3.3% 20.0%, 8.4% 6.0% and 9.4% of total bands. For 12 hr treatments they were 0% 24.0% 32.6%, 46.2% and 41.8% respectively, indicating a greater mRNA turnover rate for this treatment time. These findings were confirmed by employing primer N. The results demonstrate that a-amanitin sensitive gene expression arises at the 6-8 cell stage during bovine development while sequence analysis has provided preliminary identification of -amanitin and stage specific amplicons. Research supported by NSERC Canada.
This abstract is being presented on Sunday, August 1 at 3:15 PM at Todd 133.