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a Department of Animal Science, University of Missouri, Columbia, Missouri 65211
b Agricultural Research Service, United States Department of Agriculture, Columbia, Missouri 65211
Analysis of in vivo- and in vitro-derived control embryos revealed a decline in cyclin B1 transcripts from 5 to 33 h post-4-cell cleavage (P4CC). The quantity of cyclin B1 for the in vivo-derived embryos at 5 and 33 h P4CC was 11.26 and 4.54 attomol/embryo, respectively (P < 0.03), while the in vitro-derived embryos had 20.18 and 7.52 attomol/embryo, respectively (P < 0.03). Treatment with alpha-amanitin from 5, 10, 18, or 25 h P4CC to 33 h P4CC resulted in cyclin B1 quantities that did not differ from those in the 33-h control embryos, irrespective of time spent in the inhibitor. These findings suggest that maternal cyclin B1 transcript degradation occurred over the 4-cell stage with no detectable embryonic cyclin B1 transcripts produced.
Using reverse transcription-competitive polymerase chain reaction (RT-cPCR), the quantity of cyclin B1 transcript present over the maternal to zygotic transition was determined for both in vivo- and in vitro-derived 4-cell porcine embryos. After poly(A) RNA isolation, RT-cPCR was performed on single embryos using an introduced, truncated cyclin B1 DNA competitor. Visualization of embryonic cyclin B1 cDNA and competitor for each reaction allowed a ratio to be formed for use in transcript quantity calculations when compared to cPCR standards.
2 Correspondence: Randall S. Prather, 162 Animal Science Research Center, University of Missouri, Columbia, MO 65211. FAX: 573 882 6827; pratherr{at}missouri.edu
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