Identification and quantification of differentially expressed transcripts in in vitro-produced bovine preimplantation stage embryos.
In this study, we used mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) to analyze the mRNA expression patterns in in vitro-produced bovine 8-cell, 16-cell, morula, and blastocyst stage embryos and isolate differentially expressed amplicons. Moreover, we have used a fluorescence monitored real time quantitative PCR to quantify and analyze the expression patterns of the target differentially expressed transcripts through out the preimplantation stages from oocytes to blastocyst. For this, total RNA isolated from bovine 8-cell (n = 188), 16-cell (n = 94), morula (n = 35), and blastocyst (n = 15) were reverse transcribed and subjected to DDRT-PCR. Target differentially expressed transcripts were quantified by real time quantitative PCR. The cDNA banding pattern analysis revealed that large number of cDNA bands were conserved at 8-cell and blastocyst stage with a slight decrease at the morula stage. A total of 16 amplicons were cloned and sequenced. All expressed sequence tags (ESTs), except 1C19, showed sequence similarity with known genes or ESTs in GenBank. Sixty-two percent (10/16) of cDNA bands representing differentially expressed genes originated from 8-cell stage and the rest derived from the 16-cell, morula, or blastocyst stage. The quantitative PCR analysis has validated the expression patterns of 75% (12/16) of our transcripts to be in agreement with the results of DDRT-PCR. However, the quantitative PCR results of four transcripts showed a deviation from the pattern seen in DDRT-PCR. In conclusion, we have successfully applied mRNA DDRT-PCR to identify and isolate stage-specific expressed genes in bovine preimplantation embryos. In addition to validating the results of DDRT-PCR, quantitative real time PCR provides quantitative data on the expression of target genes.
Tesfaye
Dawit
D
Institute of Animal Breeding Science, University of Bonn, Endenicher Allee 15, Bonn, Germany. tesfaye@itz.uni-bonn.de
Ponsuksili
Siriluck
S
Wimmers
Klaus
K
Gilles
Markus
M
Schellander
Karl
K
D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D002417:Cattle; D018076:DNA, Complementary; D047108:Embryonic Development; D005314:Embryonic and Fetal Development / Q000235:genetics*; D020224:Expressed Sequence Tags; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D009028:Morula / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012333:RNA, Messenger