Comparison by restriction fragment differential display RT-PCR of gene expression pattern in bovine oocytes matured in the presence or absence of fetal calf serum.
A novel restriction fragment differential display (RFDD) RT-PCR has been used to compare patterns of mRNA expression in bovine oocytes matured in vitro in the presence (10%) or absence of fetal calf serum (FCS). Total RNA extracted from matured and denuded oocytes was processed using display Profile kit (Display System Biotech). RFDD RT-PCR products were separated on 6% polyacrylamide gel and analyzed using a Storm 860 scanner. Selected bands representing potentially differentially expressed fragments were excised from the gel and re-amplified. Re-amplified fragments with size matched to the original fragment were cloned into the TA vector and sequenced. Initially, 10 and 15 differentially expressed fragments were isolated from oocytes matured in the presence and absence of FCS, respectively. Eight out of 10 and 10 out of 15 fragments were re-amplified successfully as evidenced by size similarity to the original fragments. Finally, the size of six inserts sequenced from each group matched the size of corresponding original as well as re-amplified fragments. Sequence comparison search revealed similarity of some isolated fragments to 18s ribosomal RNA, bovine apolipoprotein A-I, bovine mitochondrion DNA, human CGI-79 mRNA, human Ab1-interactor protein, and bovine satellite DNA. The other sequenced fragments may represent novel genes. We showed that RFDD RT-PCR can be effectively applied to contrast gene expression pattern in bovine oocytes and that presence or absence of FCS during maturation interval affects gene expression pattern in matured bovine oocytes.
Rzucidlo
S J
SJ
Department of Animal and Dairy Science, University of Georgia, Athens, Georgia 30602, USA.
Gibbons
J
J
Stice
S L
SL
D000818:Animals; D001769:Blood; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D016895:Culture Media, Serum-Free; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D009865:Oocytes / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016895:Culture Media, Serum-Free