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
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
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DOI：
10.1002/mrd.1010
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发表时间：
2001-05
影响因子：
2.5
通讯作者：
S. J. Rzucidlo;J. Gibbons;S. Stice
中科院分区：
文献类型：
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作者：
S. J. Rzucidlo;J. Gibbons;S. Stice
一种新的限制性片段差异显示（RFDD）RT-PCR已被用于比较在存在（10%）或不存在胎牛血清（FCS）的情况下体外成熟的牛卵母细胞中mRNA表达的模式。从成熟和裸露的卵母细胞中提取的总RNA使用展示轮廓试剂盒（Display System Biotech）进行处理。在6%聚丙烯酰胺凝胶上分离RFDD RT-PCR产物，并使用Storm 860扫描仪进行分析。从凝胶中切下代表潜在差异表达片段的选定条带并重新扩增。将大小与原始片段匹配的再扩增片段克隆到TA载体中并测序。最初，10和15个差异表达的片段，分别从存在和不存在FCS的成熟卵母细胞中分离。10个片段中的8个和15个片段中的10个被成功地重新扩增，这通过与原始片段的大小相似性来证明。最后，从每组测序的六个插入片段的大小与相应的原始片段以及再扩增片段的大小相匹配。序列比较检索显示，一些分离片段与18 s核糖体RNA、牛载脂蛋白A-I、牛核糖体DNA、人CGI-79 mRNA、人Ab 1-interactor蛋白和牛卫星DNA相似。其他测序片段可能代表新基因。我们发现，RFDD RT-PCR可以有效地应用于牛卵母细胞中的基因表达模式的对比，并且在成熟间隔期间存在或不存在FCS会影响成熟牛卵母细胞中的基因表达模式。摩尔Reprod. Dev. 59：90-96，2001.© 2001 Wiley利斯公司
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 bysize 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. Mol. Reprod. Dev. 59:90–96, 2001. © 2001 Wiley‐Liss, Inc.