El-Halawany Nermin, Ponsuksili Siriluck, Wimmers Klaus, Gilles Markus, Tesfaye Dawit, Schellander Karl
Institute of Animal Breeding Science, University of Bonn, Bonn, Germany.
Reprod Fertil Dev. 2004;16(8):753-62. doi: 10.1071/rd04041.
The main objective of the present study was to analyse the quantitative expression pattern of genes from a subtracted blastocyst transcriptome throughout the preimplantation developmental stages of in vitro-produced bovine oocytes and embryos. For this purpose, Day 5 morula (M) cDNAs were subtracted from Day 7 blastocyst (B) cDNAs (B-M) and used to establish a B-M subtracted cDNA library, as reported previously. From the total generated clones, 19 were analysed quantitatively. The mRNA samples isolated from pools of immature oocytes (n = 150), mature oocytes (n = 150) and two-cell (n = 80), four-cell (n = 40), eight-cell (n = 20), morula (n = 6) and blastocyst (n = 3) embryos were reverse transcribed and subjected to real-time polymerase chain reaction (PCR) using sequence-specific primers and SYBR green as the DNA dye. A relative standard curve method was used to analyse the real-time data taking the morula stage as a calibrator. Applying suppression subtractive hybridisation (SSH), a total of 71 clones, which represent 33 different expressed sequence tags, were generated and available for analysis. Most transcripts were analysed for the first time in bovine embryogenesis. The real-time PCR has validated the results of SSH positively for 84% (16/19) of transcripts, whereas 16% (3/19) showed deviation in the expression pattern from the one seen during SSH. Several transcript-specific expression patterns were observed for genes that play decisive roles in bovine embryogenesis. In addition to identification, accurately quantifying the expression profiles of transcripts during development will pave the way towards understanding the molecular mechanisms of embryogenesis and their potential role in early embryo development. Most importantly, the present study has contributed to the enrichment of bovine embryo gene collection by generating new transcripts involved in bovine embryo development.
本研究的主要目的是分析体外生产的牛卵母细胞和胚胎植入前发育阶段中，来自消减囊胚转录组的基因的定量表达模式。为此，如先前报道，从第7天囊胚（B）的cDNA中减去第5天桑葚胚（M）的cDNA（B-M），并用于建立B-M消减cDNA文库。从总共产生的克隆中，对19个进行了定量分析。从未成熟卵母细胞池（n = 150）、成熟卵母细胞池（n = 150）以及二细胞（n = 80）、四细胞（n = 40）、八细胞（n = 20）、桑葚胚（n = 6）和囊胚（n = 3）胚胎中分离的mRNA样本进行逆转录，并使用序列特异性引物和SYBR green作为DNA染料进行实时聚合酶链反应（PCR）。采用相对标准曲线法，以桑葚胚阶段作为校准物来分析实时数据。应用抑制性消减杂交（SSH），共产生了71个代表33个不同表达序列标签的克隆，可供分析。大多数转录本是首次在牛胚胎发生过程中进行分析。实时PCR对84%（16/19）的转录本的SSH结果进行了正向验证，而16%（3/19）的转录本在表达模式上与SSH期间观察到的情况存在偏差。观察到了在牛胚胎发生中起决定性作用的基因的几种转录本特异性表达模式。除了鉴定之外，准确量化发育过程中转录本的表达谱将为理解胚胎发生的分子机制及其在早期胚胎发育中的潜在作用铺平道路。最重要的是，本研究通过产生参与牛胚胎发育的新转录本，为牛胚胎基因库的丰富做出了贡献。
