Identification and expression analysis of genes associated with bovine blastocyst formation
Identification and expression analysis of genes associated with bovine blastocyst formation
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DOI：
10.1186/1471-213x-7-64
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发表时间：
2007-06-08
影响因子：
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通讯作者：
Peelman, Luc J.
中科院分区：
文献类型：
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作者：
Goossens, Karen;Van Soom, Ann;Peelman, Luc J.
背景：正常的植入前胚胎发育包括第一次卵裂分裂、胚胎基因组激活、致密和囊胚形成等一系列过程，第一次谱系分化始于囊胚期滋养外胚层和内细胞团的形成。本研究的主要目的是检测、鉴定和分析与胚泡形成相关的基因，以帮助我们更好地了解这一过程。这些信息有助于改进体外胚胎生产程序。结果：构建了一个消减文库，该文库丰富了在囊胚期优先表达的转录本，而在2-细胞期和8-细胞期表达较少。获得了随机选择的65个克隆的序列信息。在植入前胚胎发育的3个阶段(2-细胞、8-细胞和囊胚)检测了12个候选基因的RNA表达水平，并与体内“金标准”胚胎的RNA表达水平进行了比较。9个转录本(75%)(KRT18、FN1、MYL6、ATP1B3、FTH1、HINT1、Slc25a5、ATP6V0B、RPL10)的RNA表达谱与消减cDNA克隆方法一致，而其余3个(25%)转录本(ACTN1、COPE、EEF1A1)在发育早期的RNA表达水平与囊胚期持平或甚至更高。此外，在体外和体内产生的胚胎之间，RNA的表达水平也有显著差异。通过免疫荧光标记，检测了KRT18、FN1和MYL6在牛着床前胚胎发育过程中的蛋白表达，并与RNA表达分析结果一致。结论：通过差减克隆获得了与胚泡形成相关的候选基因。对于几个候选基因，体内胚胎和体外产生的胚胎在基因表达上存在重要差异，反映了体外培养系统对胚胎基因表达的影响。RNA和蛋白质表达分析表明，KRT18、FN1和MYL6基因在着床前胚胎发育过程中存在差异表达，可作为牛胚泡形成的标志。
Background: Normal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation.First lineage differentiation starts at the blastocyst stage with the formation of the trophectoderm and the inner cell mass. The main objective of this study was the detection, identification and expression analysis of genes associated with blastocyst formation in order to help us better understand this process. This information could lead to improvements of in vitro embryo production procedures.Results: A subtractive cDNA library was constructed enriched for transcripts preferentially expressed at the blastocyst stage compared to the 2- cell and 8- cell stage. Sequence information was obtained for 65 randomly selected clones. The RNA expression levels of 12 candidate genes were determined throughout 3 stages of preimplantation embryo development ( 2- cell, 8- cell and blastocyst) and compared with the RNA expression levels of in vivo " golden standard" embryos using real- time PCR. The RNA expression profiles of 9 ( 75%) transcripts ( KRT18, FN1, MYL6, ATP1B3, FTH1, HINT1, SLC25A5, ATP6V0B, RPL10) were in agreement with the subtractive cDNA cloning approach, whereas for the remaining 3 ( 25%) ( ACTN1, COPE, EEF1A1) the RNA expression level was equal or even higher at the earlier developmental stages compared to the blastocyst stage. Moreover, significant differences in RNA expression levels were observed between in vitro and in vivo produced embryos. By immunofluorescent labelling, the protein expression of KRT18, FN1 and MYL6 was determined throughout bovine preimplantation embryo development and showed the same pattern as the RNA expression analyses.Conclusion: By subtractive cDNA cloning, candidate genes involved in blastocyst formation were identified. For several candidate genes, important differences in gene expression were observed between in vivo and in vitro produced embryos, reflecting the influence of the in vitro culture system on the embryonic gene expression. Both RNA and protein expression analysis demonstrated that KRT18, FN1 and MYL6 are differentially expressed during preimplantation embryo development and those genes can be considered as markers for bovine blastocyst formation.