Niemann H, Wrenzycki C
Department of Biotechnology, Institut für Tierzucht und Tierverhalten (FAL), Neustadt, Germany.
Theriogenology. 2000 Jan 1;53(1):21-34. doi: 10.1016/s0093-691x(99)00237-x.
Recent advances in molecular technology and in vitro production of bovine embryos have enabled studies of gene transcription in preimplantation embryos. On the basis of knowledge of the sequence of the selected gene, various modifications of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technology have been employed. Several lines of evidence in mouse and cattle indicate that expression patterns of genes from in vitro-produced embryos are not necessarily representative of those of in vivo embryos. An important gene that has been found to be expressed by in vivo-derived bovine blastocysts, but not in their in vitro-produced counterparts, is the Connexin43 gene that is crucial for maintenance of compaction. The bovine leukemia inhibitory factor (bLIF) and LIF-receptor-beta (LR-beta) genes were expressed by in vitro-produced embryos, but not in their in vivo counterparts. The heat shock protein gene 70.1 (Hsp 70.1) was upregulated by blastocysts produced in vitro compared to in vivo embryos, while the glucose transporter-1 mRNA (Glut-1) was downregulated by morulae produced in vitro as compared to in vivo-derived morulae. Furthermore, mRNA expression levels of a set of "marker genes" were shown to be affected by the presence or absence of serum in the culture medium. Most embryos grown under serum-free conditions had higher mRNA abundances than those cultured in serum-enriched medium. It is hypothesized that persistent alterations of the normal gene expression patterns may be responsible for the large offspring syndrome that is observed in approximately one third of the calves resulting from the transfer of in vitro-produced embryos. A primary candidate for such deviations may be an altered methylation pattern that can either lead to silencing or induction of a specific gene. Messenger RNA phenotyping of genes essential in early development provides a useful tool to assess the normality of the produced embryos and a tool to optimize in vitro culture conditions for bovine embryos.
分子技术和牛胚胎体外生产技术的最新进展使得对植入前胚胎中的基因转录进行研究成为可能。基于所选基因序列的知识，人们采用了逆转录聚合酶链反应（RT-PCR）技术的各种改进方法。小鼠和牛的多项证据表明，体外生产胚胎的基因表达模式不一定代表体内胚胎的表达模式。已发现体内来源的牛囊胚表达但体外生产的囊胚不表达的一个重要基因是连接蛋白43基因，它对维持致密化至关重要。牛白血病抑制因子（bLIF）和LIF受体β（LR-β）基因在体外生产的胚胎中表达，但在体内来源的胚胎中不表达。与体内胚胎相比，体外生产的囊胚上调了热休克蛋白基因70.1（Hsp 70.1），而与体内来源的桑椹胚相比，体外生产的桑椹胚下调了葡萄糖转运蛋白-1 mRNA（Glut-1）。此外，一组“标记基因”的mRNA表达水平显示受培养基中血清存在与否的影响。大多数在无血清条件下培养的胚胎比在富含血清的培养基中培养的胚胎具有更高的mRNA丰度。据推测，正常基因表达模式的持续改变可能是大约三分之一由体外生产胚胎移植产生的犊牛中观察到的大后代综合征的原因。这种偏差的一个主要候选因素可能是甲基化模式的改变，它可以导致特定基因的沉默或诱导。早期发育中必需基因的信使核糖核酸表型分析为评估所生产胚胎的正常性提供了一个有用的工具，也为优化牛胚胎的体外培养条件提供了一个工具。
