Giraldo Angelica M, Hylan Darin A, Ballard Casey B, Purpera Megan N, Vaught Todd D, Lynn John W, Godke Robert A, Bondioli Kenneth R
School of Animal Sciences, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803, USA.
Biol Reprod. 2008 May;78(5):832-40. doi: 10.1095/biolreprod.107.066662. Epub 2008 Jan 16.
Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of differentiated cells as the donor karyoplast. Blastocyst production and development to term of cloned embryos has been hypothesized to differ between population doublings of the same cell line as a consequence of changes in the levels of DNA methyltransferase 1 (DNMT1) and methylated DNA during in vitro culture. The objective of this study was to determine embryo production, developmental potential, and gene expression patterns of prehatched and posthatched embryos generated using donor cells with different levels of DNMT1 transcript. Day 7 embryos generated using donor cells with high and low levels of DNMT1 mRNA were transferred to recipient cows. Embryos recovered on Day 13 were morphologically characterized or used for gene expression analysis of DNMT, INFT, and MHC1. A higher proportion of 8- to 16-cell embryos developed to the blastocyst stage when cells with low levels of DNMT1 mRNA were used as donor nuclei. Day 13 NT embryos generated using donor cells with decreased levels of DNMT1 mRNA and capable of developing beyond the 8- to 16-cell stage produced a larger number of apparently developing embryos, larger conceptuses, and a higher expression of DNMT3A transcript than NT embryos reconstructed using cells with high levels of DNMT1 mRNA. However, abnormal gene expression of DNMT, INFT, and MHC1 was noted in the majority of cloned embryos, indicating inefficient nuclear reprogramming and retarded embryo development. Furthermore, aberrant DNMT1 expression may partially contribute to the inefficient nuclear reprogramming observed in cloned embryos.
有证据表明，核移植（NT）胚胎无法正常发育至少部分可归因于使用分化细胞作为供体核质体。由于体外培养过程中DNA甲基转移酶1（DNMT1）水平和甲基化DNA的变化，同一细胞系的群体倍增之间，克隆胚胎的囊胚产生和发育至足月被认为存在差异。本研究的目的是确定使用具有不同DNMT1转录水平的供体细胞产生的孵化前和孵化后胚胎的胚胎产量、发育潜力和基因表达模式。将使用高、低水平DNMT1 mRNA的供体细胞产生的第7天胚胎移植到受体母牛体内。在第13天回收的胚胎进行形态学表征或用于DNMT、INFT和MHC1的基因表达分析。当使用低水平DNMT1 mRNA的细胞作为供体核时，更高比例的8至16细胞胚胎发育到囊胚阶段。使用低水平DNMT1 mRNA且能够发育超过8至16细胞阶段的供体细胞产生的第13天NT胚胎，与使用高水平DNMT1 mRNA的细胞重建的NT胚胎相比，产生了更多明显发育的胚胎、更大的孕体和更高的DNMT3A转录本表达。然而，在大多数克隆胚胎中发现了DNMT、INFT和MHC1的异常基因表达，表明核重编程效率低下和胚胎发育迟缓。此外，异常的DNMT1表达可能部分导致克隆胚胎中观察到的核重编程效率低下。
