Betthauser Jeffery M, Pfister-Genskow Martha, Xu Hongzhi, Golueke Paul J, Lacson Jenine C, Koppang Richard W, Myers Cena, Liu Bing, Hoeschele Ina, Eilertsen Kenneth J, Leno Gregory H
Infigen, Inc., Madison, Wisconsin 53718, USA.
Mol Reprod Dev. 2006 Aug;73(8):977-86. doi: 10.1002/mrd.20493.
Successful cloning by somatic cell nuclear transfer (NT) involves an oocyte-driven transition in gene expression from an inherited somatic pattern, to an embryonic form, during early development. This reprogramming of gene expression is thought to require the remodeling of somatic chromatin and as such, faulty and/or incomplete chromatin remodeling may contribute to the aberrant gene expression and abnormal development observed in NT embryos. We used a novel approach to supplement the oocyte with chromatin remodeling factors and determined the impact of these molecules on gene expression and development of bovine NT embryos. Nucleoplasmin (NPL) or polyglutamic acid (PGA) was injected into bovine oocytes at different concentrations, either before (pre-NT) or after (post-NT) NT. Pre-implantation embryos were then transferred to bovine recipients to assess in vivo development. Microinjection of remodeling factors resulted in apparent differences in the rate of blastocyst development and in pregnancy initiation rates in both NPL- and PGA-injected embryos, and these differences were dependent on factor concentration and/or the time of injection. Post-NT NPL-injected embryos that produced the highest rate of pregnancy also demonstrated differentially expressed genes relative to pre-NT NPL embryos and control NT embryos, both of which had lower pregnancy rates. Over 200 genes were upregulated following post-NT NPL injection. Several of these genes were previously shown to be downregulated in NT embryos when compared to bovine IVF embryos. These data suggest that addition of chromatin remodeling factors to the oocyte may improve development of NT embryos by facilitating reprogramming of the somatic nucleus.
通过体细胞核移植（NT）成功克隆涉及在早期发育过程中，卵母细胞驱动基因表达从遗传的体细胞模式向胚胎形式转变。这种基因表达的重编程被认为需要对体细胞核染色质进行重塑，因此，有缺陷和/或不完全的染色质重塑可能导致NT胚胎中出现异常的基因表达和异常发育。我们采用了一种新方法，向卵母细胞中补充染色质重塑因子，并确定这些分子对牛NT胚胎基因表达和发育的影响。将核质蛋白（NPL）或聚谷氨酸（PGA）以不同浓度注射到牛卵母细胞中，在NT之前（NT前）或之后（NT后）。然后将植入前胚胎移植到牛受体中，以评估体内发育情况。显微注射重塑因子导致NPL和PGA注射胚胎的囊胚发育率和妊娠起始率出现明显差异，这些差异取决于因子浓度和/或注射时间。NT后注射NPL的胚胎产生了最高的妊娠率，相对于NT前NPL胚胎和对照NT胚胎，其也显示出差异表达基因，后两者的妊娠率较低。NT后注射NPL后有超过200个基因上调。与牛体外受精胚胎相比，这些基因中的几个先前已被证明在NT胚胎中下调。这些数据表明，向卵母细胞中添加染色质重塑因子可能通过促进体细胞核的重编程来改善NT胚胎的发育。
