Sá André Luiz, Sampaio Rafael V, da Costa Almeida Nathália Nogueira, Sangalli Juliano Rodrigues, Brito Karynne Nazaré Lins, Bressan Fabiana Fernandes, Rissino Joirge Dores, do Socorro Damasceno Santos Simone, Meirelles Flavio Vieira, Ohashi Otávio Mitio, Dos Santos Miranda Moysés
1 Laboratório de Fecundação In Vitro, Instituto de Ciências Biológicas, Universidade Federal do Pará , Belém, Brazil .
2 Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo , Pirassununga, Brazil .
Cell Reprogram. 2017 Oct;19(5):294-301. doi: 10.1089/cell.2016.0063. Epub 2017 Aug 23.
Somatic cell nuclear transfer (SCNT) success is partially hindered by the low epigenetic reprogramming efficiency of the donor cell. Previous studies suggest cellular heterogeneity among donor nuclei in regard to reprogramming potential, which precludes comparison among different strategies to increase cloning success. In this context, we evaluated the effect of using clonal cell populations (CPs) of bovine adult fibroblasts established by single-cell plating in SCNT. Different CPs were evaluated in regard to proliferation rate, senescence level, and chromosome stability, as well as for POU5F1 (POU class 5 homeobox 1) mRNA expression levels. In total, 9 of 24 CPs (37.5%) were successfully expanded in vitro up to the fourth passage and shown to proliferate following cryopreservation, at which time cell analyses were performed. The use of a CP with low senescence level, normal karyotype, and highest POU5F1 expression levels did not improve embryo development rates or quality following SCNT. As previously suggested, this study supports the notion that levels of POU5F1 expression in the donor nucleus do not impact the SCNT results. Notably, the single-cell seeding approach used herein to isolate CPs may be extended to the evaluation of additional predictor markers of reprogrammability success for SCNT in future experiments.
体细胞克隆（SCNT）的成功率部分受到供体细胞表观遗传重编程效率低下的阻碍。先前的研究表明，供体细胞核在重编程潜力方面存在细胞异质性，这使得不同提高克隆成功率策略之间无法进行比较。在此背景下，我们评估了在SCNT中使用通过单细胞铺板建立的牛成年成纤维细胞克隆细胞群体（CPs）的效果。对不同的CPs进行了增殖率、衰老水平、染色体稳定性以及POU5F1（POU类5型同源盒1）mRNA表达水平的评估。总共24个CPs中有9个（37.5%）在体外成功扩增至第4代，并在冷冻保存后显示出增殖能力，此时进行细胞分析。使用衰老水平低、核型正常且POU5F1表达水平最高的CPs并不能提高SCNT后的胚胎发育率或质量。如先前所示，本研究支持供体细胞核中POU5F1表达水平不影响SCNT结果这一观点。值得注意的是，本文中用于分离CPs的单细胞接种方法可能会在未来实验中扩展到对SCNT重编程成功的其他预测标志物的评估。
