Correct reprogramming of epigenetic marks in the donor nucleus is a prerequisite for successful cloning by somatic cell transfer (SCT). In several mammalian species, repressive histone (H) lysine (K) trimethylation (me3) marks, in particular H3K9me3, form a major barrier to somatic cell reprogramming into pluripotency and totipotency. We engineered bovine embryonic fibroblasts (BEFs) for the doxycycline-inducible expression of a biologically active, truncated form of murine Kdm4b, a demethylase that removes H3K9me3 and H3K36me3 marks. Upon inducing Kdm4b, H3K9me3 and H3K36me3 levels were reduced about 3-fold and 5-fold, respectively, compared with noninduced controls. Donor cell quiescence has been previously associated with reduced somatic trimethylation levels and increased cloning efficiency in cattle. Simultaneously inducing Kdm4b expression (via doxycycline) and quiescence (via serum starvation) further reduced global H3K9me3 and H3K36me3 levels by a total of 18-fold and 35-fold, respectively, compared with noninduced, nonstarved control fibroblasts. Following SCT, Kdm4b-BEFs reprogrammed significantly better into cloned blastocysts than noninduced donor cells. However, detrimethylated donors and sustained Kdm4b-induction during embryo culture did not increase the rates of postblastocyst development from implantation to survival into adulthood. In summary, overexpressing Kdm4b in donor cells only improved their reprogramming into early preimplantation stages, highlighting the need for alternative experimental approaches to reliably improve somatic cloning efficiency in cattle.

中文翻译：

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靶向组蛋白去甲基化可改善体细胞重编程为克隆囊胚，但不能改善植入后牛受孕。

供体细胞核中表观遗传标记的正确重编程是通过体细胞转移 (SCT) 成功克隆的先决条件。在几个哺乳动物物种中，抑制性组蛋白 (H) 赖氨酸 (K) 三甲基化 (me3) 标记，特别是 H3K9me3，形成了体细胞重编程为多能性和全能性的主要障碍。我们设计了牛胚胎成纤维细胞 (BEF)，用于多西环素诱导表达的生物活性、截短形式的鼠 Kdm4b，这是一种去除 H3K9me3 和 H3K36me3 标记的去甲基化酶。在诱导Kdm4b 时，与非诱导对照相比，H3K9me3 和 H3K36me3 水平分别降低了约 3 倍和 5 倍。供体细胞静止以前与牛的体细胞三甲基化水平降低和克隆效率提高有关。与非诱导、非饥饿的对照成纤维细胞相比，同时诱导Kdm4b表达（通过多西环素）和静止（通过血清饥饿）进一步降低了整体 H3K9me3 和 H3K36me3 水平，总共分别降低了 18 倍和 35 倍。在 SCT 之后，Kdm4b -BEFs 重编程到克隆的囊胚中的效果明显好于未诱导的供体细胞。然而，去三甲基化供体和持续的Kdm4b-胚胎培养过程中的诱导不会增加从植入到存活到成年的胚泡后发育率。总之，在供体细胞中过表达Kdm4b只会改善它们进入早期植入前阶段的重编程，突出表明需要替代实验方法来可靠地提高牛的体细胞克隆效率。