Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine.
Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine.
复制标题
M 期雪貂成纤维细胞的核转移与微管抑制剂 demecolcine 同步。
DOI：
10.1002/jez.a.234
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发表时间：
2005
期刊：
影响因子：
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通讯作者：
Engelhardt,JohnF
中科院分区：
文献类型：
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作者：
Li,Ziyi;Chen,Xin;Sun,Xingshen;Zhou,Qi;Chen,Juan;Leno,GregoryH;Engelhardt,JohnF
核移植（NT）后重建胚胎的发育似乎取决于多种因素，包括供体核和受体卵母细胞之间的细胞周期同步。在这里，我们使用微管抑制剂 demecolcine 使雪貂成纤维细胞同步于中期（M 期），以便将其细胞周期位置与受体卵母细胞在 NT 时的细胞周期位置相匹配。成纤维细胞取自 28 天的胎儿，并在 NT 前培养 1-30 天。在培养不同时间后，用 0.05 μg/ml 的 demecolcine 处理成纤维细胞培养物 3 小时或过夜（14-16 小时），以确定 M 期同步的最佳条件。经 demecolcine 处理的培养物中 G2/M 期细胞的百分比显着高于未处理培养物中的百分比（P<0.05）。通过有丝分裂抖动收集最佳同步的 M 期成纤维细胞，并评估其在 NT 中的有效性。通过电融合或显微注射重建的 M 期体细胞来源的 NT 胚胎以相似的比率进行植入并形成胎儿（分别为 5.4% vs. 3.4% 和 1.8% vs. 1.2%）；然而，NT 胚胎没有发育至足月。总而言之，这些数据说明了两个要点。首先，demecolcine 治疗有效地将雪貂成纤维细胞同步于细胞周期的 M 期；其次，这些体细胞能够在 NT 后驱动胚胎发育。我们的结果应该有助于克隆雪貂作为人类肺部疾病（例如流感和囊性纤维化）动物模型的发展。过期。动物园。 303A:1126–1234, 2005。© 2005 Wiley‐Liss, Inc.
The development of reconstructed embryos following nuclear transfer (NT) appears to be dependent upon a variety of factors, including cell cycle synchronization between the donor nucleus and recipient oocyte. Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in metaphase (M‐phase) in order to match their cell cycle position with that of the recipient oocyte at the time of NT. The fibroblasts were obtained from 28‐day fetuses and cultured for 1–30 days prior to NT. Fibroblast cultures were treated with 0.05 μg/ml of demecolcine for 3 hr or overnight (14–16 hr) after various times in culture to determine the optimal conditions for M‐phase synchronization. The percentage of G2/M‐phase cells in demecolcine‐treated cultures was significantly greater than that found in untreated cultures (P<0.05). Optimally synchronized M‐phase fibroblasts were collected by mitotic shake‐off and evaluated for their effectiveness in NT. M‐phase somatic cell‐derived NT embryos reconstituted by electrofusion or microinjection underwent implantation and formed fetuses at similar rates (5.4% vs. 3.4%, and 1.8% vs. 1.2%, respectively); however, no NT embryos developed to term. In summary, these data demonstrate two important points. First, demecolcine treatment effectively synchronizes ferret fibroblasts in M‐phase of the cell cycle; and second, these somatic cells are capable of driving embryo development following NT. Our results should facilitate the development of cloned ferrets as an animal model for human lung disease such as influenza and cystic fibrosis.J. Exp. Zool. 303A:1126–1234, 2005.© 2005 Wiley‐Liss, Inc.