Successful reproductive cloning depends on obtaining intact donor nuclei from viable cells, ideally isolated by tissue biopsy of a living donor. However, owners and veterinarians often freeze deceased animals, which eventually causes damage to cellular micro-organelles due to the formation of intracellular water crystals. In the present study, we have reported the production of viable cloned puppies using donor nuclei of cells obtained from frozen carcasses. Five cases of deceased and frozen canine specimens were presented to be cloned. Skin fibroblast cell lines were successfully established for four specimens. Significant longer time was needed for the cell growth from frozen tissues (4 days) to reach 80% confluency compared to fresh tissue and frozen tissues frozen for 1- or 2-days. Similarly, SA-βgal positive cells (death cells) were significantly higher in frozen cells for 2- or 4- days compared to samples from fresh or frozen (1 day) sources. The cloning efficiency (CE) and the pregnancy rates (PR) of frozen cells were lower than those obtained from fresh or living donors (CE 2.4 ± 1.8% vs. 0.6 ± 0.3%, PR 21.7 ± 16.1% vs. 7.7 ± 5.3% for fresh vs. frozen, respectively). Here we demonstrate is the possibility to produce healthy offspring from cell lines obtained from frozen tissue collected post-mortem.

中文翻译：

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从没有冷冻保护剂冷冻的死后组织中克隆狗

成功的生殖性克隆取决于从活细胞中获得完整的供体细胞核，最好通过活体供体的组织活检分离。然而，主人和兽医经常冷冻死去的动物，最终由于细胞内水晶体的形成而对细胞微细胞器造成损害。在本研究中，我们报告了使用从冷冻尸体获得的细胞的供体细胞核生产可行的克隆幼犬。5 例已死亡和冷冻的犬类标本被提交克隆。四个标本成功建立了皮肤成纤维细胞系。与新鲜组织和冷冻 1 天或 2 天的冷冻组织相比，冷冻组织的细胞生长需要更长的时间（4 天）才能达到 80% 的融合。相似地，与新鲜或冷冻（1 天）来源的样品相比，冷冻细胞 2 或 4 天的 SA-βgal 阳性细胞（死亡细胞）显着更高。冷冻细胞的克隆效率 (CE) 和妊娠率 (PR) 低于从新鲜或活体供体获得的细胞 (CE 2.4 ± 1.8% vs. 0.6 ± 0.3%, PR 21.7 ± 16.1% vs. 7.7 ± 5.3%分别用于新鲜和冷冻）。在这里，我们展示了从死后收集的冷冻组织中获得的细胞系产生健康后代的可能性。