Molecular Animal Breeding and Biotechnology, and Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Munich, Germany.
BMC Biotechnol. 2013 May 20;13:43. doi: 10.1186/1472-6750-13-43.
Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency.
109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring.
Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.
利用基因工程供体细胞进行体细胞核移植（SCNT）是目前用于生成用于生物医学研究的定制化猪模型的最广泛应用策略。尽管这种方法可实现与啮齿动物模型相似的遗传修饰谱，但在活克隆仔猪方面的结果却差异很大。在这项研究中，我们旨在全面分析对生成基因工程猪效率有重大影响的环境和实验因素。基于三年内进行的 274 次 SCNT 实验（总共将 18649 个重构胚胎移植到 193 个受体中）的相当大的数据集，我们评估了季节、遗传修饰类型、供体细胞来源、克隆轮数以及对早期发育的克隆胚胎进行预选对克隆效率的相对贡献。
109 个（56%）受体怀孕，其中 85 个（78%）分娩。在 318 头克隆仔猪中，243 头（76%）存活，但只有 97 头（40%）临床健康且发育正常。死产仔猪的比例为 24%（75/318），另外 31%（100/318）的克隆仔猪在出生后不久死亡。将仅包括分娩的受体包括在内，定义为每转移的 SCNT 胚胎所产后代的数量，整体克隆效率为 3.95%。在冬季使用胎儿成纤维细胞或经过添加剂基因转移的肾细胞进行的 SCNT 实验导致活产和健康后代数量最多，而在夏季进行两次或更多轮次的克隆和核转移实验则降低了健康后代的数量。
尽管各个实验室之间单个因素的影响可能不同，但我们的结果和分析策略将有助于确定和优化对旨在生成基因工程猪模型的计划中克隆成功最关键的因素。
