Viagen, Inc., 12357-A Riata Trace Parkway, Austin, TX 78727, USA.
Anim Reprod Sci. 2011 May;125(1-4):148-57. doi: 10.1016/j.anireprosci.2011.04.004. Epub 2011 Apr 21.
Somatic cell nuclear transfer (SCNT) technology has become a powerful tool for reproductive biology to preserve and propagate valuable genetics for livestock. Embryo production through SCNT involves enucleation of the oocyte and insertion of a somatic donor cell into the oocyte. These procedures lead to a few small openings on the zona pellucida that may elevate risk of viral infection for the produced SCNT embryos. The oocytes used for SCNT are mainly obtained from abattoirs where viral contamination is almost inevitable. Therefore, a systematic evaluation of risk of disease transmission through SCNT embryo production is necessary prior large scale implementation of this technology in the livestock industry. The objective of the current study was to evaluate the risk of disease transmission via SCNT embryo production and transfer by testing for the presence of porcine reproductive and respiratory syndrome virus (PRRSV) throughout the process of SCNT embryo production. The presence of PRRSV in each step of SCNT embryo production, from donor cells to pre-implantation SCNT embryo culture, was carefully examined using a real-time PCR assay with a sensitivity of five copies per-reaction. All 114 donor cell lines derived from pig skin tissue over a period of 7 years in our facility tested negative for PRRSV. Out of the 68 pooled follicular fluid samples collected from 736 ovaries, only four (5.9%) were positive indicating a small amount of viral molecule present in the oocyte donor population. All 801 Day 7 SCNT embryos produced in four separate trials and over 11,571 washed oocytes obtained in 67 batches over 10 months tested negative. These oocytes were collected from multiple abattoirs processing animals from areas with high density of pig population and correspond to a donor population of over 5828 individuals. These results indicate that the oocytes from abattoirs were free of PRRSV infection and therefore could be safely used for in vitro embryo production. Additionally, the established SCNT embryo production system, including donor cell testing, oocytes decontamination, and pathogen free embryo reconstruction and culturing, bears no risk of PRRSV transmission.
体细胞核移植 (SCNT) 技术已成为生殖生物学的一种强大工具，可用于保存和传播家畜有价值的遗传物质。通过 SCNT 进行胚胎生产涉及卵母细胞去核和将体细胞核供体细胞插入卵母细胞。这些程序会导致透明带出现几个小开口，这可能会增加产生的 SCNT 胚胎感染病毒的风险。用于 SCNT 的卵母细胞主要从屠宰场获得，那里几乎不可避免地存在病毒污染。因此，在大规模将这项技术应用于畜牧业之前，有必要对通过 SCNT 胚胎生产传播疾病的风险进行系统评估。本研究的目的是通过检测 SCNT 胚胎生产过程中猪繁殖与呼吸综合征病毒 (PRRSV) 的存在来评估通过 SCNT 胚胎生产和转移传播疾病的风险。使用实时 PCR 检测法，在每个 SCNT 胚胎生产步骤中（从供体细胞到植入前 SCNT 胚胎培养），仔细检测了 PRRSV 的存在，该检测法的灵敏度为每个反应 5 个拷贝。在我们实验室的 7 年内，从猪皮肤组织中获得的 114 个供体细胞系均未检测到 PRRSV。从 736 个卵巢中收集的 68 个卵泡液样本中，只有 4 个（5.9%）呈阳性，表明卵母细胞供体群体中存在少量病毒分子。在四个独立的试验中生产的 801 个第 7 天的 SCNT 胚胎和在 10 个月内从 67 批中获得的超过 11571 个洗涤卵母细胞均呈阴性。这些卵母细胞来自多个屠宰场，这些屠宰场处理来自猪群密度高的地区的动物，对应于超过 5828 个人的供体群体。这些结果表明，来自屠宰场的卵母细胞没有 PRRSV 感染，因此可以安全地用于体外胚胎生产。此外，建立的 SCNT 胚胎生产系统，包括供体细胞检测、卵母细胞去污以及无病原体胚胎重建和培养，没有 PRRSV 传播的风险。
