Department of Animal, Dairy, and Veterinary Sciences, Utah State University.
Reproductive Sciences, Beckman Center for Conservation Research, San Diego Zoo Wildlife Alliance.
J Vis Exp. 2022 Jul 6(185). doi: 10.3791/64060.
Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes' zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve's regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (± standard deviation) of 137,904 ± 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 ± 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 ± 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P < 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.
种间体细胞核移植（iSCNT）可用于拯救濒危物种，但重构胚胎中存在两种不同的线粒体 DNA（mtDNA）群体：一种存在于受体卵质中，另一种存在于供体细胞中。这种线粒体异质性可能导致胚胎和胎儿的发育问题。手工克隆方案包括卵母细胞二分法，它可以用来降低 mtDNA 拷贝数，从而降低重构胚胎中的线粒体异质性程度。去透明带的成熟牛卵母细胞的离心产生了卵母细胞一个极的可见线粒体密集区带。卵母细胞的透明带被暴露在蛋白酶溶液中去除。使用微刀片进行二分法，去除可见的线粒体部分。qPCR 用于定量从整个卵母细胞和二分卵质体提取的 DNA 样本中的 mtDNA，提供二分法前后 mtDNA 拷贝数的比较。使用循环阈值值、标准曲线回归线公式和包含 mtDNA PCR 产物和基因组 PCR 产物各自大小的比率来计算拷贝数。一个牛卵母细胞的平均 mtDNA 拷贝数（±标准偏差）为 137904 ± 94768（n = 38）。一个线粒体耗竭的卵质体的平均 mtDNA 拷贝数为 8442 ± 13806（n = 33）。富含线粒体的卵质体中存在的平均 mtDNA 拷贝数为 79390 ± 58526 mtDNA 拷贝（n = 28）。这些计算平均值之间的差异表明，与原始卵母细胞相比，离心和随后的二分法可以显著降低线粒体耗竭卵质体中的 mtDNA 拷贝数（P < 0.0001，通过单向方差分析确定）。mtDNA 的减少应降低重构胚胎中的线粒体异质性程度，可能促进标准胚胎和胎儿发育。补充供体细胞的线粒体提取物对于实现胚胎的成功发育也可能是必不可少的。
