Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer.
Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer.
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DOI：
10.1186/1471-213x-7-141
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发表时间：
2007-12-21
影响因子：
--
通讯作者：
Steinborn, Ralf
中科院分区：
文献类型：
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作者：
Burgstaller, Joerg P.;Schinogl, Pamela;Dinnyes, Andras;Mueller, Mathias;Steinborn, Ralf
据报道，体细胞核移植(SCNT)获得的克隆羊Dolly和其他9个绵羊克隆的线粒体DNA(MtDNA)只由受体卵母细胞mtDNA组成，核供体细胞没有检测到任何mtDNA贡献。在牛、鼠和猪中，几个或大部分克隆显示核供体mtDNA的传递导致线粒体异质性。为了阐明供体mtDNA在绵羊克隆中的不同传播模式，我们分析了从胎儿成纤维细胞克隆的7个胎儿和5只羔羊的mtDNA组成。用于SCNT的3个胎儿成纤维细胞供体细胞的线粒体DNA拷贝数较低(A：753±54，B：292±33，C：561±88)。用定量扩增难扩增突变系统(ARMS)聚合酶链式反应(ARMS-qPCR)测定供受者卵母细胞线粒体DNA的比例。为了定量检测频率低于0.1%的SNP变异体，我们建立了限制性内切酶介导的选择性定量聚合酶链式反应(REMS-qPCR)。我们首次报道了SCNT来源的绵羊克隆中的受体卵母细胞/核供体mtDNA异质性的第一例(n=4胎，n=3只羔羊)，表明没有物种效应阻碍绵羊核供体mtDNA传递给体细胞克隆后代。大多数异质克隆表现出低水平的异质性(0.1%~0.9%，n=6)，表明亲本mtDNA的中性传递。1例出现高度异质性(6.8%~46.5%)。该克隆具有不同的受体卵母细胞来源的mtDNA基因，与供体相比有三个罕见的氨基酸变化，包括在一个进化保守的位置上的一个替换。我们的研究使用了最先进的mtDNA定量技术，如ARMS-qPCR和新型的REMS-qPCR，首次证明了供体mtDNA进入体细胞克隆的过程。在检测的12个克隆中有7个检测到mtDNA异质性，除1例外，所有克隆的核供体细胞对mtDNA的贡献都不到1%，表明中性分离。
The mitochondrial DNA (mtDNA) of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT) was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts. The three fetal fibroblast donor cells used for SCNT harboured low mtDNA copy numbers per cell (A: 753 ± 54, B: 292 ± 33 and C: 561 ± 88). The ratio of donor to recipient oocyte mtDNAs was determined using a quantitative amplification refractory mutation system (ARMS) PCR (i.e. ARMS-qPCR). For quantification of SNP variants with frequencies below 0.1% we developed a restriction endonuclease-mediated selective quantitative PCR (REMS-qPCR). We report the first cases (n = 4 fetuses, n = 3 lambs) of recipient oocyte/nuclear donor mtDNA heteroplasmy in SCNT-derived ovine clones demonstrating that there is no species-effect hindering ovine nucleus-donor mtDNA from being transmitted to the somatic clonal offspring. Most of the heteroplasmic clones exhibited low-level heteroplasmy (0.1% to 0.9%, n = 6) indicating neutral transmission of parental mtDNAs. High-level heteroplasmy (6.8% to 46.5%) was observed in one case. This clone possessed a divergent recipient oocyte-derived mtDNA genotype with three rare amino acid changes compared to the donor including one substitution at an evolutionary conserved site. Our study using state-of-the-art techniques for mtDNA quantification, like ARMS-qPCR and the novel REMS-qPCR, documents for the first time the transmission of donor mtDNA into somatic sheep clones. MtDNA heteroplasmy was detected in seven of 12 clones tested, whereby all but one case revealed less than 1% mtDNA contribution from the nuclear donor cell suggesting neutral segregation.