Generation of transmitochodrial cybrids using a microfluidic device
Generation of transmitochodrial cybrids using a microfluidic device
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使用微流体装置生成传输细胞杂种
DOI：
10.1016/j.yexcr.2022.113233
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发表时间：
2022
影响因子：
3.7
通讯作者：
Yoshikazu Yonemitsu
中科院分区：
文献类型：
--
作者：
Ken-Ichi Wada*;Kazuo Hosokawa;Yoshihiro Ito;Mizuo Maeda;Yui Harada;Yoshikazu Yonemitsu
线粒体克隆是实现同质线粒体DNA(MtDNA)突变的一种有前景的方法。我们之前开发了一种微流控装置，通过在线粒体DNA完整的细胞和无线粒体DNA的细胞(ρ0)之间通过微隧道促进细胞质连接来执行单个线粒体的转移。在本研究中，我们描述了一种使用微流控装置产生传递线粒体杂交体的方法。用微流控装置实现Hela细胞与胸苷激酶缺陷型ρ0143B细胞之间的线粒体转移后，用不含丙酮酸和尿苷(PU)、添加5-溴-2‘-脱氧尿苷的培养液进行选择性培养。得到的细胞含有HeLa线粒体DNA和143B核，但这些细胞中都没有143B线粒体DNA和HeLa核。此外，这些细胞的乳酸产量低于亲本ρ0143B细胞，细胞生长所需的PU营养缺陷性消失。这些结果表明使用微流控装置成功地产生了传递线粒体胞质。此外，在微流控装置中，我们通过去除未融合的ρ0细胞和基于嘌呤霉素的选择，成功地在添加PU的条件下选择性地收获了产生的传递线粒体杂交体。
Mitochondrial cloning is a promising approach to achieve homoplasmic mitochondrial DNA (mtDNA) mutations. We previously developed a microfluidic device that performs single mitochondrion transfer from a mtDNA-intact cell to a mtDNA-less (ρ0) cell by promoting cytoplasmic connection through a microtunnel between them. In the present study, we described a method for generating transmitochondrial cybrids using the microfluidic device. After achieving mitochondrial transfer between HeLa cells and thymidine kinase-deficient ρ0143B cells using the microfluidic device, selective culture was carried out using a pyruvate and uridine (PU)-absent and 5-bromo-2′-deoxyuridine-supplemented culture medium. The resulting cells contained HeLa mtDNA and 143B nuclei, but both 143B mtDNA and HeLa nuclei were absent in these cells. Additionally, these cells showed lower lactate production than parent ρ0143B cells and disappearance of PU auxotrophy for cell growth. These results suggest successful generation of transmitochondrial cybrids using the microfluidic device. Furthermore, we succeeded in selective harvest of generated transmitochondrial cybrids under a PU-supplemented condition by removing unfused ρ0cells with puromycin-based selection in the microfluidic device.