Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model
Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model
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DOI：
10.1371/journal.pone.0235856
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发表时间：
2020-07-10
期刊：
影响因子：
3.7
通讯作者：
Ambrosio, Carlos Eduardo
中科院分区：
文献类型：
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作者：
de Oliveira, Vanessa Cristina;Mariano Junior, Clesio Gomes;Ambrosio, Carlos Eduardo
为了增加知识，必须深入研究大型动物模型中的基因编辑，以便将来在转化医学和食品生产中应用。线粒体转录因子A（TFAM）是HMGB亚家族的成员，与mtDNA启动子结合。该基因维持线粒体DNA，并且对于启动线粒体DNA转录是必需的。最近，我们通过CRISPR/Cas 9技术破坏牛成纤维细胞中的TFAM基因产生了一种新的细胞系。我们证明了CRISPR/Cas9设计通过杂合突变克隆的产生是有效的。在这种情况下，一旦该基因调节mtDNA复制特异性，该研究旨在确定编辑后的细胞是否能够在体外维持，并评估它们在培养物中连续传代后是否存在mtDNA拷贝和线粒体膜电位的变化。将编辑后的细胞在培养物中扩增，我们进行了生长曲线、倍增时间、细胞活力、线粒体DNA拷贝数和线粒体膜电位测定。编辑过程没有使细胞培养不可行，即使与对照相比细胞生长速率和活力降低，因为我们观察到当在补充有尿苷和丙酮酸的培养基中培养时细胞生长良好。它们还表现出典型的成纤维细胞样外观。用于确定mtDNA拷贝数的RT-qPCR显示，在不同细胞传代中，与未编辑的克隆（对照）相比，编辑的克隆减少。用Mitotracker绿色和红色的细胞染色表明与未编辑的细胞相比，编辑的细胞中红色荧光减少。因此，通过表征，我们证明了TFAM基因对线粒体维持至关重要，因为它干扰了不同细胞传代中线粒体DNA拷贝数的稳定性和膜电位，证实了杂合编辑细胞中线粒体活性的降低。
Gene editing in large animal models for future applications in translational medicine and food production must be deeply investigated for an increase of knowledge. The mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters. This gene maintains mtDNA, and it is essential for the initiation of mtDNA transcription. Lately, we generated a new cell line through the disruption of the TFAM gene in bovine fibroblast cells by CRISPR/Cas 9 technology. We showed that the CRISPR/Cas9 design was efficient through the generation of heterozygous mutant clones. In this context, once this gene regulates the mtDNA replication specificity, the study aimed to determine if the post-edited cells are capable of in vitro maintenance and assess if they present changes in mtDNA copies and mitochondrial membrane potential after successive passages in culture. The post-edited cells were expanded in culture, and we performed a growth curve, doubling time, cell viability, mitochondrial DNA copy number, and mitochondrial membrane potential assays. The editing process did not make cell culture unfeasible, even though cell growth rate and viability were decreased compared to control since we observed the cells grow well when cultured in a medium supplemented with uridine and pyruvate. They also exhibited a classical fibroblastoid appearance. The RT-qPCR to determine the mtDNA copy number showed a decrease in the edited clones compared to the non-edited ones (control) in different cell passages. Cell staining with Mitotracker Green and red suggests a reduction in red fluorescence in the edited cells compared to the non-edited cells. Thus, through characterization, we demonstrated that the TFAM gene is critical to mitochondrial maintenance due to its interference in the stability of the mitochondrial DNA copy number in different cell passages and membrane potential confirming the decrease in mitochondrial activity in cells edited in heterozygosis.