Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model

Author(s):Show less -

de Oliveira, Vanessa Cristina ^[1] ; Mariano Junior, Clesio Gomes ^[1] ; Belizario, Jose Ernesto ^[2] ; Krieger, Jose Eduardo ^[3] ; Bressan, Fabiana Fernandes ^[1] ; Santos Roballo, Kelly Cristine ^{[1, 4]} ; Fantinato-Neto, Paulo ^[1] ; Meirelles, Flavio Vieira ^[1] ; Chiaratti, Marcos Roberto ^[5] ; Concordet, Jean-Paul ^[6] ; Ambrosio, Carlos Eduardo ^[1]

Affiliation

^[1] Univ Sao Paulo, Fac Anim Sci & Food Engn, Dept Vet Med, Sao Paulo - Brazil

^[2] Univ Sao Paulo, Inst Biomed Sci, Dept Pharmacol, Sao Paulo - Brazil

^[3] Univ Sao Paulo, Sch Med, Inst Heart, Lab Genet & Mol Cardiol, Sao Paulo - Brazil

^[4] Univ Wyoming, Coll Hlth Sci, Sch Pharm, Laramie, WY 82071 - USA

^[5] Univ Fed Sao Carlos, Dept Genet & Evolut, Sao Carlos, SP - Brazil

^[6] Museum Natl Hist Nat, CNRS UMR7196, INSERM U1154, Lab Struct & Instabil Genomes, Paris - France

• Document type: Journal article
• Source: PLoS One; v. 15, n. 7 JUL 10 2020.
• Web of Science Citations: 0

Abstract

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. (AU)

• FAPESP's process: 17/08896-4 - Potential of application of post edited cells modified TFAM gene by CRISPR Cas 9 technology in bovine model
• Grantee: Vanessa Cristina Oliveira Nogueira de Pontes
• Support Opportunities: Scholarships in Brazil - Post-Doctoral