Generation of a heteroplasmic mouse line transmitting a non-coding variant affecting mitochondrial DNA replication

• Grant number: 25/13867-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: March 01, 2026
• End date: February 28, 2027
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Marcos Roberto Chiaratti
• Grantee: Angelica Camargo dos Santos
• Supervisor: Patrick Francis Chinnery
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
• Institution abroad: University of Cambridge, England
• Associated to the scholarship: 22/15264-2 - Impact of MFN2 deficiency and maternal obesity on the oocyte and the transmission of metabolic syndromes, BP.DR

Abstract

Single nucleotide variations (mtSNV) are present in the human mitochondrial DNA (mtDNA) ranging in frequency from ~10 to 15 fold higher than that seen in the nuclear DNA, being able to affect mitochondrial function and different physiological parameters, beside determining important rare diseases. Despite this, little is known about the mechanisms regulating the inheritance of mtSNVs, which are maternally-exclusively transmitted. In part, this lack of understating is due to the inexistence of animal models for mtDNA mutations, a scenario that only recently has changed with the advent of new methods allowing mtDNA editing through DddA-derived cytosine base editing (DdCBE). Therefore, the aim of the present proposal is to establish a new line of heteroplasmic mice transmitting a non-coding variant affecting mtDNA replication. The chosen mutation is a variant that in humans affect the ability of the light-strand origin (OriL) to initiate replication. DNA encoding DdCBE pairs will be synthesised commercially and optimised for on- and off-target activity in cultured mouse cells. To generate heteroplasmic mice, we will inject one-cell embryos (pro-nuclear zygotes) with mRNA coding for the DdCBE pair, implant modified embryos into pseudopregnant, surrogate mothers to obtain F0 offspring. To verify germline transmission of the resulting mtDNA mutation, we will cross females F0 mouse a wild-type C57BL6/J male to obtain F1 pups. We will carry out mtDNA off-targeting analysis by analysing mtDNA-wide C*G-to-T*A mtSNVs and comparing to WT animals.