Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains

Abstract

Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals.

Mitofusin 1 is required for oocyte growth and communication with follicular somatic cells

“…Floxed mice (Mfn fl/fl ) 26 were obtained from the Mutant Mouse Regional Resource Centers (Davis, CA, USA; Cat# 029901‐UCD, and MMRRC Cat# 029902‐UCD, ), whereas Zp3 ‐CRE transgenic mice 27 were supplied by the Jackson Laboratory (Bar Harbor, ME, USA; Cat# JAX:003651, :003651). Before use, Zp3 ‐CRE mice were mated with heteroplasmic C57BL/6N females containing mtDNA of NZB/BINJ and C57BL/6N origin 28 …”

“…Ear biopsies were digested for 3 hours at 55°C using a solution containing 50 mM KCl, 10 mM Tris‐Cl, 2 mM MgCl 2 , 0.1 mg/mL gelatin, 0.45% Igepal CA‐630, 0.45% Tween 20, and 125 µg/mL proteinase K (Thermo Fisher Scientific, Waltham, MA, USA). After digestion, samples were incubated at 95°C for 10 minutes, centrifuged at 10 000 g for 5 minutes and the supernatant used for genotyping 28 …”

The biochemistry of feed efficiency, energy metabolism, and mitochondrial function, an animal and molecular approach

“…Once mitochondrial function depends on an orchestrated communication between nuclear DNA and mtDNA, which encodes 13 peptides involved in oxidative phosphorylation (OXPHOS) Bottje and Carstens, 2012), we investigated the amounts of mtDNA in liver, muscle and adipose tissue of Nellore bulls. Quantification of mtDNA copy number was estimated in relation to a single copy nuclear gene, providing a rapid and sensitive analysis (Chiaratti et al, 2010;Machado et al, 2015). The results found in the current study is in accordance with experiments measuring mtDNA copy number in the hepatic and adipose tissue of dairy cows .…”

“…Total DNA was isolated using the Qiagen DNeasy Tissue kit (Valencia, CA, USA) according to manufacturer instructions. Subsequently, the relative quantification of mtDNA was performed by a quantitative real-time PCR (qPCR) method as previously described (Chiaratti et al, 2010;Machado et al, 2015).…”

High incidence of heteroplasmy in the mtDNA of a natural population of the spider crab Maja brachydactyla

“…However, this general assumption of uniparentally transmitted, homoplasmic and non-recombining mitochondrial genomes is becoming more and more controversial [3][4][5]. Until now, the presence of different sequences of mtDNA within a cell or individual was a rare phenomenon in animals, but new detection methods based on NGS (Next-Generation Sequencing) or qPCR (quantitative Polymerase Chain Reaction) are allowing their detection [6][7][8][9]. Nowadays, heteroplasmy has been reported in many organisms such as insects [10,11], crustaceans [12,13], molluscs [14], fishes [15,16], frogs [17] birds [18,19], mice [20] and humans [21,22], being the paternal leakage the primary cause of it.…”