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.
Highlights
In mammals, the mitochondrial genome encodes 13 polypeptides that are essential subunits of the enzyme complexes in the oxidative phosphorylation pathway [1,2]
As the mtDNA haplotype of the NZB/BINJ (NZB) strain differs by dozens of nucleotides from the mtDNA haplotype of most laboratory inbred strains [8,14,15], including BALB/cByJ and C57BL/6 (B6), NZB mice are often used as a source of polymorphic mtDNA [5,6,7,8,9,10,11,12]
We sought to investigate whether primers designed based on allelic refractory mutation detection system (ARMS) technology could be used for quantification of heteroplasmy by quantitative PCR (qPCR)
Summary
The mitochondrial genome (mtDNA) encodes 13 polypeptides that are essential subunits of the enzyme complexes in the oxidative phosphorylation pathway [1,2]. Since mutations in mtDNA have been implicated in several maternally-inherited human diseases [2], there is a growing interest in developing animal models to address the mechanisms underlying mitochondrial inheritance. Heteroplasmic mice are generated by mixing cytoplasm from two zygotes that differ in their mtDNA haplotype (i.e., NZB and B6 strains), followed by embryo transfer to foster mothers [6,7,11]. Since heteroplasmic mice are viable, the level of NZB mtDNA can be tracked down to study mitochondrial inheritance in different cell types and across generations [5,6,7,8,9,10,11,12,13]
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