Droplet digital PCR reveals that transgene copy number determines lifespan in SOD1G93A ALS mice

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease resulting in muscle weakness and eventually paralysis. Pre-clinical ALS models are a useful tool to understand mechanisms of disease development and to test therapeutic candidates. Approximately 20% of ALS cases have a genetic component, and of those, approximately 20% are attributed to mutations in the superoxide dismutase 1 (SOD1) gene.

Multiple mouse models have been generated to study the different mutations in ALS, however, the most popular and well-characterized is the transgenic SOD1G93A mouse model. In this model, copies of the mutated human SOD1 (hSOD1) gene are inserted into the mouse genome and the number of copies correlates with severity of disease. To obtain accurate and reproducible data, it is recommended that animals are screened for their copy number prior to enrollment in a study.

However, there is no widely-accepted standardized protocol for testing SOD1G93A copy number and the absolute copy number of hSOD1 insertion in mice has never been quantified. Here, we isolated DNA from a cohort of SOD1G93A animals (JAX Strain: 004435) and validated a method for absolute quantification of transgene copy number via droplet digital PCR (ddPCR). We found that both ddPCR and the current standard, qPCR, reliably determined genotype, however, ddPCR copy numbers ranged from 51.68 to 119.5 whereas qPCR reported 23 to 44.65 copies.

We demonstrate that ddPCR copy number correlated significantly with disease phenotype and progression and in comparing ddPCR to real-time PCR, we determined ddPCR to be a more accurate and reliable way to measure SOD1G93A copy number. We would like to thank Hillary Hoyt for her contributions in animal husbandry related to these experiments. Graphics were created with BioRender.com.

This study was supported by the US Department of Defense: DOD GRANT13938213. Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA Department of Genetic and Cellular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA Ashley L. Harkins, Sukanya Iyer, Maria Predtechenskaya, Meghan Blackwood & Allison M.

Keeler Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA Morningside Graduate School of Biomedical Sciences, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA NeuroNexus Institute, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA Correspondence to Robert H. The authors declare no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.