A High-throughput qPCR-based Method to Genotype the SOD1G93A Mouse Model for Relative Copy Number

Abstract

The most commonly used mouse model in ALS preclinical research expresses multiple copies of the human SOD1 (G93A) transgene. During the course of breeding, successive generations of mice can lose copies of the transgene. Because shorter lifespan of these mice is dependent on transgene copy number, it is essential to ensure that no low-copy, and therefore longer-lived, mice are included in preclinical studies. Existing techniques for SOD1 G93A mouse genotyping are broadly based on creating a standard curve using a reference gene and deducing the relative amount of SOD1 by comparison with the standard curve. This type of technique is used in Alexander et al. (2004), Vieira et al. (2017 and Maier et al. (2018). However, it is not described in detail (see Note 1). This paper provides a detailed protocol for determining the relative copy number of the human SOD1 transgene.Briefly, the protocol involves first the extraction of high-quality genomic DNA from mouse ear tissue, creation of a genomic DNA concentration-based standard curve, and qPCR analysis of up to 88 samples at once alongside the standard curve with Gapdh as a reference gene. Analysis involves the normalization of each unknown sample using the standard curve followed by determination of the copy number of the sample relative to the cohort median. This protocol has been optimized to produce highquality genomic DNA and consistent results, and the relative copy number cutoffs have been optimized and validated empirically by comparison of relative copy number and mouse lifespan.

PERK modulation, with GSK2606414, Sephin1 or salubrinal, failed to produce therapeutic benefits in the SOD1G93A mouse model of ALS

“…The colony was maintained by Biomere (Worcester, MA) by crossing hemizygous C57BL6-SJL sires harboring the SOD1 transgene with non-transgenic C57BL6-SJL dams. SOD1G93A mice and wild-type litter mates were shipped to ALS TDI at 35–45 days of age, then genotyped for the expression of the transgene as previously described [ 43 ], and allowed at least one week to acclimate to ALS TDI’s animal facility (12-h light/dark cycle at a temperature of 18–23°C and 40–60% humidity) before being assigned to a study. Male mice were singly housed, because of aggressive behavior, while female mice were housed in pairs.…”

PERK modulation, with GSK2606414, Sephin1 or salubrinal, failed to produce therapeutic benefits in the SOD1G93A mouse model of ALS

“…The colony was maintained by Biomere (Worcester, MA) by crossing hemizygous C57BL6-SJL sires harboring the SOD1 transgene with non-transgenic C57BL6-SJL dams. SOD1G93A mice and wild-type litter mates were shipped to ALS TDI at 35–45 days of age, then genotyped for the expression of the transgene as previously described 44 , and allowed at least one week to acclimate to ALS TDI’s animal facility (12-h light/dark cycle at a temperature of 18–23 °C and 40–60% humidity) before being assigned to a study. Male mice were singly housed, because of aggressive behavior, while female mice were housed in pairs.…”