The Twitcher (Twi) mouse is a neurological Krabbe disease (KD, or globoid cell leukodystrophy) spontaneous mutant line. The genome of the Twi mouse presents a single nucleotide polymorphism (SNP), leading to an enzymatically inactive galactosylceramidase (GALC) protein and causing KD. In this context, mouse Twi genotyping is an essential step in KD research. To date, the genotyping method used is labor-intensive and often drives ambiguous results. Here, we evaluate a novel protocol for the genotype determination of GALC mutation status in Twi mice based on the allele-discrimination real-time polymerase chain reaction (PCR).

DNA is extracted from Twi mice (n = 20, pilot study; n = 120, verification study) and control group (n = 10, pilot study; n = 30 verification study) and assessed by allele-discrimination real-time PCR to detect SNP c.355G>A.

Using the allele-discrimination PCR, all the samples are identified correctly with the genotype GG (wild-type, WT), GA (heterozygote, HET), or AA (homozygote, HOM) by the first analysis and no animals were not genotyped.

We demonstrated that this novel method can be used to distinguish timely, accurately and without ambiguity among HOM, WT, and HET animals. This protocol represents a great opportunity to increase accuracy and speed in KD research.

DNA samples were obtained from tail biopsies using Euroclone spinNAker Universal Genomic DNA mini kit. Pilot study and validation study data were acquired with Applied Biosystems Real-Time PCR System. GALC activity data was acquired with the microplate GloMax fluorescence reader. Body weight data were collected with a scale. Latency to fall data was acquired with the rotarod apparatus (Ugo Basile). 

Data files are uploaded as an OpenDocument Spreadsheet (. ods) format. The .ods files can be opened with some spreadsheet applications, such as OpenOffice.org Calc and Google Docs.