Reliable and fast genotyping protocol for galactosylceramidase (GALC) in the Twitcher (Twi) mouse

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 ..., 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.抽搐小鼠（Twitcher mouse，缩写Twi）是自发性神经型克拉伯病（Krabbe disease，KD，又称球状细胞脑白质营养不良）的自发突变品系小鼠。该品系小鼠的基因组存在单核苷酸多态性（single nucleotide polymorphism，SNP），导致半乳糖苷神经酰胺酶（galactosylceramidase，GALC）蛋白丧失酶活性，进而引发KD。在此研究背景下，Twi小鼠的基因分型是KD相关研究的必要环节。迄今为止，所采用的基因分型方法不仅操作繁琐耗时，且常得到歧义性检测结果。本研究基于等位基因鉴别实时聚合酶链反应（allele-discrimination real-time polymerase chain reaction，PCR），开发了一种可用于Twi小鼠GALC突变状态基因分型的全新方案，并对其性能进行了评估。本研究分别从Twi小鼠（预实验：n=20；验证实验：n=120）与对照组小鼠（预实验：n=10；验证实验：n=30）中提取基因组DNA，通过等位基因鉴别实时PCR检测SNP位点c.355G>A。采用该等位基因鉴别PCR方法，所有样本均可被准确鉴定为GG型（野生型，WT）、GA型（杂合子，HET）或……。基因组DNA通过尾组织活检获取，并使用Euroclone spinNAker Universal Genomic DNA mini试剂盒提取。预实验与验证实验的相关数据均通过应用生物系统公司（Applied Biosystems）实时PCR系统采集。GALC活性数据通过GloMax微孔板荧光读数仪采集。体重数据通过电子秤采集。跌落潜伏期数据通过旋转棒实验装置（Ugo Basile）采集。本研究数据集以开放文档电子表格（OpenDocument Spreadsheet，.ods）格式上传。.ods格式文件可通过多款电子表格软件打开，例如OpenOffice.org Calc与Google Docs。