Using Real-Time (TaqMan) PCR to Genotype Offspring of Transgenic CF-null Mice- A Core Facility of the UK Cystic Fibrosis Gene Therapy Consortium.

Smith RL, Davies LA, Painter H, Varathalingam A, Gill DR, Hyde SC

Molecular Therapy, 11 S1 S141

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The American Society of Gene Therapy Annual Conference, St Louis, 2005

Cystic fibrosis (CF) is a common and fatal recessive disease, which is caused by dysfunction of a chloride ion channel, termed the CF transmembrane conductance regulator (CFTR).

Several transgenic mouse models have been created to assess the effectiveness of a variety of gene transfer vectors for CF in the respiratory epithelium of the nose and lung (Snouwaert et al , 1992). Use of these animal models requires a sensitive, accurate and fast method for genotyping neonatal mice in order that individual animals may be selected for experiments.

The speed of the assay is important as many CF transgenic mouse models have poor survival rates under standard housing conditions. We have established a Core Testing Service using the ABI7700 Sequence Detector (TaqMan , Applied Biosystems Inc, Foster City, USA) to differentiate wild-type, homozygous Cftr tm1UNC and heterozygous littermates by simultaneously detecting mCFTR exon 10, and the vector that replaces exon 10 in the transgenic mice. These sequences were confirmed by cloning of the target DNA to ensure primer specificity.

Genotyping can be performed on a range of samples, including ear clips, tail clips and blood, from immediately after birth. Samples are collected in the core facility and the DNA extracted using the DNEasy mini kit (Qiagen Ltd, Crawley, UK). DNA is amplified using the TaqMan instrument. Fluorescent detection avoids the use of gel electrophoresis and potential observer error. The sensitivity of the assay is such that only 20ng DNA is necessary for reproducible genotyping. Up to 28 mice can be assayed at once, and results are typically available in less than 24h. The assay is capable of differentiating between allele copy numbers in homozygotes and heterozygotes, and Figure 1 shows the differing Cts in detection of the wild type allele in CF-null, heterozygous and wild type mice. Detection of the Cftr tm1UNC allele followed a similar pattern.

The assay has been seen to be in complete agreement with other assessments of genotype including weight abnormality. PCR in real time is a rapid, reliable and sensitive method to assess genotype and well suited for genotyping a range of transgenic animal models.