A novel genotyping method to determine copy number in a mouse line commonly used for inducible transgene expression in brain and spinal cord [version 2; peer review: 1 approved, 2 approved with reservations]

Abstract

Background: The NEFH-tTA mouse has the human neurofilament heavy polypeptide promoter directing tetracycline-controlled transactivator protein (tTa) expression to the brain and spinal cord, allowing tissue-specific and doxycycline-suppressible expression of a target gene. Current genotyping protocols can only differentiate between wild-type and transgenic animals. Being able to differentiate between hemizygous and homozygous animals would be beneficial in experiment planning and reducing animal numbers. Methods: We have identified the insertion site of the NEFH-tTA transgene via targeted locus amplification and next-generation sequencing. This was then used to design a multiplex PCR assay to distinguish between hemizygous and homozygous mice. Results: The NEFH-tTA transgene is located on chromosome 12. Our genotyping method can identify hemizygous and homozygous mice. Conclusions: The NEFH-tTA transgenic mouse line is a useful tool for studying a wide range of diseases including frontotemporal dementia and motor neuron disease, as well as other neurodevelopmental, neuromuscular or neurodegenerative disorders. We have designed and utilised a novel genotyping assay to distinguish between hemizygous and homozygous mice, involving a simple PCR assay. This is easily adaptable to a laboratory-specific protocol or machine, and will allow refinement of breeding strategies and a reduction in the number of animals that cannot be used in experiments.

Keywords

• NEFH-tTA
• frontotemporal dementia
• FTD
• ALS
• targeted locus amplification
• genotyping
• eng