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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.

A novel genotyping method to determine copy number in a mouse line commonly used for inducible transgene expression in brain and spinal cord