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.
Highlights
The NEFH-tTA mouse has the human neurofilament heavy polypeptide promoter directing tetracycline-controlled transactivator protein expression to large-calibre axons of the brain and spinal cord, allowing tissue-specific and doxycyclinesuppressible expression of a target gene1
The line was generated via pronuclear injection of a plasmid with the human NEFH promoter isolated from BAC clone (CHORI: RP1191J21) to drive expression of the tetracycline transactivator gene, which randomly integrated into the genome1
While a genotyping protocol is available to distinguish between wild-type and transgenic animals1, ideally a colony of homozygous animals would be maintained for experimental breeding with another transgenic line with the gene of interest under the control of a tetracycline response element (TRE)
Summary
1. Virginia Lee, University of Pennsylvania, Philadelphia, USA Mandana Hunter, University of Pennsylvania, Philadelphia, USA. Any reports and responses or comments on the article can be found at the end of the article. Keywords NEFH-tTA, frontotemporal dementia, FTD, ALS, targeted locus amplification, genotyping, mouse. Abbreviations NEFH, neurofilament heavy polypeptide promoter; TG, transgene; tTa, tetracycline-controlled transactivator protein; TRE, tetracycline response element; TLA, targeted locus amplification
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