A hexanucleotide repeat (GGGGCC)n expansion in the human C9ORF72 gene has recently been identified as a causative mutation in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). It accounts for 25 to 37% of familial and 4-7% of sporadic ALS cases making the C9ORF72 mutation the most prevalent genetic determinant for ALS. The biological function of the C9ORF72 gene is unknown, and the mechanism behind how the repeat expansion causes neurodegeneration is a question of great importance. Mouse models of human genetic disorders are critical research tools for understanding disease pathologies as well as for testing compounds in pre-clinical trials. The proposed research aims to generate a suite of C9ORF72-related mouse models and make them available to the scientific community as expeditiously as possible. To complement the human C9ORF72 BAC transgenics already underway in our lab, we are proposing to genetically engineer the following sets of mice: Knockout mice with transcript-specific deletions in the mouse ortholog of the C9ORF72 gene, as well as a complete knockout (Aim1). These mice will be generated through the application of zinc finger nuclease technologies, allowing us to generate the mice more quickly than by traditional homologous recombination. Knock-in mice containing the hexanucleotide repeat (Aim 2). The first knock-in will contain the repeat generated by cell-free cloning into the C9ORF72 mouse ortholog. This cloning strategy reduces the need for bacterial-based cloning, which can cause repeats to contract during the engineering process. The second knock-in will contain the repeat driven by a strong ubiquitous promoter with a GFP reporter. This construct will be targeted to the Gt(ROSA)26Sor locus and designed to allow for the expression of the repeat in a tissue-specific and temporal manner.
The third aim of the grant is to carefully monitor somatic and germ line instability. Finally, the fourth aim is to create embryonic stem (ES) cells that are capable of differentiating into motor neurons or other neuronal subtypes from the most pertinent models mentioned above. These ES cells have the potential to be very useful in cell-based assays. As is the case for many diseases, more than one genetically engineered mouse model is required to help fully understand disease mechanism and pathology. By creating a suite of genetically standardized mouse models for immediate distribution to the scientific community, we hope to be able to efficiently provide the necessary tools for researchers to advance the field of C9ORF72-associated ALS and FTD research.
Recent discoveries have shown that a mutation in the gene C9ORF72 accounts for an astonishing one-third of all inherited cases of Amyotrophic Lateral Sclerosis (ALS). ALS is a relentless and invariably fatal neurodegenerative disease resulting in paralysis and death;the lifetime prevalence of ALS in the population is approximately 1 in 300. This research application is aimed at engineering mouse models that will aid in understanding the cellular mechanisms of how mutations in C9ORF72 lead to ALS.