Published: July 14th, 2016
A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.
Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.
Age-associated diseases are becoming increasingly prevalent in modern society. As medical science improves and life expectancy increases, the population continues to age and the burden of these diseases grows. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging can scientists begin to identify potential therapeutic targets and develop strategies for intervention. Common age-related conditions include neurodegenerative disorders such as Parkinson's disease (PD) and age-related macular degeneration (AMD). PD is the most co....
Ethics statement: Studies involving animals were conducted in compliance with the National Institutes of Health recommendations in the Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committee (IACUC) at Yale University.
1. Behavioral Testing of Genetically Modified Mice
Note: All mice should be subjected to the same testing regimen to limit differences in habituation to handling. Tests should be performed at the same time of day.......
This section describes examples of the results obtainable using these methods. In the hind-limb test, the number of pull attempts made and the latency to fall are summed over two consecutive tests for each day. This test can be used to compare genetically different groups to distinguish mice with reduced neuromuscular strength. HtrA2tm1jhoh (HTRA2 KO) mice in Figure 1A-B demonstrate no change in the number of pulls and latency to fall .......
Robust treatments are needed to limit the impact of debilitating conditions related to human aging, but they remain elusive for many conditions. To identify potential therapeutic targets and develop strategies for intervention, the causes and pathology of diseases associated with aging must first be understood. Not all genetically modified mice immediately present with clear phenotypes that are related to the disease of interest, even if those genes have previously been linked to the condition in human studies. Therefore.......
Funding for this research came from Rosebay Medical Foundation and a Yale Medical School Dean's Research Fund (JH). We thank Dr. Claire Koenig for help with behavioral experiments. Genetically engineered mouse lines were generated at Ozgene (Perth, Australia).....
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