A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 each time.

1. Neonatal Hind Limb Test
   Note: The hind limb test is conducted daily from postnatal day (P) 4 to P10 to evaluate proxi....

Results

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. HtrA2^tm1jhoh (HTRA2 KO) mice in Figure 1A-B demonstrate no change in the number of pulls and latency to fall .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol	Decon (Fisher Scientific)	435541
50 ml conical tube	Fisher Scientific	1443222
cotton balls	Walmart
heat mat	Sunbeam	0000756-500-000
Holding tray (ice cube tray)	Walmart
Electronic stopwatch	GOGO	PC396
Plexiglass box	constructed in workshop		12" by 12"
Vixia HF R400 Camcorder	Canon	8155B004
9 oz Clear Cups	Walmart
1/4 inch wire mesh	Home Depot	204331884 (online) / 554219 (in store)	12" by 12"
Bubble wrap	VWR	470092-416
Straight specimen forceps	VWR	82027-438
Fine-tip dissecting forceps	VWR	82027-408
Fine scissors	VWR	82027-578
Paraformaldehyde 16% solution	Electron Microscopy Sciences	15710
10x phosphate buffered saline pH 7.4	American Bioanalytical	AB11072-04000
Sucrose	JT Baker	4072-01
superfrost slides	Fisher Scientific	12-550-15
Hematoxylin Stain Solution	Fisher Scientific (Ricca)	353016
Eosin Y Stain Solution	Fisher Scientific (Ricca)	2845-32
Tris hydrochloride	Sigma	T3253
Tris	American Bioanalytical	AB02000-01000
Nicotinamide adenine dinucleotide, reduced disodium salt hydrate	Sigma	N8129
Nitrotetrazolium Blue chloride	Sigma	N6876
Acetone	JT Baker	9006-05
Sodium phosphate monobasic monohydrate	Sigma	S9638
Sodium phosphate dibasic heptahydrate	Sigma	S9390
Sodium succinate dibasic hexahydrate	Sigma	S2378
VectaMount aqueous mounting medium	Vector Labs	H-5501-60
Cover glass	Fisher Scientific	12-545-M	60 mm x 24 mm
AxioImager A1 microscope	Zeiss
Video camera tripod	Amazon
Optimal Cutting Temperature (OCT)	Fischer Scientific	23730571
Cryostat Sectioning  Machine	Leica	CM1900	Discontinued but since replaced by CM1950