Funding for this research came from Rosebay Medical Foundation and a Yale Medical School Dean&#39;s Research Fund (JH). We thank Dr. Claire Koenig for help with behavioral experiments. Genetically engineered mouse lines were generated at Ozgene (Perth, Australia).

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...

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...

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&#39;s disease (PD) and age-related macular degeneration (AMD). PD is the most common movement disorder caused by neurodegeneration in humans. Most PD patients show symptoms such as resting tremor, bradykinesia and rigidity after 50 years of age. Early onset has also been observed in approximately 10% of cases.
AMD is the leading cause of blindness in the elderly, progressively damaging photoreceptors and the retinal pigment epithelium (RPE) in the eye. Central vision is impaired but the peripheral vision is generally unaffected. There are two forms of AMD. In the &#34;dry&#34; form, extracellular protein deposits known as drusen form between the RPE and Bruch&#39;s membrane (BM), leading to geographic atrophy and blurring of central vision. The more severe &#34;wet&#34; form results from neovascularization from the choroid across BM into the RPE and photoreceptor layers and can lead to hamorrhaging beneath the retina that causes permanent damage to retinal tissue. 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) on chromosome 1 and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located1-5. Combinations of these alleles increase the likelihood of AMD in a dose dependent manner and specific SNPs can be preferentially associated with either the wet or dry forms of AMD3-6.
CFH acts as a negative regulator of the alternative pathway (AP) of the complement system by inhibiting the activation of C3. A single nucleotide polymorphism (SNP) has been linked to increased risk of AMD, causing exchange of tyrosine 402 in exon 9 with histidine due to a T to C substitution1. In AMD it is believed that the AP is misregulated because of a loss of function of CFH but whether the SNP plays a causal role is unclear. One hypothesis is that the positively charged histidine is thought to negate the ability of CFH to bind to interacting proteins C-reactive protein and heparin sulfate1,7. In vitro studies of CFH Y402H provide conflicting results over functional differences between the variants, and in vivo work in Cfh-/- mice expressing humanized CFH is ongoing8. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. HtrA1 is a serine protease but ARMS2 is poorly characterized. The linkage disequilibrium between SNPs in the AMD-associated locus has made it difficult to determine the contributions to risk of individual mutations of the genes in this region, but recent work has suggested that it is overexpression of HtrA1 rather than ARMS2 that leads to neovascularization and subretinal protein deposits9-11. However, the close proximity of the genes in this locus may allow for interactions that cannot be studied using randomly inserted transgenes.
In addition to AMD, the HtrA family of serine proteases has been associated with many human diseases. All HtrA proteins contain a serine protease domain followed by at least one C-terminal PDZ domain. HtrA1, HtrA3 and HtrA4 share the greatest homology, consisting of a signal peptide, insulin-like growth factor binding domain, a Kazal protease inhibitor domain, the serine protease domain and a PDZ domain. HtrA2 has a different N-terminus, composed of a mitochondrial localization sequence, transmembrane domain and inhibitor of apoptosis binding domain followed by the protease and PDZ domains12-16. Mammalian HtrA1 is regulated by substrate-induced remodeling in the active site of its protease domain17-20, and HtrA2 can also be modulated by interaction between the serine protease and PDZ domains that suppresses protease activity21. Interestingly, the PDZ domain does not appear to confer similar regulation to HtrA316. The HtrA proteases can also be regulated by extrinsic factors: it was recently demonstrated that there exists a regulatory interaction between HtrA1 and protoporphyrins22 and HtrA2 can be regulated by phosphorylation upon activation of the p38 MAP kinase pathway in a PINK1-dependent manner23. The deletion of individual members of the HtrA family in mice has been documented, however mechanistic effects mostly are unclear partly due to lack of visible phenotypes.
HtrA1 plays an important function in protein quality control and its misregulation or mutation has been associated with many different human diseases including arthritis, cancer and an increased risk of AMD3,4,24-32. Loss of HtrA2 function in neural tissues has been associated with PD phenotypes in humans and mice, while its loss from non-neural tissues results in accelerated aging33-37. HtrA3 dysregulation has been associated with diseases including preeclampsia and certain types of cancer38,39. Up-regulation of HtrA4 has been observed in the placentas of preeclampsia patients but knockout mice do not display an overt phenotype40,41. The lack of phenotypes observed in some knockout mice has been postulated to be a result of compensation between the HtrA family member: it is thought that both HtrA4 and HtrA1 interact with the TGF-B family of proteins, allowing for compensation by HtrA1 upon deletion of HtrA441. Similarly, it is thought that since HtrA1 and HtrA3 have a high degree of domain homologies they might have complementary functions42. However, it has been suggested that HtrA proteins may have partially antagonistic roles, competing to regulate common targets43.
To further investigate these risk factors three humanized knock-in mouse lines were generated. In Cfhtm1(CFH*9)jhoh and Cfhtm2(CFH*9)jhoh, exon 9 of the Cfh gene is replaced with exon 9 of the human homologue. Cfhtm1(CFH*9)jhoh encodes the non-disease associated tyrosine residue at position 402, whereas Cfhtm2(CFH*9)jhoh carries the Y402H, risk-associated SNP. In ARMS2tm1jhoh the human ARMS2 sequence was targeted to a region upstream of HtrA1. A loxP-flanked STOP sequence placed upstream of the gene sequence but downstream of the included UbiC promoter was excised by crossing to OzCre mice, which express Cre recombinase under the control of the Rosa26 promoter, as previously described34. In addition to these knock-in lines, conditional knockout alleles for Cfh and HtrA1 (Cfhtm1jhoh and HtrA1tm1jhoh), as well as the other known HtrA family members were generated: HtrA2 (HtrA2tm1jhoh), HtrA3 (HtrA3tm1jhoh) and HtrA4 (HtrA4tm1jhoh). The germ-line knockouts were created by crossing OzCre mice to animals engineered to flank specific exons with loxP sites, such that deletion causes a frame shift and/or deletion of the active domain (Cfh; exon 3, HtrA1; exons 2-3, HtrA2: exons 2-4, HtrA3; exon 3, HtrA4; exons 4-6)34,41. A neural deletion of HtrA2, deleted using Cre recombinase under control of the Nestin promoter (HtrA2flox;Tg(Nes-cre)1Kln/J), has also been described34. Only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes, presenting with Parkinsonian phenotypes.
Since some of these genes of interest are posited to be localized to mitochondria11,44-47, and deletion of HtrA2 generated Parkinsonian phenotypes, a phenotyping regimen with a mitochondrial and neurological focus is described here and representative results from the tests of interest are provided. In order to examine genetically engineered mice produced to investigate human, age-related disease thoroughly and systematically an initial phenotyping schedule was established, consisting of a series of tests related to the two main diseases of interest: AMD and Parkinson&#39;s.

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a phenotyping regimen for genetically modified mice used to study genes implicated in human diseases of aging

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.

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 ...

Watch this Scientific Journal Video about A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging at JoVE.com

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

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 ...

The overall goal of this testing regimen is to establish a series of simple tests that can be used as an initial phenotyping pipeline to direct subsequent detailed investigations into behavioral phenotypes and the structure and function of tissues. Whether the behavioral and cellular phenotypes of human diseases are recapitulated in the mouse model. The main advantage of this technique is that the tests can be performed in laboratories with no prior experience in such experiments without significant investment yet still informed further detail studies.Conduct the Hind Limb Test daily from post-natal day four to 10 to evaluate proximal hind limb muscle strength, weakness and fatigue. To begin, let the mice acclimate to the test area for 30 minutes. Meanwhile prepare a new 500 milliliter conical tube by washing it out with 70%ethanol and then pushing one or two cotton balls into the bottom of the tube.Secure this tube in a vertical position for the test. When not being tested, keep the pups with the dam to maintain their temperature. After the half-hour acclimation, remove a pup from the cage and record its weight.Then gently suspend the neonate by its hind limbs over the rim of the tube, so the pup is facing the cotton. Then count the number of pull attempts made by the pup and measure its latency to fall. After waiting 30 seconds, repeat the test.After the second test, label the pup with a marker. Then rub the pup gently with bedding from the home cage and return it to the dam. Because older P8 pups can not be marked identify them with a toe clipping.Before proceeding to the next pup, wipe the test tube down with 70%ethanol. Administer the Weanling Observation Test daily from P19 to P21 to score movement and general activity levels. Begin with letting the mice acclimate to the test area for 30 minutes.While the pups acclimate, prepare the arena. Mark a plexiglass testing box with a six-by-six grid of two inch squares. Before using the box, clean it with 70%ethanol.Then position a video camera so a side view of the whole box fills the field of view. Standardize the appearance of the surroundings to avoid introducing novelty. Before the test, transfer weanling to a clean holding cup and record its weight.Then began video recording. First, film a test identification card with the mouse identification number, its age and the current date. Then transfer the mouse from the holding cup to the center square.For three minutes, count the number of lines crossed by both front paws. After three minutes, return the mouse to a clean cage and end the video recording. Between every test, clean the box with 70%ethanol.From the videos, count the rearing events, which is when a mouse stands with both front paws clear of support. Also count the grooming events, which occur when both front paws are engaged in cleaning. Conduct the Grip Strength Test on alternate days between P22 and P26 to assess neuromuscular strength.As usual, give the mice 30 minutes to adapt to the test area. The testing apparatus is a plexiglass box with a mesh grid. Clean both with 70%ethanol.Then line the bottom of the box with a layer of bubble wrap for padding and cover the bubble wrap with paper to absorb excretions. Proceed to separate the test mice and load them into individual cups. Now, place the first mouse onto the center of the wire mesh and slowly invert the mesh to 10 to 20 centimeters above the soft surface of the box.Measure the latency to falling or end the test after 60 seconds, if the mouse does not fall. Then return the first mouse to the holding cup. Between tests, wipe the mesh with 70%ethanol and replace the paper over the bubble wrap.Then conduct the test on the two other mice in the group. After each mouse is tested once, repeat the process twice more for three trials per mouse. If there are fewer than three mice in a group, make sure that there is an inter-trial recovery time of at least five minutes.For an NADH Diaphorase Stain combine equal volumes of freshly made NADH solution and freshly made NBT solution. Then add one milliliter of staining solution to each slide and incubate the slides in a humidified chamber at 37 degrees Celsius for 30 minutes. For a Succinate Hydrogenase Stain, prepare a solution of 0.1 molar sodium succinate, 1.25 molar NBT and 0.2 molar phosphate buffer.Then add one milliliter to each slide. Pack the slides into a humidified chamber and incubate the slides at 37 degrees Celsius for an hour. After incubating with either stain, aspirate the staining solution and wash the slides three times with distilled water.Then wash the slides through a series of acetone baths that increase and decrease in concentration. With each concentration of acetone, perform three washes for two minutes per wash. Lastly, rinse the slides three times in distilled water.Then mount the slides with an aqueous medium. Mice without HTRA2 expression were examined using the described battery of tests and compared with litter mate controls. No change in the number of pulls or the latency to fall was seen between P4 and P6.Between P7 and P10, a reduction in neuromuscular strength was noted.In the activity test, there was reduced activity of HTRA2 knockout mice. HTRA2 knockout mice also showed a reduction in strength in the wire mesh grip test. H and E staining show that the HTRA2 knockout mice had no apparent changes in retinal structure compared to controls at P35.Finally, histochemical staining for electron transport chain complex activity was used to identify changes in respiratory enzyme functions. There was decreased staining in the HTRA2 knockout mice compared to controls. This was noted in the cerebellum and striatum, but not the cortex.After watching this video, you should have a good understanding of how to assess the activity and neuromuscular strength of genetically modified mice, as well as how to combine these studies with cellular axes. Thanks for watching and good luck with your experiments.

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8.1K vistas.  Yale University School of Medicine. The overall goal of this testing regimen is to establish a series of simple tests that can be used as an initial phenotyping pipeline to direct subsequent detailed investigations into behavioral phenotypes and the structure and function of tissues. Whether the behavioral and cellular phenotypes of human diseases are recapitulated in the mouse model. The main advantage of this technique is that the tests can be performed in laboratories with no prior experience in such experiments without sign...