En Fænotype Regimen for genetisk modificerede mus bruges til at studere Gener impliceret i 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
Alder-associerede sygdomme bliver mere og mere udbredt i det moderne samfund. Som lægevidenskaben forbedrer og levetiden stiger, fortsætter befolkningen alder og byrden af disse sygdomme vokser. Effektive behandlinger er nødvendige for at mindske virkningerne af invaliderende betingelser, men forblive undvigende i mange tilfælde. Kun ved at forstå årsagerne til og patologi af sygdomme forbundet med aldring kan forskerne begynde at identificere potentielle terapeutiske mål og udvikle strategier for intervention. Fælles aldersrelaterede tilstande indbefatter neurodegenerative lidelser, såsom Parkinsons sygdom (PD) og aldersrelateret maculadegeneration (AMD). PD er den mest almindelige bevægelse lidelse forårsaget af neurodegeneration i mennesker. De fleste PD patienter viser symptomer såsom hvile rysten, bradykinesi og stivhed efter 50 års alderen. Tidlig debut er også blevet observeret hos ca. 10% af tilfældene.
AMD er den hyppigste årsag til blindhed hosældre, gradvis skader fotoreceptorer og retinale pigment epitel (RPE) i øjet. Centrale syn forringes men perifere syn er generelt upåvirket. Der er to former af AMD. I "tørre" form ekstracellulære protein indskud kendt som drusen formular mellem RPE og Bruchs membran (BM), hvilket fører til geografisk atrofi og sløring af det centrale syn. De mere alvorlige "våde" formularresultater fra neovaskularisering fra årehinden tværs BM ind RPE og fotoreceptor lag og kan føre til hamorrhaging under nethinden, der forårsager permanent skade på retinalt væv. Genome sammenslutning undersøgelser har tidligere identificeret loci, der er forbundet med forøget modtagelighed for denne sygdom og identificeret to regioner af interesse: komplement faktor H (CFH) på kromosom 1 og 10q26-locuset, hvor aldersrelateret makulopati modtagelighed 2 (ARMS2) og høj temperatur krav faktor A1 (HtrA1) gener er placeret 1-5 </sup>. Kombinationer af disse alleler øge sandsynligheden for AMD på en dosisafhængig måde og specifikke SNP'er kan fortrinsvis forbundet med enten våde eller tørre former af AMD 3-6.
CFH fungerer som en negativ regulator af det alternative forløb (AP) af komplementsystemet ved at inhibere aktiveringen af C3. En enkelt-nukleotid polymorfisme (SNP) er blevet forbundet med øget risiko for AMD, forårsager udveksling af tyrosin 402 i exon 9 med histidin på grund af en T til C substitution 1. I AMD menes det, at AP er misregulated grund af et tab af funktion af CFH men om SNP spiller en kausal rolle er uklar. En hypotese er, at den positivt ladede histidin menes at negere evne CFH til at binde til interagerende proteiner C-reaktivt protein og heparin sulfat 1,7. In vitro undersøgelser af CFH Y402H give modstridende resultater i løbet funktionelle forskelle mellem varianter, og i vivo arbejde i <em> CFH – / – mus, der udtrykker humaniseret CFH er i gang 8. ARMS2 ligger opstrøms for HtrA1 i primat genom, skønt genet er fraværende hos mus. HtrA1 er en serinprotease men ARMS2 er dårligt karakteriseret. Den bindingsuligevægt mellem SNP'er i AMD-associeret locus har gjort det vanskeligt at bestemme bidragene til risiko for individuelle mutationer af generne i denne region, men nyligt arbejde har antydet, at det er overekspression af HtrA1 stedet ARMS2 som fører til neovaskularisering og subretinal protein indskud 9-11. den tætte nærhed af generne i dette locus, kan dog tillade for interaktioner, der ikke kan studeres under anvendelse tilfældigt indsatte transgener.
Foruden AMD har HtrA familien af serinproteaser blevet forbundet med mange humane sygdomme. Alle HtrA proteiner indeholder en serinprotease domæne efterfulgt af mindst én C-terminalt PDZ-domæne. HtrA1, HtrA3 og HtrA4 deler grspiser homologi, der består af et signalpeptid, insulin-lignende vækstfaktor bindende domæne, en Kazal proteaseinhibitor domæne, serinproteasedomænet og en PDZ-domæne. HtrA2 har en forskellig N-terminal, som består af et mitokondrisk lokaliseringssekvens, transmembrandomænet og inhibitor af apoptose bindingsdomæne efterfulgt af protease- og PDZ-domæner 12-16. Mammal HtrA1 reguleres af substrat-induceret remodellering i det aktive sted i sit proteasedomæne 17-20, og HtrA2 kan også moduleres ved vekselvirkning mellem serinproteasen og PDZ-domæner, der undertrykker proteaseaktivitet 21. Interessant, har PDZ-domænet ikke ud til at give lignende regulering til HtrA3 16. HtrA proteaser kan også reguleres ved ydre faktorer: det for nylig blev vist, at der eksisterer en regulerende samspil mellem HtrA1 og protoporphyriner 22 og HtrA2 kan reguleres ved phosphorylering ved aktivering af p38 MAP-kinasepathway i en Pink1-afhængig måde 23. Sletningen af enkelte medlemmer af HtrA familien i mus er blevet dokumenteret, men mekanistiske effekter meste er uklare dels på grund af mangel på synlige fænotyper.
HtrA1 spiller en vigtig funktion i protein kvalitetskontrol og dens fejlagtig regulering eller mutation er blevet associeret med mange forskellige humane sygdomme, herunder arthritis, cancer og en øget risiko for AMD 3,4,24-32. Tab af HtrA2 funktion i neurale væv er blevet associeret med PD fænotyper hos mennesker og mus, mens dens tab fra ikke-neurale væv resulterer i accelereret ældning 33-37. HtrA3 dysregulering er blevet forbundet med sygdomme, herunder præeklampsi og visse former for kræft 38,39. Opregulering er blevet observeret af HtrA4 i placenta af præeklampsi patienter, men knockout-mus ikke vise en åbenlys fænotype 40,41. Manglen på fænotyper observeret hos nogle knockout-mus har væretpostuleret at være et resultat af kompensation mellem HtrA familiemedlem: det menes, at både HtrA4 og HtrA1 interagere med TGF-B familie af proteiner, der giver mulighed for erstatning HtrA1 ved sletning af HtrA4 41. Tilsvarende er det, at da HtrA1 og HtrA3 har en høj grad af domænenavne homologier de måtte have supplerende funktioner 42. Det er imidlertid blevet foreslået, at HtrA proteiner kan have delvis antagonistiske roller, konkurrerer om at regulere fælles mål 43.
For yderligere at undersøge disse risikofaktorer tre humaniseret knock-in mus linjer blev genereret. I CFH TM1 (CFH * 9) jhoh og CFH TM2 (CFH * 9) jhoh, exon 9 i CFH genet er erstattet med exon 9 i humane homolog. CFH TM1 (CFH * 9) jhoh koder for ikke-sygdom forbundet tyrosin rest ved position 402, mens CFH TM2 (CFH * 9) jhoh bærer Y402H, risiko-associeret SNP. IARMS2 tm1jhoh den menneskelige ARMS2 sekvens blev målrettet til en region opstrøms for HtrA1. En loxP-flankeret STOP sekvens placeret opstrøms for genet sekvens, men nedstrøms for den medfølgende Brancheorganisationer promotoren blev udskåret ved at krydse til OzCre mus, som udtrykker Cre-rekombinase under kontrol af den Rosa26 promotoren, som tidligere beskrevet 34. Ud over disse knock-i linjer, blev betingede knockout alleler for CFH og HtrA1 (CFH tm1jhoh og HtrA1 tm1jhoh), samt de andre kendte HtrA familiemedlemmer genereret: HtrA2 (HtrA2 tm1jhoh), HtrA3 (HtrA3 tm1jhoh) og HtrA4 ( HtrA4 tm1jhoh). De kim-line knockouts blev skabt ved at krydse OzCre mus til dyr manipuleret til at flankere specifikke exons med loxP sites, således at sletning forårsager en ramme skift og / eller sletning af det aktive domæne (CFH, exon 3, HtrA1; exon 2-3, HtrA2: exoner 2-4, HtrA3; exon 3, HtrA4; exonerne 4-6) 34,41. En neurale sletning af HtrA2, slettes ved hjælp Cre rekombinase under kontrol af Nestin promotor (HtrA2 FLOX, Tg (Nes-CRE) 1Kln / J), er også blevet beskrevet 34. Kun mus, der manglede HtrA2, enten systemisk eller i neurale væv, viste klare fænotyper, præsenterer sig med Parkinson fænotyper.
Da nogle af disse gener af interesse postuleret at være lokaliseret til mitokondrier 11,44-47, og sletning af HtrA2 genereret Parkinson fænotyper, en fænotype regime med en mitokondrie og neurologisk fokus beskrives her og repræsentative resultater fra test af interesse leveres . For at undersøge gensplejsede mus produceret til at undersøge menneskelige, aldersrelaterede sygdomme grundigt og systematisk en indledende fænotypebestemmelse tidsplan blev etableret, bestående af en række forsøg vedrørende de to vigtigstesygdomme af interesse: AMD og Parkinsons.
Protocol
Representative Results
Discussion
Der er behov for Robuste behandlinger til at begrænse virkningen af invaliderende tilstande relateret til menneskets aldring, men de forbliver undvigende for mange betingelser. For at identificere potentielle terapeutiske mål og udvikle strategier for intervention, skal årsagerne til og patologi af sygdomme forbundet med aldring først blive forstået. Ikke alle genetisk modificerede mus umiddelbart til stede med klare fænotyper, der er relateret til sygdommen af interesse, selv om disse gener tidligere …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Finansieringen af denne forskning kom fra Rosebay Medical Foundation og en Yale Medical School Dean Research Fund (JH). Vi takker Dr. Claire Koenig om hjælp med adfærdsmæssige eksperimenter. Gensplejsede muselinjer blev genereret ved Ozgene (Perth, Australien).
Materials
| 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 | |
| 9oz 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 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 |
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