Name: antibody transfection into neurons as a tool to study disease pathogenesis
Uploaded: 2012-09-26T13:00:00
Description: Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Dette arbejde er støttet af Office of Research og udvikling, Medical Research Service, Department of Veterans Affairs. Denne undersøgelse blev finansieret af en VA Merit anmeldelse Award (til MCL) og University of Tennessee Health Science Center Multipel sklerose Research Fund.

1. Antistofmærkning (figur 1) <ol><li> Gøre 0,1 M NaHCO3-puffer: <ul type="circle"><li> 8,4 g NaHCO3 </li><li> 29,2 g NaCI </li><li> 1 liter H2O </li></ul></li><li> Bring buffer op til en pH-værdi på 8,4 med denne opløsning (10,6 g Na2 CO 3, 29,2 g NaCl, 1 l H 2 0). </li><li> Tilføj 35 pi Atto550 NHS til 70 pi anti-hnRNPA1 og 500 pi NaHCO3-puffer og roter i 1 time ved stuetemperatur. Efter den timelange rotation, injicere blandingen i e...

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For at teste hypoteser om specifikke molekyler og deres ekspression, er gener og andre vektorer almindeligvis transficeres i celler. Det unikke ved denne procedure er evnen til let at transficere antistoffer i målceller. Vi udførte denne metode i fem separate forsøg hver i dubletter. Transfektionseffektivitet resultater svarede blandt alle eksperimenter. Vigtigt er det, at fremgangsmåden muliggør optagelse af antistoffer i cellen uden at ændre antistof struktur eller funktion. Yderligere, ved anvendelse af fluores...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Fluorescerende mikroskop AxioObserver A1 </td><td> Zeiss </td><td></td></tr><tr><td> AxioVision udgave 4,2 </td><td> Zeiss </td><td></td></tr><tr><td> Anti-rhnRNPA1 </td><td> Abcam </td><td> Ab4791 </td></tr><tr><td> FITC-mærket anti-Rigg </td><td> OZ Biosciences </td><td> AI20100 </td></tr><tr><td> Atto550 NHS-ester </td><td> Sigma </td><td> 92.835 </td></tr><tr><td> Ab-deliverin </td><td> OZ Biosciences </td><td> AI20100 </td></tr><tr><td> DMEM/F12 +10% FBS +1% antibiotika </td><td> Gibco </td><td> 11320-033 </td></tr><tr><td> Poly-D-lysin 8-brønds kultur Slides </td><td> BD </td><td> 354.632 </td></tr><tr><td> Multi-Purpose Rotator </td><td> Videnskabelig Industries Inc </td><td> Model 151 </td></tr><tr><td> DAPI Mounting Media </td><td> Millipore </td><td> S7113 </td></tr><tr><td> PBS </td><td> Ambion </td><td> 9626 </td></tr><tr><td> Dialysator </td><td> Thermo Fisher Scientific </td><td> 66.380 </td></tr><tr><td> Amicon Ultra-0.5 </td><td> Millipore </td><td> UFC501096 </td></tr><tr><td> Paraformaldehyd (4% opløsning) </td><td> Elektronmikroskopi Sciences </td><td></td></tr><tr><td> Nano-Drop </td><td> Thermo Fisher Scientific </td><td></td></tr></tbody></table>

antibody transfection into neurons as a tool to study disease pathogenesis

Antistoffer giver mulighed for at få ny indsigt i forskellige arrangementer, der finder sted i levende systemer. Evnen til at producere meget specifikke antistoffer mod målproteiner har givet mulighed for meget præcise biologiske spørgsmål, der skal behandles. Vigtigere, er antistoffer blevet impliceret i patogenesen af ​​en række humane sygdomme, herunder systemisk lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multipel sclerose (MS) og human T-lymfotropisk virus type 1 (HTLV-1) associeret myelopati / tropisk spastisk paraparese (HAM / TSP) 1-9. Hvordan antistoffer forårsage sygdom er et område med igangværende undersøgelse, og data tyder på, at samspillet mellem antistoffer og forskellige intracellulære molekyler resulterer i inflammation, ændres cellulær messaging, og apoptose 10. Det er blevet vist, at patienter med MS og HAM / TSP producerer autoantistoffer til det intracellulære RNA-bindende protein heterogen ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. De seneste data viser, at antistoffer mod både intra-neuronale og overfladeantigener er patogene 3, 5-9, 11. Således en procedure, der muliggør studiet af intracellulært antistof: ville proteininteraktioner give stor indsigt i sygdommen patogenese. Gener er almindeligt transficeret ind i primære celler og cellelinier i kultur, har imidlertid transfektion af antistoffer i celler blevet hæmmet af ændring af antistofstrukturen eller dårlig transfektionseffektivitet 12. Andre metoder til transfektion omfatter antistof transfektion baseret på kationiske liposomer (bestående af DOTAP / DOPE) og polyethylenimines (PEI), som begge resulterede i en ti-fold fald i antistof-transfektion sammenlignet med kontroller 12. Fremgangsmåden udføres i vores undersøgelse ligner kationisk lipidmedieret metoder og anvender et lipid-baseret mekanisme til dannelse af ikke-kovalente komplekser med antistoffer via elektrostatiske og hydrophobic interaktioner 13. Vi udnyttede Ab-deliverin reagens, som er en lipidformulering kan indfange antistoffer gennem ikke-covalente elektrostatiske og hydrofobe interaktioner og levere dem i celler. Således kemiske og genetiske koblinger er ikke nødvendige til afgivelse af funktionelle antistoffer i levende celler. Denne metode har gjort det muligt for os at udføre forskellige antistof sporing og protein lokalisering eksperimenter, samt analyser af de molekylære konsekvenser af intracellulært antistof: protein interaktioner 9. I denne protokol vil vi vise, hvordan at transficere antistoffer til neuroner hurtigt, reproducerbart og med en høj grad af transfektionseffektivitet. Som et eksempel, vil vi bruge anti-hnRNP A1 og anti-IgG-antistoffer. For let kvantificering af transfektionseffektiviteten vi anvendte anti-hnRNP A1 antistoffer mærket med Atto-550-NHS og FITC-mærket IgG. Atto550 NHS er en ny etiket med høj molekylær absorption og kvante yienergimærkningsdirektivet. Excitationskilde og fluorescerende filtre til Atto550 svarer til Cy3 (eksempel 556 Em. 578). Desuden har Atto550 høj fotostabilitet. FITC-mærket IgG blev anvendt som kontrol til at vise, at denne metode er alsidig og ikke farve afhængig. Denne fremgangsmåde og de data, der genereres vil bidrage til forståelse af den rolle, som antistoffer mod intracellulære målantigener kan spille i patogenesen af ​​sygdomme hos mennesker.

Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

En hurtig fremgangsmåde til undersøgelse af vekselvirkninger og virkningerne på molekylære mekanismer i forbindelse med tilstedeværelsen af ​​antistoffer i et intracellulært miljø, er beskrevet. Fremgangsmåden involverer transfektion af antistoffer i levende celler under anvendelse af et ikke-kovalent kompleksdannelse baseret på en lipidformulering. Teknikken kan tilpasses til udødeliggjorte cellelinier og primære celler.

Antistof Transfektion i neuroner som et værktøj til at studere Disease Patogenese

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

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