Direkte injeksjon av en Lentiviral vektor høydepunkter flere Motor veier i ryggmargen rotte
Summary
Denne protokollen demonstrerer injeksjon av en retrogradely transportable viral vektor i rotte ryggmargen vev. Vektoren er tatt opp på synapse og fraktet til celle kroppen av målet neurons. Denne modellen er egnet for retrograd sporing av viktige spinal veier eller målretting celler for gene terapi programmer.
Abstract
Innføre proteiner av interesse i cellene i nervesystemet er utfordrende på grunn av medfødte biologiske barrierer som begrenser tilgang til de fleste molekyler. Injeksjon direkte i ryggmargen vev utenom disse barrierene, gir tilgang til cellen legemer eller synapser der molekyler kan innlemmes. Kombinerer viral vektor teknologi med denne metoden gir mulighet for innføring av målet gener i nervøse vev for å genterapi eller tarmkanalen sporing. Her er et virus som er utviklet for svært effektiv retrograd transport (HiRet) innført på synapser av propriospinal interneurons (PNs) å oppmuntre bestemt transport til nerveceller i ryggmargen og hjernestammen kjerner. Målretting PNs utnytter mange tilkoblinger de mottar fra motor trasé som rubrospinal og reticulospinal områder, samt deres samtrafikk med hverandre gjennom ryggmargen segmenter. Representant sporing HiRet vektoren med constitutively aktive grønne fluorescerende protein (GFP) viser Hi-Fi detaljer cellen legemer, axons og dendrittiske arbors i thorax PNs og reticulospinal nerveceller i pontine reticular dannelsen. HiRet inneholder også til hjernestammen stier og PNs men viser alder avhengig integrering i corticospinal skrift neurons. Oppsummert er ryggmargen injeksjon ved hjelp av viral vektorer en egnet metode for innføring av proteiner av interesse i nevroner i målrettede traktater.
Introduction
Viral vektorer er viktige biologiske verktøy som kan innføre genetisk materiale i cellene for å kompensere for defekte gener, upregulate viktig vekst proteiner eller produserer markør proteiner som fremhever struktur og synaptic tilkoblinger av sine mål. Denne artikkelen fokuserer på direkte injeksjon av en svært effektiv retrogradely transportable lentiviral vektor i ryggmargen rotte for å markere større motor veier med fluorescerende sporing. Denne metoden er også svært hensiktsmessig for axonal regenerasjon og gjenvekst studier å innføre proteiner rundt i forskjellige befolkningsgrupper neurons og har vært brukt til taushet neurons for funksjonell kartlegging studier1,2.
Mange av de anatomiske detaljene av spinal motor var belyst gjennom direkte injeksjon studier med klassisk tracers som BDA og fluoro-gull3,4,5,6,7 , 8. disse tracers anses gull standard, men kan ha visse ulemper som opptak av skadet axons eller axons i passasjen i hvit saken rundt en injeksjon området9,10,11 . Dette kan føre til feil tolkninger av veien kan være en ulempe i gjenfødelse studier der fargestoff absorpsjon av skadet eller kuttet axons kan forveksles med regenererer fiber under senere analyse12.
Lentiviral vektorer er populære i gene terapi studier, som de gir stabil, langsiktig uttrykk i neuronal bestander13,14,15,16,17,18 ,19. Men tradisjonelt pakket lentiviral vektorer kan ha begrenset retrograd transport og kan utløse immunsystem respons når de brukes i vivo4,20,21. En høyeffektiv retrograd transport vektor kalt HiRet er produsert av Kato et al. ved å endre viral konvolutten med en rabiat virus glykoprotein å opprette en hybrid vektor som forbedrer retrograd transport22,23.
Retrograd sporing introduserer en vektor i synaptic plass av en målet Nevron, slik at det å bli tatt av cellens axon og transportert til celle kroppen. Vellykket transport av HiRet har blitt demonstrert fra neuronal synapser i hjernen av mus og primater23,24 og muskler i motor neurons22. Denne protokollen demonstrerer injeksjon i lumbal ryggmargen, spesielt rettet mot synaptic terminalene på propriospinal interneurons og hjernestammen neurons. PNs motta tilkoblinger fra mange ulike spinal veier og kan dermed brukes til å målrette en mangfoldig befolkning av nerveceller i ryggmargen og hjernestammen. Merket nerveceller i denne studien representerer kretser innervating motor neuron bassengene om hindlimb funksjon. Robust merking er sett i ryggmargen og hjernestammen, inkludert Hi-Fi detaljer om dendrittiske arbors og axon terminaler. Vi har også brukt denne metoden i tidligere studier innen cervical ryggmargen merke propriospinal og hjernestammen reticulospinal veier25.
Denne protokollen demonstrerer injeksjon av en viral vektor i lumbal ryggmargen av rotte. Som sett i filmen 1, snittet skal identifisere L1 vertebra på siste vrborden. Dette er brukt som et caudal landemerke for en 3-4 cm snitt som eksponerer muskulaturen over L1-L4 ryggmargen. Laminectomies av dorsal aspekter av T11-T13 ryggvirvlene utføres og en skrå nål er rettet 0,8 mm lateral fra midtlinjen og senket 1,5 mm dypt inn i grå substans å injisere virus.
Protocol
Representative Results
Discussion
Genetisk manipulasjon av nerveceller i hjernen og ryggmarg har tjent til å markere sensoriske, motoriske og autonome trasé via fluorescerende sporing og å utforske gjenvekst potensialet i nevrale trakter etter skade27,28, 29 , 30 , 31 , 32 , 33. direkte injeksjon av en retrogradely transp…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbeidet ble finansiert av en bevilgning fra National Institute av nevrologiske lidelser og slag R01 R01NS103481 og Shriners sykehus for Pediatric forskning tilskudd SHC 84051 og SHC 86000 og Forsvarsdepartementet (SC140089).
Materials
| #10 Scalpel Blades | Roboz | RS-9801-10 | For use with the scalpel. |
| 1 mL Syringes | Becton, Dickinson and Company | 309659 | For anesthetic IP injection, potential anesthetic booster shots, and antibiotic injections. |
| 10mL Syringes | Becton, Dickinson and Company | 309604 | For injecting saline into the animal, post-surgery. |
| 4.0 Chromic Catgut Suture | DemeTECH | NN374-16 | To re-bind muscle during closing. |
| 48000 Micropipette Beveler | World Precision Instruments | 32416 | Used to bevel the tips of the pulled glass capillary tubes to form functional glass needles. |
| 5% Iodine Solution | Purdue Products L.P. | L01020-08 | For use in sterilzation of the surgical site. |
| 70% Ethanol | N/A | N/A | For sterilization of newly prepared glass needles, animal models during surgical preparation, and surgeon's hands during surgery, as well as all other minor maintainances of sterility. |
| Anesthetic (Ketamine/Xylazine Solution) | Zoetis | 240048 | For keeping the animal in the correct plane of consciousness during surgery. |
| Antibiotic (Cefazolin) | West-Ward Pharmaceuticals | NPC 0143-9924-90 | To be injected subcutaneously to prevent infection post-surgery. |
| Bead Sterilizer | CellPoint | 5-1450 | To heat sterilize surgical instruments. |
| Bonewax | Fine Science Tools | 19009-00 | To seal up bone in the case of bone bleeding. |
| Cauterizer | Fine Science Tools | 18010-00 | To seal any arteries or veins severed during surgery to prevent excessive blood loss. |
| Digital Scale | Okaus | REV.005 | For weighing the animal during surgical preparation. |
| Flexible Needle Attachment | World Precision Instruments | MF34G-5 | For cleaning glass needles and loading red oil into glass needles. |
| Gelfoam | Pfizer | H68079 | To seal up bone in the case of bone bleeding. |
| Glass Capillary Tubes | World Precision Instruments | 4878 | For pulled glass needles – should be designed for nanoliter injectors. |
| Hair Clippers | Oster | 111038-060-000 | For clearing the surgical site of hair. |
| Hemostats | Roboz | RS-7231 | For general use in surgery. |
| Kimwipes | Kimtech | 34155 | For general use in surgery. |
| Medium Point Curved Forceps | Roboz | RS-5136 | For general use in surgery. |
| Micromanipulator with a Vernier Scale | Kanetec | N/A | For precise targeting during surgery. |
| Microscissors | Roboz | RS-5621 | For cutting glass whisps off of freshly pulled glass capillary tubes. |
| Microscope with Light and Vernier Scale Ocular | Leitz Wetzlar | N/A | Used to visualize and measure beveling of pulled glass capillary tubes into functional glass needles. |
| MicroSyringe Pump Controller | World Precision Instruments | 62403 | To control the rate of injection. |
| Nanoliter 2000 Pump Head Injector | World Precision Instruments | 500150 | To load and inject virus in a controlled fashion. |
| Needle Puller | Narishige | PC-100 | To heat and pull apart glass capillary tubes to form glass needles. |
| Ophthalamic Ointment | Dechra Veterinary Products | RAC 0119 | To protect the animal's eyes during surgery. |
| Parafilm | Bemis | PM-996 | To assist with loading virus into the nanoinjector. |
| PrecisionGlide Needles (25G x 5/8) | Becton, Dickinson and Company | 305122 | For use with the 1mL and 10 mL syringes to allow injection of the animal model. |
| Rat Tooth Forceps | Roboz | RS-5152 | For griping spinous processes. |
| Red Oil | N/A | N/A | To provide a front for visualization of virus entering tissue during injection. |
| Retractors | Roboz | RS-6510 | To hold open the surgical wound. |
| Rimadyl Tablets | Bio Serv | MP275-050 | For pain management post-surgery. |
| Rongeurs | Roboz | RS-8300 | To remove muscle from the spinal column during surgery. |
| Scalpel Blade Handle | Roboz | RS-9843 | To slice open skin and fat pad of animal model during surgery. |
| Scissors | Roboz | RS-5980 | For general use in surgery. |
| Stainless Steal Wound Clips | CellPoint | 201-1000 | To bind the skin of the surgical wound during closing. |
| Staple Removing Forceps | Kent Scientific | INS750347 | To remove the staples, should they be applied incorrectly. |
| Sterile Cloth | Phenix Research Products | BP-989 | To provide a sterile surface for the operation. |
| Sterile Cotton-Tipped Applicators | Puritan | 806-WC | To soak up blood in the surgical wound while maintaining sterility. |
| Sterile Gauze | Covidien | 2146 | To clean the surgical area and surgical tools while maintaining sterility. |
| Sterile Saline | Baxter Healthcare Corporation | 281324 | For use in blood clearing, and for replacing fluids post-surgery. |
| Surgical Gloves | N/A | N/A | For use by the surgeon to maintain sterile field during surgery. |
| Surgical Heating Pad | N/A | N/A | For maintaining the body temperature of the animal model during surgery. |
| Surgical Microscope | N/A | N/A | For enhanced visualization of the surgical wound. |
| Surgical Stapler | Kent Scientific | INS750546 | To apply the staples. |
| T/Pump Heat Therapy Water Pump | Gaymar | TP500C | To pump warm water into the water convection warming pad. |
| Water Convection Warming Pad | Baxter Healthcare Corporation | L1K018 | For use in the post-operational recovery area to maintain the body temperature of the unconscious animal. |
| Weighted Hooks | N/A | N/A | To hold open the surgical wound. |
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