Name: subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye
Uploaded: 2012-11-25T14:00:00
Description: Watch this Scientific Journal Video about Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye at JoVE.com

Forskning for at forebygge blindhed, Experimental assistance fra Takayuki Nagasaki; Denne forskning er i overensstemmelse med ARVO erklæring for anvendelse af dyr i Oftalmologiske og Visual Research. KJW understøttes af NIH tilskud 5T32EY013933 og 5T32DK007647-20. VBM understøttes af NIH bevilling K08EY020530.

1. Saml Hjælpemidler til subretinal Injection <ol><li> Købe eller lave en 100 um diameter nål fra et glaskapillarrør. Dette kan gøres manuelt ved hjælp af en Sutter P-97 pipette puller eller anden lignende anordning. Enden af ​​kapillærrøret skal opvarmes og trækkes indtil det når den ønskede diameter (100 um). En mindre diameter nål kan anvendes til genterapivektorer, men dette er den anbefalede diameter for celleinjektion uden skade på cellerne eller øjet. Sammenlignet med stål nåle, har glaskapi...

<ol>
	<li>Abe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+retina+using+pigment+epithelial+cell+transplantation.">Regeneration of the retina using pigment epithelial cell transplantation.</a> Nihon Ganka Gakkai Zasshi. 106, 778-803 (2002).</li><li>Jacobson, S. G., Cideciyan, A. V., Ratnakaram, R., Heon, E., Schwartz, S. B., Roman, A. J., Peden, M. C., Aleman, T. S., Boye, S. L., Sumaroka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+Leber+congenital+amaurosis+caused+by+RPE65+mutations:+safety+and+efficacy+in+15+children+and+adults+followed+up+to+3+years.">Gene therapy for Leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years.</a> Arch. Ophthalmol. 130, 9-24 (2011).</li><li>Schwartz, S. D., Hubschman, J. P., Heilwell, G., Franco-Cardenas, V., Pan, C. K., Ostrick, R. M., Mickunas, E., Gay, R., Klimanskaya, I., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> Lancet. 379, 713-720 (2012).</li><li>Wang, N. K., Tosi, J., Kasanuki, J. M., Chou, C. L., Kong, J., Parmalee, N., Wert, K. J., Allikmets, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+reprogrammed+embryonic+stem+cells+improves+visual+function+in+a+mouse+model+for+retinitis+pigmentosa.">Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa.</a> Transplantation. 89, 911-919 (2010).</li><li>Song, B. J., Tsang, S. H., Lin, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene Ther. Mol. Biol. 11, 229-262 (2007).</li><li>Tosi, J., Sancho-Pelluz, J., Davis, R. J., Hsu, C. W., Wolpert, K. V., Sengillo, J. D., Lin, C. S., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus-mediated+expression+of+cDNA+and+shRNA+slows+degeneration+in+retinitis+pigmentosa.">Lentivirus-mediated expression of cDNA and shRNA slows degeneration in retinitis pigmentosa.</a> Exp. Biol. Med. 236, 1211-1217 (2011).</li><li>Tucker, B. A., Park, I. H., Qi, S. D., Klassen, H. J., Jiang, C., Yao, J., Redenti, S., Daley, G. Q., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+adult+mouse+iPS+cell-derived+photoreceptor+precursors+restores+retinal+structure+and+function+in+degenerative+mice.">Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.</a> PLoS One. 6, e189992 (2011).</li><li>Mahajan, V. B., Mondino, B. J., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A high-throughput Mouse Eye Phenomics System. , (2010).</li><li>Chang, B., Hawes, N. L., Hurd, R. E., Davisson, M. T., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42, 517-525 (2002).</li><li>Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fundus+photography+and+angiography:+a+catalogue+of+normal+and+mutant+phenotypes.">Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes.</a> Mol. Vis. 5, 22 (1999).</li><li>Won, J., Shi, L. Y., Hicks, W., Wang, J., Hurd, R., Naggert, J. K., Chang, B., Nishina, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+resources+for+vision+research.">Mouse model resources for vision research.</a> J. Ophthalmol. 2011, 391384 (2011).</li></ol>

Denne video teknik indeholder en vejledning i at udfylde subretinal injektion kirurgiske procedure med succes, og sikre, at genterapivektoren eller stamceller er placeret på det sted, nødvendigt for effektivt at behandle oftalmiske sygdom. Denne teknik giver mulighed for målretning af retinale celler, såsom RPE eller fotoreceptorer, fordi de bringer de genterapivektorer eller stamcelle afledt væv i nærheden af ​​disse celler. Tidligere metoder involverede intravitreale injektioner, hvor fluidet er anbragt i de...

<table border="1"><tbody><tr><td> Navn </td><td> Firma </td><td> Catalog </td><td> Kommentarer </td></tr><tr><td> 0,8-1,10 x 100 mm kapillarrør (glas) </td><td> Kimble Glass, Inc. </td><td> 34502 99 </td><td></td></tr><tr><td> Flaming / Brown Mikropipette Puller </td><td> Sutter Instrument </td><td> P-97 </td><td> Narishige microforge kan anvendes i stedet. Catalog # MF-900 </td></tr><tr><td> Sigmacote </td><td> Sigma Aldrich </td><td> SL2-25ML </td><td> Silicone </td></tr><tr><td> Dubecco s Fosfatbufferet saltvand med calciumchlorid og magnesiumchlorid </td><td> Gibco-Invitrogen </td><td> 14040-133 </td><td></td></tr><tr><td> Safety-Lok 25 3/4G x 12 &quot;Blood Collection Set </td><td> BD Vacutainer </td><td> 367.298 </td><td></td></tr><tr><td> 1 ml Sub-Q 265/8G Slip-Tip Sprøjte </td><td> Becton-Dickinson </td><td> 309.597 </td><td></td></tr><tr><td> 0,5-10 pi Finnpipette II Tilpasningslån Volume pipette </td><td> Fisherbrand </td><td> 21-377-815 </td><td></td></tr><tr><td> 1-200 ul Natural facetslebne Tips </td><td> USA Scientific, Inc. </td><td> 1111-1700 </td><td></td></tr><tr><td> Discovery.V8 stereomikroskop </td><td> Zeiss </td><td> MC1500 </td><td></td></tr><tr><td> 60 mm x 15 mm Style behandlet polystyren Cell Culture Dish </td><td> Corning Incorporated </td><td> 430.166 </td><td></td></tr><tr><td> Vannas Lige saks </td><td> Storz oftalmiske </td><td> E3383 S </td><td></td></tr><tr><td> Krumme Dressing Tang med takker Delicate </td><td> Storz oftalmiske </td><td> E1408 </td><td></td></tr><tr><td> 15 Degree Microsurgery Knife </td><td> Wilspå Ophthalmic Corp </td><td> 091.204 </td><td></td></tr><tr><td> Ketamin </td><td> Ketaset III </td><td> NADA # 45-290 </td><td></td></tr><tr><td> Xylazin </td><td> Lloyd Laboratories </td><td> NADA # 139-236 </td><td></td></tr><tr><td> Bupivacain (marcain) </td><td> AstraZeneca </td><td> N / A </td><td></td></tr><tr><td> Buprenorphin </td><td> Sigma Aldrich </td><td> B9275 </td><td></td></tr></tbody></table>

subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye

Tabet af synet påvirker omkring 3,4 millioner mennesker i USA og forventes at stige i de kommende år. 1 Senest har genterapi og stamceller transplantationer blevet centrale terapeutiske redskaber til behandling af blindhed som følge af retinal degenerative sygdomme. Adskillige former for autolog transplantation for aldersrelateret makulær degeneration (AMD), såsom iris pigment epithelial celletransplantation, har skabt opmuntrende resultater, og humane kliniske forsøg er begyndt for andre former for gen-og stamceller. 2 Disse omfatter RPE65 generstatning behandling af patienter med Lebers medfødt amaurosis og en RPE cell transplantation anvendes humane embryonale stamceller (ES)-celler i Stargardt sygdom. 3-4 nu, at der er genterapivektorer og stamceller til rådighed til behandling af patienter med retinale sygdomme, er det vigtigt at verificere disse potentielle behandlinger i dyremodeller før anvendelseING dem i humane studier. Musen er blevet en vigtig videnskabelig model til test af terapeutiske effektivitet af genterapivektorer og stamcelletransplantation i øjet. 8/5 I denne video artikel præsenterer vi en teknik til at injicere genterapivektorer eller stamceller i det subretinale rum af muse øjet samtidig minimere skader på det omgivende væv.

En tegning af musen øjet er vist med store strukturer mærket til reference, med pile viser de steder for både intravitreale og subretinal injektion kirurgiske procedurer (pilespidser, figur 1). Genterapivektorer, såsom lacZ lentivirusvektoren (fig. 2), kan injiceres ved hjælp af disse placeringer. Derudover kan stamceller, såsom muse embryonale stamceller (figur 3), også transplanteres på disse steder i muse øjet. 1 ml af e...

Watch this Scientific Journal Video about Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye at JoVE.com

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

Denne kirurgiske teknik illustrerer injektion af genterapivektorer og stamceller i det subretinale rum af muse øjet.

Subretinal injektion af genterapivektorer og stamceller i mus Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

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