Research to Prevent Blindness; Bartly J. Mondino M.D., Director of the Jules Stein Eye Institute, UCLA; and Ramiro Ramirez-Solis, Jacqui White, and Jeanne Estabel at the Sanger Institute, Wellcome Trust Genome Campus. This research complies with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research.

1. Blunt Dissection: Enucleation of the mouse eye in unfixed specimens
<ol>
 <li>Pull apart the eyelids to improve exposure and access to the posterior globe (eyeball) surface. </li>
 <li>Place a curved dressing forcep behind (under) the globe in the orbit (eye socket). The Mahajan Sharptip dressing forcep is a custom instrument with pointed tips to make this step easier (see materials and reagents table).</li>
 <li>Close the forcep and grasp the orbital connective tissue and optic nerve behind the globe while being ca...

<ol>
	<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>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+high-throughput+Mouse+Eye+Phenomics+System.">A high-throughput Mouse Eye Phenomics System.</a> Cold Spring Harbor Laboratories, Mouse Genetics Meeting. , (2010).</li><li>Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Research Methods For Mutant Mice. , (2002).</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>Anderson, M. G., Smith, R. S., Hawes, N. L., Zabaleta, A., Chang, B., Wiggs, J. L., 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=Mutations+in+genes+encoding+melanosomal+proteins+cause+pigmentary+glaucoma+in+DBA/2J+mice.">Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice.</a> Nat. Genet. 30, 81-85 (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, 22-22 (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, 391384-391384 (2011).</li><li>Pinto, L. H., Vitaterna, M. H., Siepka, S. M., Shimomura, K., Lumayag, S., Baker, M., Fenner, D., Mullins, R. F., Sheffield, V. C., Stone, E. M., Heffron, E., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+from+screening+over+9000+mutation-bearing+mice+for+defects+in+the+electroretinogram+and+appearance+of+the+fundus.">Results from screening over 9000 mutation-bearing mice for defects in the electroretinogram and appearance of the fundus.</a> Vision. Res. 44, 3335-3345 (2004).</li><li>Heckenlively, J. R., Winston, J. V., Roderick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+mouse+retinal+degenerations.+I.+Correlation+of+indirect+ophthalmoscopy,+electroretinograms,+and+histology.">Screening for mouse retinal degenerations. I. Correlation of indirect ophthalmoscopy, electroretinograms, and histology.</a> Doc. Ophthalmol. 71, 229-239 (1989).</li><li>Tsang, S. H., Gouras, P., Yamashita, C. K., Kjeldbye, H., Fisher, J., Farber, D. B., Goff, S. P. <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+in+mice+lacking+the+gamma+subunit+of+the+rod+cGMP+phosphodiesterase.">Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase.</a> Science. 272, 1026-1029 (1996).</li><li>Tsang, S. H., Woodruff, M. L., Jun, L., Mahajan, V., Yamashita, C. K., Pedersen, R., Lin, C. S., Goff, S. P., Rosenberg, T., Larsen, M., Farber, D. B., Nusinowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+carrying+the+H258N+mutation+in+the+gene+encoding+the+beta-subunit+of+phosphodiesterase-6+(PDE6B)+provide+a+model+for+human+congenital+stationary+night+blindness.">Transgenic mice carrying the H258N mutation in the gene encoding the beta-subunit of phosphodiesterase-6 (PDE6B) provide a model for human congenital stationary night blindness.</a> Hum. Mutat. 28, 243-254 (2007).</li></ol>

Most transgenic mice exist in laboratories that do not examine the eyes. Our video technique illustrates a simple, standardized method for remote surgical skill transfer to optimize tissue acquisition from laboratories with little experience with the eyes. This video technique helps overcome a major pitfall in high-throughput phenotyping, which is the use of a limited number of expert sites due to non-standardized tissue collection and fixation methods that prevent meaningful comparative phenotypic and molecular studies....

<table border="1">
 <tbody>
 <tr>
 <th>Name</th>
 <th>Company</th>
 <th>Catalog Number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>Curved Dressing Forcep</td>
 <td>Storz Ophthalmics</td>
 <td>E1408</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mahajan Sharptip dressing forcep</td>
 <td>Storz Ophthalmics</td>
 <td>E1406 (REF SP7-64520)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
	<td>Curved Westcott Scissors</td>
	<td>Storz Ophthalmics</td>
	<td>E3321 WH</td>
	<td>&nbsp;</td>
 </tr>
 <tr>
 <td>15&deg; BD Beaver Microsurgical Blade</td>
 <td>Becton-Dickinson</td>
 <td>374881</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.22 Fine-Castroviejo Suturing Forceps</td>
 <td>Storz Ophthalmics</td>
 <td>E1805</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.12 Colibri forceps</td>
 <td>Storz Ophthalmics</td>
 <td>2/132</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>30-gauge needle</td>
 <td>Becton-Dickinson</td>
 <td>305128</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biohazard Mailer</td>
 <td>Fisher</td>
 <td>03-523-4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Parafilm</td>
 <td>Fisher</td>
 <td>13-374-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass scintillation vials</td>
 <td>Wheaton</td>
 <td>4500413033</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS, pH 7.4</td>
 <td>Invitrogen</td>
 <td>70011-044</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>16% Paraformaldehyde</td>
 <td>Electron Microscopy Sciences</td>
 <td>15700</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2.5% Paraformaldehyde/ 2.5% Glutaraldehyde in 0.1M sodium phosphate buffer</td>
 <td>Electron Microscopy Sciences</td>
 <td>15700 &amp; 16300</td>
 <td>Mixed in laboratory</td>
 </tr>
 <tr>
 <td>50% Glutaraldehyde</td>
 <td>Electron Microscopy Sciences</td>
 <td>16300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.25% Formvar</td>
 <td>Electron Microscopy Sciences</td>
 <td>15810</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Copper Slot Grid</td>
 <td>Electron Microscopy Sciences</td>
 <td>M2010-CR</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>4% Osmium Tetroxide</td>
 <td>Electron Microscopy Sciences</td>
 <td>19140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-SOD3 antibody</td>
 <td>Abcam</td>
 <td>Ab21974</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Goat anti-rabbit Alexa Fluor 488</td>
 <td>Invitrogen</td>
 <td>A11070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spurr's embedding resin</td>
 <td>Electron Microscopy Sciences</td>
 <td>14300</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

mouse eye enucleation for remote high-throughput phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

Watch this Scientific Journal Video about Mouse Eye Enucleation for Remote High-throughput Phenotyping at JoVE.com

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

mouse-eye-enucleation-for-remote-high-throughput-phenotyping

Research

JoVE Journal

Medicine

20.1K 조회수.  University of Iowa. 분해 기술은 높은 처리량 화면에서 phenotyping을 수행 할 조직 고정에 대한 마우스 눈의 핵의 제거를 보여줍니다.