Subretinal Delivery of Genetic Agents in Mice

Subretinal injections (1 μl) in mice were performed using a Pico-Injector (PLI-100, Harvard Apparatus) or using a 35-gauge needle (Ito Co. Fuji, Japan). In vivo transfection of plasmids expressing Alu sequences (pAlu)^{67 (link),68 (link)}, empty control vector (pNull), Flag-cGAS (pFlag-cGAS), Flag-GFP, mouse mature IL-18 (pIL-18ss)^{57 ,69 (link)}, wildtype mouse gasdermin D (pGSDMD-WT), the p30 cleavage incompetent mutant mouse gasdermin D ((pGSMDD-D276A)^{19 (link)}, IFN-β (Origene Cat# MR226101), or mtDNA (10 ng) was achieved using 10% Neuroporter (Genlantis) as previously described^{4 (link),5 (link)}. In vitro transcribed Alu RNA (0.15–0.3 μg/μl), IFN-β neutralizing antibody (10 ng; Abcam Cat# ab24324), control isotype IgG, recombinant IL-18 (100 ng/μl, MBL Cat#B002-5), or IFN-β (500 mUnit/μl, PBL Cat#12410-1) were administered via subretinal injection^{4 (link),5 (link)}. Similarly, to knock down Dicer1, 1 μl of cholesterol conjugated siRNA (1 μg/μl) targeting mouse Dicer1 or scrambled control siRNAs were injected (Dicer1 siRNA sense- CUCUGUGAGAGUUGUCCdTdT; Control siRNA sense- UAAGGCUAUGAAGAGAUdTdT). The eye used for active versus control injection was chosen randomly.

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Kerur N., Fukuda S., Banerjee D., Kim Y., Fu D., Apicella I., Varshney A., Yasuma R., Fowler B.J., Baghdasaryan E., Marion K.M., Huang X., Yasuma T., Hirano Y., Serbulea V., Ambati M., Ambati V.L., Kajiwara Y., Ambati K., Bastos-Carvalho A., Ogura Y., Terasaki H., Oshika T., Kim K.B., Hinton D.R., Leitinger N., Cambier J.C., Buxbaum J.D., Kenney M.C., Jazwinski S.M., Nagai H., Hara I., West A.P., Fitzgerald K.A., Sadda S.R., Gelfand B.D, & Ambati J. (2017). cGAS drives non-canonical inflammasome activation in age-related macular degeneration. Nature medicine, 24(1), 50-61.

Publication 2017

PLI-100 IFN-β Neuroporter

Cat 1 Cgas Cholesterol Cleavage Dicer1 Il 18 Isotype Mouse Mouse gasdermin d Mtdna Needle Neutralizing antibody Plasmids Sirna Transfection

Corresponding Organization : University of Virginia

Other organizations : University of Kentucky, Doheny Eye Institute, University of Tsukuba, Center for Digital Image Evaluation, Icahn School of Medicine at Mount Sinai, Nagoya City University, Nagoya University, Southern California Eye Institute, University of Southern California, University of Colorado Denver, University of California, Irvine, Tulane University, Kobe University, Wakayama Medical University, Texas A&M University, University of Massachusetts Chan Medical School

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Variable analysis

independent variables

Subretinal injections (1 μl) in mice performed using a Pico-Injector (PLI-100, Harvard Apparatus) or using a 35-gauge needle (Ito Co. Fuji, Japan)
In vivo transfection of plasmids expressing Alu sequences (pAlu), empty control vector (pNull), Flag-cGAS (pFlag-cGAS), Flag-GFP, mouse mature IL-18 (pIL-18ss), wildtype mouse gasdermin D (pGSDMD-WT), the p30 cleavage incompetent mutant mouse gasdermin D (pGSMDD-D276A), IFN-β (Origene Cat# MR226101), or mtDNA (10 ng)
In vitro transcribed Alu RNA (0.15–0.3 μg/μl), IFN-β neutralizing antibody (10 ng; Abcom Cat# ab24324), recombinant IL-18 (100 ng/μl, MBL Cat#B002-5), or IFN-β (500 mUnit/μl, PBL Cat#12410-1) administered via subretinal injection
Knockdown of Dicer1 using cholesterol conjugated siRNA (1 μg/μl) targeting mouse Dicer1 or scrambled control siRNAs

dependent variables

Not explicitly mentioned

control variables

10% Neuroporter (Genlantis) used for in vivo transfection of plasmids
Control isotype IgG administered via subretinal injection
Scrambled control siRNAs for Dicer1 knockdown

positive controls

Empty control vector (pNull)
Flag-GFP
Control isotype IgG

negative controls

Scrambled control siRNAs for Dicer1 knockdown

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