Mice experiments were performed after obtaining Institutional Animal Ethics Committee approvals. The in vivo siRNA targeting the CLIP170 (5-GGAGAAGCAGCAGCACAUUTT-3) or scrambled siRNA (5-UUCUCCGAACGUGUCACGUTT-3) were synthesized from Ambion. In vivo siRNA was complexed with the in vivo siRNA delivery reagent, Invivofectamine 2.0 (Invitrogen) according to the manufacturer’s protocols. One hundred microliters of siRNA complex that contained 50 μg of siRNA, was injected into 6 weeks old female C57BL/6 mice through tail vein. To examine the silencing of endogenous CLIP170 in the liver, 48 h after the siRNA delivery, Kupffer cells were harvested from the mice employing a gradient centrifugation protocol as described by Li et al., 2014 (29 ). Adhered Kupffer cells were lysed and subjected to immunoblotting using anti-CLIP170 antibody. Spleens were also harvested and dispersed in RPMI1640 in 30 mm culture dishes. Spleen homogenate was then filtered through a cell strainer and seeded into 6-well plates. Splenocytes were allowed to adhere for 2 hours followed by cell lysis and immunoblotting.
In vivo sirna
Manufactured by Thermo Fisher Scientific
In vivo siRNA is a laboratory tool designed for the delivery of small interfering RNA (siRNA) molecules in living organisms. It facilitates the study of gene function and gene silencing in biological systems.
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2 protocols using in vivo sirna
1
In Vivo Silencing of CLIP170 in Mice
2
Targeted siRNA Delivery to Tumor-Associated Macrophages
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Mice were intravenously injected with B16 tumor cells, 4 days after tumor injection, nanoparticles containing 50 µg control or Il9r siRNA (Ambion in vivo siRNA) were injected intravenously in the tumor-bearing mice every 72 h. Targeted liposomal nanoparticles were prepared using the extrusion method detailed in previous reports32 (link),33 (link),55 (link). All lipid, and lipid-conjugate components were individually prepared and purified, then combined at the desired stoichiometric ratios in chloroform and dried to form a lipid film. Lipid films were then hydrated siRNA containing buffer and extruded to form the liposomes. A SIRPα-targeting peptide was used to target tumor-associated macrophage and its sequence was reported in Rodriguez et al.56 (link).
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