To knockdown Ptn in Pericyte-Cre mice, we used Ptn-specific chemically modified, 21-mer, double-stranded Ambion® In Vivo siRNA (ThermoFisher), following our previously described approach8 (link). As Ptn in mice is only expressed in the brain and not in peripheral tissues, as shown by the mouse genome project38 (link), and is enriched in pericytes, as we (Fig. 4a –c ; Fig S8d ,e ) and others5 (link),24 (link),39 (link) show, to short term silence Ptn we administered centrally siPtn or scrambled control siRNA by bilateral intracerebroventricular injection.
Ambion in vivo sirna
Manufactured by Thermo Fisher Scientific
Sourced in France, Japan, United States
Ambion® In Vivo siRNA is a laboratory product designed for the delivery of small interfering RNA (siRNA) molecules into living organisms for the purpose of gene silencing. The core function of this product is to facilitate the introduction of siRNA into target cells or tissues in vivo.
Automatically generated - may contain errors
Lab products found in correlation
In vivo jetpei, by Polyplus Transfection (1 mentions)
Invivofectamine 3, by Thermo Fisher Scientific (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Hematoxylin qs, by Vector Laboratories (1 mentions)
Invivofectamine 3.0 reagent, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Dl dithiothreitol, by Merck Group (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
8 protocols using ambion in vivo sirna
1
Pericyte-specific Ptn knockdown in mice
2
In vivo siRNA Silencing of PAR1 and PAR3
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For in vivo PAR1 and PAR3 silencing, we used F2r-specific (4457308) and F2rl2-specific (4457298) chemically modified 21-mer double-stranded Ambion in vivo siRNA, respectively (Thermo Fisher Scientific). This Ambion technology has established effectiveness and stability, as we reported previously in plasminogen (Montagne et al., 2018 (link)) and pleiotrophin (Nikolakopoulou et al., 2019 (link)), by showing that ICV delivery of their respective siRNAs effectively suppressed their local gene expression in brain within 24 h of ICV administration, with the effect lasting for >2 wk after a single injection when used with Invivofectamine reagent. Ambion in vivo siRNA was reconstituted in Invivofectamine 3.0 reagent and diluted in PBS; a final dose of 0.1 nmol in 1 µl was delivered ICV to both lateral ventricles over 5 min using a Hamilton syringe, as previously reported (Montagne et al., 2018 (link); Nikolakopoulou et al., 2019 (link)). To confirm the knockdown efficiency, the CC was dissected and prepared for PAR1 and PAR3 immunoblotting 1 wk after ICV siRNA administration. WM stroke was induced 1 wk after PAR1 and PAR3 silencing.
3
Pericyte-specific Ptn knockdown in mice
4
In Vivo siRNA Delivery Using jetPEI
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siRNA (Ambion In Vivo siRNA, Thermo Fisher Scientific) was delivered in vivo using in vivo-jetPEI (Polyplus-transfection, Illkirch, France) according to the manufacturer's protocols. In brief, 40 μg siRNA duplex was mixed with 6.4 μl in vivo-jetPEI in a final volume of 200 μl of 10% glucose. The mixture was incubated for 15 min at room temperature and was delivered to the mice by intravenous injection through the femoral vein.
5
Systemic and Central Knockdown of Plg in F7/F7 Mice
6
SREBP1c Silencing in Obese Mouse Model
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Eight-week-old male C57/B6J mice (CLEA Japan) were fed an amylin liver MASH diet—enriched in fat (40% kcal, Primex partially hydrogenated vegetable oil shortening), fructose (22% by weight), and cholesterol (2% by weight)—for 20 weeks (Research Diets). For the last 4 weeks, Ambion in vivo siRNA (Thermo Fisher Scientific, Tokyo, Japan) with the antisense of SREBP1c target sequence 5′-UACAUCUUUAAAGCAGCGGGT-3′ were combined with invivofectamine 3.0 (Thermo Fisher Scientific) to form complexes following the manufacturer’s instructions. The complex was injected into MASH model mice by means of the tail vein at 20 µg per mouse every three days for 4 weeks. PBS was used as a control. The experimental protocol is outlined in Supplemental Figure S1C, http://links.lww.com/HC9/A615 .
7
Hepatocyte siRNA Delivery Protocol
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Livers from C57BL/6J mice were digested by collagenase perfused through the portal vein using a two-step collagenase perfusion method. Hepatocytes were collected as pellets by centrifugation at 50 × g for 5 min. Hepatocytes were cultured in DMEM containing 10% FBS on collagen-I coated plates. siRNA studies were performed using the siRNAs specified below. The siRNAs were purchased from Thermo Fisher Scientific (Ambion® In Vivo siRNA; Ambion, Thermo Fisher Scientific) and incorporated chemical modifications for superior serum stability with in vivo delivery. Mouse Inhbe siRNA (siINHBE) had the following sequence:
5’-CAGCTTTGCTACCATCATA-3’ (sense). Non-targeting siRNA (siNON) was also used, and this siRNA had no significant homology with any known gene in mouse, rat, or human (Ambion® In Vivo Negative Control #1 siRNA). The siRNAs were transfected into hepatocytes using Invivofectamine 2.0 transfection reagent (Invitrogen, Thermo Fisher Scientific) 1 day after plating.
8
Optimized Lipid-Mediated Delivery of siRNA for PCSK9 Inhibition
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All following reagents and solvents were purchased from Merck Sigma-Aldrich (Darmstadt, Germany) and were of LC-MS grade, unless otherwise specified: urea, sodium deoxycholate (DOC), Trisma hydrochloride (Tris), DL-dithiothreitol (DTT), iodoacetamide (IAA), N-acetyl-L-cysteine (NAC), ethylenediaminetetraacetic acid (EDTA), water, acetone, acetonitrile, and formic acid (FA). Gold LC-MS sequencing-grade trypsin was purchased from Promega (Madison, WI, USA). Invivofectamine 3.0 reagent was purchased from Thermo Scientific (Rockford, IL, USA). Oil Red O was offered by Merck Sigma-Aldrich, while Hematoxylin QS was ordered from Vector Laboratories (Burlingame, CA, USA). Atorvastatin was bought from Terapia Ranbaxy (Cluj-Napoca, Romania). Ambion In Vivo siRNA for PCSK9 was custom designed for inhibiting the rabbit PCSK9 gene (Gene ID: 100338756) using the Ambion by Life Technologies, now a part of Thermo Scientific (Carlsbad, CA, USA) Silencer Select algorithm and Ambion In Vivo chemical modifications. The designed sense sequence (5′—>3′) was GUCGCUUUCUUAGCAAGAAtt, while the antisense sequence was UUCUUGCUAAGAAAGCGACag. The annealed reagent was thereafter HPLC purified and used in a 1.2:1 siRNA: lipid ratio as recommended by the manufacturer.
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