慢病毒Injection
一疫向、靜脈慢病毒注射
Lentiviral supernatant was thawed at 37°C and diluted in 0.9% saline (injection, USP; APP, Schaumburg, IL) and polybrene (8 μg/ml final concentration; Sigma, St. Louis, MO, USA) to give a dose of 1.6 × 107 or 1.6 × 108 TU in a 50-μl injection volume.
ref——In Vivo Transduction by Intravenous Injection of a Lentiviral Vector Expressing Human ADA into Neonatal ADA Gene Knockout Mice: A Novel Form of Enzyme Replacement Therapy for ADA Deficiency
二、腦組織慢病毒注射：
Injection of lentivirus for a single injection. Place the injection micropipette into the holder of the stereotaxic arm. Fill the micropipette with 3 μL lentivirus. Then slowly lower the micropipette to the desired z coordinate of the injection site. Apply pressure steadily via a pump or syringe to inject 100~500 μL lentivirus at >1E+9 TU/mL within 60~90 seconds.
ref——Infection Protocol for Recombinant Lentivirus
三、脂肪組織慢病毒注射：
- Then, lentiviral injection in iWAT and pgWAT was performed at week 11. To inject lentiviral particles in both iWAT and pgWAT, mice were anesthetized by isoflurane before dissection of the skin. The lentiviral preparation (1 × 107–8 plaque-forming units in a volume of 100 μl) was injected into the right and left pgWAT and iWAT depot, which were distributed first in 6 injections of 10 μl for each pgWAT, and then 4 injections of 8 μl for each iWAT (as showed below in Fig. 1A–B). Each mouse was injected with ~185 μl of lentiviral preparation.
ref——Latorre J, Lluch A, Ortega FJ, Gavaldà-Navarro A, Comas F, Morón-Ros S, Rodríguez A, Becerril S, Villarroya F, Frühbeck G, Ricart W, Giralt M, Fernández-Real JM, Moreno-Navarrete JM. Adipose tissue knockdown of lysozyme reduces local inflammation and improves adipogenesis in high-fat diet-fed mice. Pharmacol Res. 2021 Feb 5:105486.
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In this protocol 1000 ng/25 μl (BAT) and 1000 ng/30 μl (WATi) of lentivirus were injected into each fat lobe (Fig 2).
ref——Balkow A, Hoffmann LS, Klepac K, Gl?de A, Gnad T, Zimmermann K, Pfeifer A. Direct lentivirus injection for fast and efficient gene transfer into brown and beige adipose tissue. J Biol Methods. 2016 Jul 16;3(3):e48.
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Lentiviral vectors coding for A2A (LVA2A) were used to overexpress A2A in WATi; lentiviral vectors coding for GFP were used as control. 10-week old male C57Bl/6 mice randomized for weight were anaesthetized with isofluorane, a short incisionπ was made in the flank and viral particles (350 ng lentiviral particles/fat pad (p24 antigen Elisa, ZeptoMetrix)) were directly injected into the iWAT depots and animals were put on HFD for 6 weeks.
ref——Gnad T, Scheibler S, von Kügelgen I, Scheele C, Kili? A, Gl?de A, Hoffmann LS, Reverte-Salisa L, Horn P, Mutlu S, El-Tayeb A, Kranz M, Deuther-Conrad W, Brust P, Lidell ME, Betz MJ, Enerb?ck S, Schrader J, Yegutkin GG, Müller CE, Pfeifer A. Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors. Nature. 2014 Dec 18;516(7531):395-9.
