We utilized the AAV Helper‐Free System (AAV Helper‐Free System, Stratagene) for viral production using a triple‐transfection, helper‐free method and purified it as described in a previous study.23 The interference sequences were as follows: shRNA: 5′‐CCGGTTATGTGTATGGGATTCTAAACTCGAGTTTAGAATCCCATACACATAATTTTTG‐3′; and control shRNA, 5′‐TTCTCCGAACGTGTCACGT‐3′. Briefly, nude mice were anaesthetized with an isoflurane/air mix (3% for initial induction and 1.5–2% for maintenance). Three hundred nanolitres of either AAV5‐shRAB31 or AAV5‐shCtrl was injected into the Coleman fat with LRG‐1 according to different groups as mentioned before. The injections were performed using a 34‐gauge needle (World Precision Instruments) attached to a 10‐μl NanoFil microsyringe (Nanofil, World Precision Instruments).
34 gauge needle
Manufactured by World Precision Instruments
The 34-gauge needle is a precision laboratory instrument designed for delicate applications. It features a small diameter and thin wall construction, allowing for precise fluid handling and sample extraction. The needle is made of high-quality materials to ensure durability and consistent performance in a variety of laboratory settings.
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3 protocols using 34 gauge needle
1
AAV-Mediated Delivery of shRNA in Mice
2
Mechanic Stretch-Induced Skin Remodeling
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Briefly, WT mice (8 weeks) were anaesthetized with an isofluorane/air mix (3% for initial induction and 1.5–2% for maintenance). Three hundred nanoliters of either AAV5-shLRG-1 or AAV5-shCtrl were injected into the subcutaneous of mice back skin 5 days before mechanic stretch and continued till the end of mechanic loading. The injections were performed using a 34-gauge needle (World Precision Instruments) attached to a 10 µL-NanoFil microsyringe (Nanofil, World Precision Instruments).
3
AAV-Mediated Smad7 Silencing in Mice
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We utilized the AAV Helper-Free System (AAV Helper-Free System, Stratagene) for viral production using a triple-transfection, helper-free method and purified it as described in a previous study. The interference sequences were as follows: Smad7 shRNA: 5ʹ-CACCGCTTTCAGATTCCCAACTTCTCGAAAGAAGTTGGGAATCTGAAAGC-3ʹ and control shRNA, 5′-TTCTCCGAACGTGTCACGT-3′. Mice were anaesthetized with an isoflurane/air mix (3% for initial induction and 1.5-2% for maintenance). Three hundred nanoliters of either AAV5-shSmad7 or AAV5-shCtrl was injected into the subcutaneous of mice dorsal skin 5 days before mechanical loading and continuously injected during the loading period according to different groups. The injections were performed using a 34-gauge needle (World Precision Instruments) attached to a 10-μL NanoFil microsyringe (Nanofil, World Precision Instruments).
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