Hydrodynamic Gene Delivery: A Technique for In Vivo Transfection of Exogenous DNA in Murine Hepatocytes via Tail Vein Injection

Protokoll

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparing Solution for Hydrodynamic Tail Vein Injection

NOTE: Prepare the plasmid constructs for constitutive and inducible gene expression by cloning technique using competent bacteria.  

1. Prepare sterile 0.9% saline for injection (do not use PBS). Use volume corresponding to about 10% of mouse body weight. Example: for a mouse weighing 20 g, prepare 2 mL of solution.
2. Prepare injection vectors that were purified using an endotoxin-free plasmid purification kit.
3. Add 10 µg or 15 µg of endotoxin-free sleeping beauty vector construct (use 10 µg, from step 4 use 15 µg, respectively) and 1 µg of endotoxin-free pc-HSB5 per mL of sterile saline.
4. Store solution for up to 4 h at 4 °C. Do not freeze.

2. Performing Hydrodynamic Tail Vein Injection

1. Use a restrainer for tail vein injection (commercial or prepared from a 50 mL conical tube with holes for breathing and for the tail). Fill bottom of the tube with tissue paper.
2. For injection, use mice of about 8–10 weeks of age with weights of 20–25 g.
3. Weigh mice before injection and prepare injection volume according to body weight (corresponding to 10% of body weight). Prepare a sterile 3 mL-syringe with a 27 G-needle for injection and fill with the required volume.
4. Place the mouse into the restrainer. Adjust the amount of tissue paper to leave only minimal space for movement but enough space for breathing.
5. Ensure that the mouse is breathing regularly.
6. Warm the tail using an infrared lamp for 30-60s. Carefully watch for signs of overheating.
7. Clean the tail with an alcohol swab.
8. Insert the needle almost horizontally into either one of the two lateral tail veins close to the base of the tail.
   NOTE: If placed successfully, a small amount of blood might flow back into the cone of the needle. It is not recommended to actively aspirate as any additional movement of the needle can result in its displacement and/or injury of the vein.
9. Inject the total volume into the tail vein within 8-10 s.
10. Immediately remove the mouse from the restrainer. Compress injection wound for at least 30 s or until any bleeding subsides.
11. Place the mouse into a separate cage. Once the mouse has recovered from the procedure (about 30–60 min), transfer the mouse back to its original cage. Check on the mouse regularly for the next 24 h.
    NOTE: Mild sedation of the mouse is routinely observed for up to 2 h after injection.

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Materialien

Name	Company	Catalog Number	Comments
NaCl 0,9%	Braun	3200905	Carrier for intravenous injections
Falcon Conical Tube 50ml	Corning Life Science	352095
Infrared Lamp	N/A	N/A	For warming of mouse tail
Plasmids for cloning of sleeping beauty-transposon vectors for HTVI.
pTC	n/a		Vector for constitutive gene expression
pEN_TTmcs	Addgene	#25755	Entry vector for inducible gene expression
pEN_TTGmiRc2	Addgene	#25753	Entry vector for inducible miRshRNA expression with coexpression of GFP
pEN_TTmiRc2	Addgene	#25752	Entry vector for inducible miRshRNA expression without coexpression of GFP
pTC ApoE-Tet	Addgene	#85578	Expression vector for inducible gene or miR-shRNA expression with ApoE.HCR.hAAT promotor
pTC-CMV-Tet	Addgene	#85577	Expression vector for inducible gene or miR-shRNA expression with CMV promotor