Protocol/Condensation of DNA with PEI and Analysis

Protocol for Condensation of DNA with PEI and Analysis

Materials and Reagents

• Filtered (0.22 μm) 5% glucose solution.
• Autoclaved 0.9% NaCl solution.
• DNA resuspended in water at a final

concentration of <0.5 μg/μL.

• PEI solutions diluted extremporaneously

to 10 mM.

• Sterile polypropylene tubes (1.5 mL).
• Vortex mixer.
• Electrophoresis equipment for checking

complexation (not required each time, only for the first round of experiments). Procedure 1. Plasmid DNA is diluted in sterile (0.22 μm filtered) 5% glucose to the chosen concentration (usually 0.5–2 μg/μL). After vortex mixing, the appropriate amount of a 0.1 M PEI solution is added, and the solution is vortex mixed again. The required amount of PEI, according to DNA concentration and number of equivalents needed, is calculated by taking into account that 1 μg

• Sodium pentobarbital (Sanofi, Paris,

France) diluted to 10% to anesthetize adult animals (70 mg/kg weight, i.p.).

• Recovery chamber. Animals should be

kept under an infrared lamp or a specially constructed heated cage until fully recovered from anesthesia and surgery.

Procedure

1. Obviously, all the procedures described herein that involve animals and their care must be conducted in conformity with appropriate institutional guidelines that are in accordance with national and international laws and polices. 2. Adult (1–2 month old) mice are anesthetized using sodium pentobarbital diluted to 10% in 0.9% NaCl. Animals are anesthetized by an i.p. injection (70 mg/kg). Adult mice or rats are placed in the stereotaxic apparatus. An incision is made to expose the cranial skull and a hole made with a 21 gauge needle at chosen stereotaxic coordinates. Between 0.5 and up to 5 μL of complexes in 5% glucose are injected slowly (<5 min) with a Hamilton syringe adapted on a stereotaxic apparatus, small volumes are used for intrathecal injection and larger volumes for intraventricular injection. The needle is left in place for 5 minutes postinjection, to limit backflow from the injection site. 3. Newborn mice are anesthetized by hypothermia on ice, and 1 μL of complexes are injected with a microcapillary adapted on a micromanipulator. The head of the anesthetized pup is held manually for direct microinjection. Again, injections should be as slow as possible, and the capillary left in place for at least a minute to limit backflow. 4. Animals are left in a recovery chamber until active. When optimizing PEI delivery, newborns are returned to the dam for 24 hours before sacrifice, and adults are kept for 72 hours before sacrifice, as expression is usually maximal at these time points.

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