In vivo delivery protocol

FANA Delivery in Mice: Protocol

Note: The following is a general protocol for the use of FANA in mouse models. The protocol can be adapted for use in other mammalian or in vivo models.

• 1. Prepare a FANA stock solution by reconstituting lyophilized ASOs at the desired concentration.

  • Resuspend FANA ASOs using the appropriate volume of sterile water or saline buffer.

  • We recommend a stock solution of 5-10 mg/ml. The approximate molecular weight of a 21 nt singlestranded FANA oligo is 6000-7000 g/mol (sequence-dependent). 100 nmol of FANA oligos is equivalent to 650 µg.

  • Pipette solution up and down 3-5 times while avoiding introduction of bubbles. Let the vial sit at room temperature for 5-10 min. Briefly centrifuge to collect solution to bottom of the tube.

• 2. Calculate FANA concentrations for in vivo delivery.
  

  • For in vivo systemic delivery, we recommend a dose between 3 – 30 mg/kg in mice. If topical (organ, intratumoral, etc) delivery is desired, the concentration can be adjusted accordingly, considering the volume of the organ or tumor.

  • We recommend you perform a dose response using 2-3 working concentrations to determine the most optimum concentration for your specific application.

  • If long-term gene silencing is desired, multiple doses of FANA can be administered.

• 3. Administer FANA oligos to mice.

  • FANA oligos can be administered via different routes: intravenous (IV), intraperitoneal (IP), intradermal (ID), intratumoral (IT), intranasal (IN), intratracheal, hydrodynamic tail injection, inhalation, or local organ delivery.

• 4. Assay for gene knockdown in your target tissues.

  • Note: Uptake of fluorescently-labeled FANA can be observed as early as 24 hr, but full knockdown in vivo is best assessed at 72 hr to several days post FANA treatment.