Adeno-Associated Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 10

The mechanism by which this complex enters cells is not
known in detail, although it has been suggested that it is first drawn
into acidifying endosomes before DNA is transferred to the
nucleus. The transfer of DNA across the nuclear membrane must
occur during mitosis. Therefore, expression of the gene of interest
will not occur until the cells have passed through this phase.
Although this technique has minimal cellular toxicity and is
both simple and inexpensive, the relatively low level of transgene
expression using some cell lines prompted development of other
techniques.
1. Thaw plasmids to ensure complete DNA resuspension.
Prewarm the following solutions to 37 °C:
2.5 M CaCl 2
2× HBS, pH 7.05 (the pH is critically important)
Complete DMEM media with 5 % FBS, Pen-Strep, and
l l-glutamine.
2.5 M CaCl 2 : 36.75 g CaCl 2 · 2H 2 O (Sigma C7902) in 100 ml
dH 2 O, sterilized by iltration through a 0.22 ʼm ilter. Store
at −20 °C.
2 × HBS : 280 mM NaCl, 1.5 mM Na 2 HPO 4 , 50 mM HEPES.
Adjust to pH 7.05 with NaOH and sterilize by filtration. Store
at −20 °C.
(Note: The pH of HBS is extremely important as even relatively
small deviations can negatively affect transfection efficiency)
2. Prepare three 50 ml tubes (Tube 1, each will serve for trans-
fection of 10 dishes) and add 1.25 ml CaCl 2 to each tube plus
the calculated amounts for each of the three plasmids: helper
plasmid, packaging plasmid, cis plasmid, e.g., pCMV-GFP.
Make up to 12.5 ml with sterile water.
Prepare three more tubes (Tube 2) and add 12.5 ml of
2 × HBS.
Place Tube 1 on a holder and gently bubble it slowly by
expelling air using pipette and electronic pipette aid. Then add
the content of Tube 2 drop-wise into Tube 1 using a 1 ml
pipette. Once all solution has been added, allow the mixed
solution to settle for 10 min at 37 °C. The mixture should
appear slightly opaque and milky.
3. To a tissue culture flask, add 200 ml of prewarmed serum-free
media and then add 25 ml transfection mixture.
Aspirate the medium from the 30 dishes and add 20 ml of
the medium with DNA precipitates into each dish. Mix the
suspension as you go along to prevent the DNA precipitates
from dropping to the bottom of the container. Be careful not
to displace the cells from the dishes.
4. Transfer the dishes to the incubator and leave for 48 h.