Biomedical Engineering Reference
In-Depth Information
PROTOCOL 11.1 Co-Transfection of FG293 Cells with Ad5 Shuttle
Vector pDC516 and pBHGfrt Genomic Plasmid
Equipment and reagents
•
FG293 human embryonic kidney cells (Microbix Systems Inc., http://microbix.com)
•
Cell culture medium I: minimal essential medium, glutamine, 10% fetal calf serum (FCS)
(all Sigma)
•
Cell culture medium II: minimal essential medium, glutamine, penicillin/streptomycin,
10% newborn calf serum (NBS) (all Sigma)
•
T25 tissue culture flasks (Falcon; Beckton Dickinson)
•
pBHGfrt genomic adenovirus plasmid (Microbix Systems Inc.)
•
pDC516 containing cDNA insert of interest (Microbix Systems Inc.)
•
HEPES-buffered saline (HeBS) (5 g 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES)-free acid, 8 g NaCl, 0.37 KCl, 1 g glucose, make up to 1 l and pH to 7.1) (see
note a)
2.5
M
CaCl
2
a
•
•
Phosphate-buffered saline (PBS)++ (PBS supplemented with 0.01% CaCl
2
and 0.01%
MgCl
2
)
•
100% glycerol sterilized by autoclaving.
Method
1 Day 0: set up six T25 flasks of FG293 cells from growing stocks.
2 Day 1: change the medium to cell culture medium I and incubate cells for 4 h at
37
◦
C.
3 Set up 5 vol of DNA precipitate:
1vol: 5
μ
g pBHGfrt (genomic adenovirus plasmid)
3
μ
g plasmid containing insert (e.g. pDC516-ICAM-1 [53])
500
μ
lHeBS
50
μ
l2.5
M
CaCl
2
(added dropwise)
Leave to precipitate for 20-30min at room temperature.
4 Add 500
μ
l of precipitate per flask in five flasks (without removing medium) and
leave for 24 h at 37
◦
C (or up to 48 h). One flask will remain untransfected as a
control.
5 Day 2: remove medium and replace with fresh cell culture medium I.
6 Thereafter replace medium twice per week with cell culture medium II and observe cells
every 2 days.
