Biomedical Engineering Reference
In-Depth Information
4 Carry out three hygromycin selections for a period of 3-5 days, to allow cell
recovery.
5 Select stable lines incorporating the
CMV-HYTK
gene in DMEM plus 2% Methocel,
containing hygromycin.
6 Pick individual clones after 2-3 weeks and grow in a medium containing
hygromycin.
7 Check the integrity of transgene by polymerase chain reaction (PCR) amplification and
Southern blotting using GFP as probe.
8 Identify clones containing a single copy
b
of the L1-HYTK-L1 cassette by Southern
blots.
c
Notes
a
Cells can also be electroporated using the Gene Pulser II cell electroporator.
b
Linearization of plasmid is recommended for more efficient isolation of single-copy integrants,
followed by dephosphorylation prior to transfection.
c
Alternatively, it is also possible to determine the site of integration by linear amplification-
mediated-polymerase chain reaction (LAM-PCR) [35].
PROTOCOL 7.2
Isolation of Exchanged Transgenes
Equipment and reagents
•
Cre recombinase expression plasmid pBS185 (GIBCO)
•
DMEM (Gibco)
•
FBS (Wisent)
•
Fluorescent flow cytometry (Becton Dickinson)
•
PCR machine (MasterCycler, Eppendorf)
•
Methocel (Fluka)
•
Ganyclovir (Sigma)
•
Hygromycin B (Roche)
Method
1 For erythroid cell lines, co-transfect the linearized plasmids (the test and Cre expression
plasmids) with Lipofectamine for 4 h, in the presence of DMEM cell culture media
without serum (Figure 7.1a).
d
2 Culture the clones (containing the
CMV-HYTK
gene) in selection medium with
0.75mg/ml of hygromycin B.
e
3 Three days after the transfection, perform the negative selection over a period of 10
days, in the presence of 50 ng/ml of gancyclovir.
f
