Biomedical Engineering Reference
In-Depth Information
(a)
HyTk
13
14
20
LoxP
LoxP
pBLL1
RMCE-
HD3
-
Random integration
Hygromycin selection
HyTk
LOXP
LoxP
HyTk
HyTk
LoxP
LoxP
LoxP
LoxP
+ Cre
GFP
D
GFP
LoxP
LoxP
LoxP
sil
enh
LoxP
α
Gancyclovir
selection
GFP
D
GFP
LoxP
sil
enh
LoxP
LoxP
LoxP
α
(b)
HD3wt
RMCE-HD3-13
Line 1
Line 2
Line 3
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Figure 7.1
Site-specific chromosomal integration in mammalian cells. (a) Schematic represen-
tation of recombinase-mediated cassette exchange procedure. Stable and single-copy integrants
are generated and isolated in the chicken erythroblast HD3 cell line (RMCE-HD3-13, -14 and
-20) that are randomly integrated in the HD3 genome. The test vector is co-transfected with
an expression vector for Cre-recombinase. Cells are subjected to gancyclovir and subsequently
individual monoclonal stable lines are isolated. (b) Isolation of individual clones through flow
cytometry analysis. Cells were grown sequentially for 3 days in the presence of gancyclovir,
3 days without selection and again 3 days with gancyclovir selection. Despite all these pre-
cautions, cell sorting remains the most suitable complementary strategy. The diagram shows a
non-transfected wild-type (HD3wt) FACS profile and three independent clones associated with
the same integration site (RMCE-HD3-13) [30, 31, 34].
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