Biomedical Engineering Reference
In-Depth Information
Homologous
recombination event
2 mg/ml Geneticin
selection
DT40 cells
Micronuclei
cell population
collect
cells
Transfected plasmid
by electroporation
Enucleation
Add colcemid and incubate
for 24
Collect the
sample from
the gradient
48 h
−
Equilibrate and
ultracentrifuge for
80 min at 20
Add
cytochalasin B
Mix
−
25
°
C
Add Percoll to cells
with media and
cytochalasin B in
oakridge tubes
Centrifuge
and wash the
pellet with
serum free
DMEM
Slowly drip1 ml of
50% PEG 1500
Micronuclei
K562 cells
Resuspend the
pellet in 50 ml of
K562 media and
incubate at 37
+
°
C
for 48 h.
Centrifuge
and wash
Centrifuge and
resuspend in geneticin
media
Colonies should be
visible in 3
−
4 weeks
Figure 7.2
A flow diagram describing experimental procedure for the generation of microcell
hybrids and modification of chromosomal DNA using homologous recombination in chicken pre-B
cell line, DT40. Homologous recombination events are selected and transferred into a mammalian
cell line (K562 cells). The cell donor may be obtained from either mammalian cells or directly from
chicken DT40 cells. For example, the first donor cells could be from human origin, transferred and
modified in DT40 cells by the process of homologous recombination, and eventually the modified
human chromosome is transferred a second time for analysis in an appropriate mouse cell line.
(See Plate 7.2.)
