Biomedical Engineering Reference
In-Depth Information
The critical point when using DT40 cell line is that chromosomes from other species can
be transferred into these cells, where targeting efficiencies of the transferred chromosomes
are often of significantly higher magnitude than in other vertebrate cells. Thus, genetic
manipulation can be done in DT40 cells in the presence of appropriate selectable markers,
and then the modified chromosome can again be transferred into another cell line with
an appropriate or convenient genetic background where gene expression studies can be
performed (Figure 7.2).
Thus, a typical experiment takes into account two steps that involve microcell fusion of
particular human chromosomes containing the genetic domain of interest and a previously
integrated selectable marker gene. However, it is important to emphasize that, for each
microcell fusion event, it is extremely important to verify the integrity of the transferred
chromosomes, since during these procedures the loss of entire chromosomal regions had
been observed with some frequency [38, 40]. To this end, systematic PCR analysis on entire
chromosome with previously defined genetic markers, complemented with fluorescent
in situ
hybridization (FISH), is necessary to confirm chromosome integrity. Microcell fusion proce-
dures coupled with homologous recombination techniques have been successfully applied to
chromatin studies, particularly to the human and mouse
β
-globin loci [38, 40]. In summary,
microcell fusion coupled with the DT40 cells' capacity to perform homologous recombi-
nation represents a clear alternative to knock-out mice and, more broadly, the spectrum of
cell types that can be analyzed.
Microcell fusion protocol takes advantage of the ability of donor cells (DT-40 chicken
lymphoid cell line) to become micronucleated. Protocols 7.3, 7.4 and 7.5 are general proto-
cols for microcell transfer into K562 cells (human erythroleukemic cell line). The frequency
for this fusion is 30-40%. As DT40
human microcell fusions are relatively inefficient, a
large number of donor microcells is required for successful transfer [36, 37].
×
PROTOCOL 7.3
Microcell Fusion: Micronucleation of Donor Cells
Equipment and reagents
•
DMEM (Gibco)
•
FBS (Wisent)
•
Chicken serum (Gibco)
•
10% tryptone phosphate buffer (Sigma)
•
Penicillin/streptomycin (Invitrogen)
•
Geneticin (Invitrogen)
•
Colcemid, demecolcine (Sigma), powder is stored desiccated and protected from light at
−
20
◦
C. Stock solution of 1mg/ml in saline is stable for at least 6 months at
−
20
◦
Cif
protected from light.
•
Hoechst dye33258, bisbenzimide (cat. B-2883, Sigma) powder is stored at room
temperature. Saline stock solution at 50 mg/ml is stable indefinitely at room
temperature.
500ml spinner bottle
•
Cell electroporator (Gene Pulser II, BioRad)
•
