Gene Expression in Mammalian Cells - Genomics: Essential Methods - page 164

• Cell culture stirrer (Thermolyne, type 45600 cellgro stirrer)
• Bench-top centrifuge (Labofuge 400R, Heraeus)
• ALV-induced bursal lymphoma DT40 cell line from chicken (ATCC CRL-2111)
Method
1 Generate recombinant plasmids with homologous sequences of the genomic region
under study on each side of a selectable marker gene. j
2 Transfect the recombinant plasmid by electroporation into 1 × 10 7 DT40 cells to
generate stable clones and select them further with 2 μ g/ml of geneticin. k
3 After stable DT40 cells are obtained, check the recombination event and the integrity of
the recombinant vector by PCR and Southern blot.
4 For micronucleation of recombinant DT40 cells, seed 250ml of DMEM containing 10%
FBS with 50ml of DT40 cells (1-3 × 10 7 cells /ml) in a 500ml spinner bottle.
5 Incubate at 37 ◦ C for 24 h.
6 Add Colcemid l at 0.5 μ g/ml and incubate the cells at 37 ◦ C for 24 h to induce
micronucleation.
7 Harvest the micronucleate cells by collecting in 50ml sterile, polypropylene tubes.
Leave one aliquot (5 ml) to count with a hemocytometer and another one for
visualization of micronucleate cells. m
8 Centrifuge the micronuclei cells at 1000 g in a bench-top centrifuge and pool the pellets
in 50 ml of DMEM/10% FBS.
Notes
j The neomycin-resistant gene should be flanked on each side by loxP site-specific recombinant
motifs and homologous sequences should be of at least 1.5 kb in length and devoid of repetitive
sequences.
k 25 μ g of linearized plasmid is needed to obtain satisfactory transfection efficiencies in DT40
cells [37].
l Colcemid is used to induce micronucleation interfering with the microtubule formation. Thus,
chromosomes are scattered in the cell and nuclear envelope is formed around one or several
chromosomes forming micronuclei. The micronuclei containing the chromosome of interest are
then isolated in the presence of geneticin selection.
m For visualization, pellet the cells and fix them in at least three changes of 3 : 1 (v/v)
methanol : acetic acid. Place a drop of the suspension on a clean, dry microscope slide and
air dry. Overlay with Hoechst solution ( ∼ 0.5 μ g/ml) and stain for 1-3 min. Rinse with water
and mount a cover slip with mounting medium. Micronucleate cells can be visualized under
UV illumination (excitation 365 nm, emission 480 nm); they are easily distinguished from
mononucleate cells (Figure 7.2).