DT40: Basic culture and common problems

DT40
Basic culture and common problems

DT40
• ALV-induced bursal lymphoma from female (ZW) F1
White Leghorn chicken 1
• similar phenotype to human Burkitt’s lymphomas:
p53 null and degregulated MYC expression
• triplication of chromosome 2
taken from Winding & Berchtold (2001)
J. Immunol. Meth. 249, 1-16
ER, endoplasmatic reticulum;
M, mitochondria;
N, nucleus;
V, viral particles.
• c. 10µm in diameter in G1, 12µm in G2
• famously high frequency of targeted to
random integration 2
1. Baba T.W. et al. (1985) Cell lines derived from avian lymphocytes exhibit two distinct phenotypes Virology 144, 139-151.
2. Buerstedde J.-M. and Takeda, S. (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines Cell
67, 179-188.

DT40 culture conditions
• base medium
• serum
• antibiotics
• 2-mercaptoethanol?
• CO2
• temperature

DT40 growth characteristics
doubling time 11 hours at 37˚C

DT40 growth characteristics
doubling time 11 hours at 37˚C

DT40 growth characteristics
doubling time 11 hours at 37˚C
Edmunds et al. Molecular Cell, 2008, 30, 519-29

DT40 growth characteristics
• ideally aim for density between 2 x 10 5
and 1x10 6 /ml...this really means splitting
every day but with care you can reduce
this to every 48 hours
97% viable
17% viable

• infection
DT40 common culture
problems
• DT40 is happy with most common antibiotics e.g. penicillin, streptomycin.
• Gentamicin and ciprofloxacin can be useful in extremis.
• Fluconazole for fungal infection
• For us, mycoplasma infection is very uncommon; can be treated with
ciprofloxacin or BM-cyclin (Roche) although latter is quite toxic

• clumping
DT40 common culture
problems
• exclude low-grade infection, especially with fungi
• sometimes happens in conditions of low or poor serum
• ?sometimes antibody mediated
• sticking to plastic
• can certainly be antigen-receptor mediated
(Cumbers et al. Nat. Biotechnol 2002, 20, 1129-34.)
• usually pretty low affinity - can be largely knocked off
• however, difficult to ‘cure’ - if a problem, probably have to find another clone

Culture of DT40 on
methylcellulose
• useful for immobilising DT40 for e.g. colony
survival assays
• autoclave 10g of methylcellulose in
a 1L bottle with stir bar
• fully disperse methylcellulose in
warm water
• add 2x medium bit-by-bit with
energetic shaking
• top up to IL and stir in cold room
overnight
• the medium should be crystal clear
- precipitates of methylcellulose are
bad news...

Cloning DT40
• cloning by limiting dilution
• cloning by cell sorter
• ensure cells are in tip-top
condition; generally no need for
conditioned medium
• in both cases this is very
straightforward but is improved by
using round-bottomed 96-well
plates
• consider increasing serum to 15%
total if cloning efficiency is low

• Electroporation
Getting DNA into DT40
• High energies (e.g. 250V, 950µF in a 4mm cuvette) favour random integration
through formation of DNA breaks
• For gene targeting a lower energy, higher voltage approach is better (e.g.
550V, 25µF in a 4mm cuvette)
• generally 20 million cells per transfection works well
• 10 - 30µg DNA in water (volume

Getting DNA into DT40
• Amaxa nucleofection
• works very well for transient transfection - standard lipid-based transfection
(e.g. Lipofectamine and FuGene) have been hopeless in our hands;
electroporation is inefficient
• needs special electroporator, solutions and cuvettes - expensive
• somewhat limited in number of cells that can be transfected
• Solution T and program B23 work well
• cells hate being in nucleofector solution, so fiddly to do large numbers if you
want it to work (although there are plate-based options around)

• Transduction
Getting DNA into DT40
• allows introduction of transgene without the
cellular stress associated with electroporation
• highly efficient using VSV-G pseudotyped MMTVbased
amphotropic virus
• requires Class II containment facilities (normally
DT40 is Class I)
• first step is virus production in 293 cells. The virus is
produced by co-transfection of three plasmids: one
encodes gag & pol, one the VSV-G pseudotyped env and
the third your gene of interest linked to a viral
packaging signal.
• virus is harvested in supernatant and can be frozen
• the viral supernatant can then be used to infect DT40
cells
Randow & Sale, DT40 handbook

Stable selection
• Commonly used selection markers
Drug
G418
Puromycin
Resistance
gene
Final
concentration
Notes
neomycin
phosphotransferase 2 mg/ml slow; cells may need feeding
puromycin
acetyltransferase
0.5 µg/ml
generally clean; cells can take
several days to die
Blasticidin S blasticidin S deaminase 20 µg/ml rapid with clean selection
Histidinol histidinol dehydrogenase 20 µg/ml
Hygromycin B
hygromycin
phosphotransferase
1.4 mg/ml
Mycophenolic acid E. coli XGPRT 15 µg/ml
Zeocin
extremely rapid with clean
selection
batch to batch variation in amount
of active compound
need to add xanthine and
hypoxanthine; quick and clean
Streptoalloteichus.
hindustanus Ble 300 µg/ml works by causing DNA breaks

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Marker recycling with Cre
recombinase
Amaxa transfection of Cre recombinase expression
plasmid
next day, plate out at 1, 3, 5 viable cells per well
grow up and select clones from plates with fewest
positive wells i.e. from 1c.p.w. if it has worked
pick clones to 24-well plate containing normal medium
once established in 24-well plate, replica plate to selective
media, keeping cells thin to ensure clear selection...and
remember to keep the plate with no antibiotic!

• Freezing mix
• Freezing tubes
Cryopreservation
• 90% serum / 10% DMSO
• slow down, quick up
• Freezing plates
• Storage
• Recovery after freezing

Questions?