Transcript Class 21

DNA transfection
Last updated: Nov. 21, 2011 1:10 AM
Transfection agents:
CaPO4 (co-precipitates with DNA)
Electroporation (naked DNA, high voltage pulse transient holes)
Lipofection (multilamellar liposomes)
Polybrene (detergent)
DNA
Ballistic (DNA-coated gold particles)
DEAE-dextran (toxic, OK for transient)
polybrene
Poly-ethylenimine (PEI, cheap)
Effectene (non-liposomal lipid)
DNA
Must traverse cytoplasm. Much engulfed in lysosomes.
Inhibition of lysosomal function often helps (chloroquin).
Linear PEI
Co-integration of high MW DNA . Can reach 2000 KB.
Separate plasmids transfected together  same site (co-integration).
Separate transfections  separate locations
Random or semi-random (many) integration sites (unless targeted)
Low but real homologous recombination rate.
DEAE= diethyl-amino-ethyl (positively charged)
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Transient transfection
vs.
cloned genes
unintegrated DNA.
unnatural ?
super-physiological expression
levels (per transfected cell) ?
Permanent transfection:
chromosomally integrated
position effects ?
(so analyze a pool of many to
average)
Transient -> 10-90% transfection efficiency (stain)
Permanents more like 0.001 transfectants per μg DNA per cell (~high).
i.e., 106 treated cells -> 1000 colonies; could be much less for certain
types of cells
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One the most dramatic first applications of gene transfection from total DNA:
Transfer of the growth-transformed phenotype: ability to grow in multilayers or
in suspension in soft agar: (Weinberg; Wigler)
DNA from tumor transfected into growth-controlled mouse 3T3 cells.
Look for foci (one = focus).
Make a library from growth-transformed transfectant.
Screen for human Alu repeat.
Verify that cloned DNA yields a high frequency of focus-forming transfectants.
Isolate cDNA by hybridization to the cloned genomic DNA.
Sequence. Identify gene/protein: = a dominant oncogene.
Ras, a signaling protein in a transducing pathway for sensing growth factors
Growth in soft agar
Mouse 3T3 cells
Transformed Mouse 3T3 cells
transfected with an EGFreceptor gene
foci
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Viral transduction of genes
Lentivirus (a retrovirus)
env
gag
pol
RNA +strand genomes (2)
Also, adenovirus (DNA)
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DNA
Packaging signal
on RNA
Viral structural
proteins
contributed by
packaging cell line
(gag, env, pol, etc.)
Reverse transcribes
to cDNA, integrates
Infects cells at high efficiency
http://en.dogeno.us/2009/11/concentrate-retrovirus-carrying-vsv-g-envelope-by-ultracentrifugation/
ES cells and transgenic mice.
Selection for homologous recombinants via the loss of HSV TK genes (Capecchi):
----------– hsvtk – homol. region – drugR – homol. region – hsvtk –---------Non-homologous recombination favors ends: tk is inserted, conferring sensitivity to the
drug gancyclovir (HSVtk specific, not a substrate for human tk)
Resistant to
gancyclovir
HSV-TK gene is removed during
homologous recombination, but
remains joined during nonhomologous recombination.
Unlike mammalian TK, HSVTK
converts gancyclovir to a toxic
product
Die in
gancyclovir
M. Capecchi, Nature Medicine 7, 1086 - 1090 (2001)
Generating mice with targeted mutations
HSV = Herpes simplex virus
tk = thymidine kinase
FIAU = equivalent to gancyclovir
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Gene knockouts via homologous recombination
Most K.O. work in ES cells  mice  homozygosis via F1 breeding
Little work in cultured lines:
Myc double sequential K.O. = viable, ~sick (J. Sedivy, Genes & Dev. 1998. 12: 3797-3802 )
Splicing factor (ASF) double K.O. see next graphic.
ASF = alternative splicing factor
ES cells = embryonic stem cells
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Double knockout of the ASF gene, a vital gene, by homologous recombination
Chicken DT40 cells (high rate of
homologous recombination
+
Human
ASF
neo
ASF
ASF
ASF
ASF
Human
ASF
neo
One ASF gene
allele disrupted by
homologous
recombination
hol
neo
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ASF-
ASF
hol
Human
ASF
Select for HolR,
Screen by
Southern blotting
Tet-off promoter
Hol = histidinol resistance; pur = puromycin resistance
Drug resistance genes here chosen for illustration.
neo
pur
Human
X ASF
Wang, Takagaki, and Manley,
Targeted disruption of an
essential vertebrate gene:
ASF/SF2 is required
for cell viability. Genes Dev.
1996,10:2588-99.
Cell dies without ASF
(follow events biochemically)
pur
Both alleles have been
disrupted in some
purR, holR cells
neo
+tet
ASF shut off
pur
ASFASF-
Human
ASF
cell viable
(covered by human ASF gene
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Histidinol dehydrogenase detoxifies histidinol, confers histidinol resistance
protein synthesis
NAD+
Histidinol dehydrogenase
inhibits protein synthesis
(competitive inhibitor of histidinyltRNA synthetase)
Protein synthesis stops at a
histidine codon
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Gene amplification for high level production in CHO dhfr- cells.
DHFR system (dihydrofolate reductase):
Selection for resistance to marginal levels of methotrexate
Folate
DHFR
DHFR
tetrahydrofolate
“FH4”
dihydrofolate
“FH2”
MDR
Methotrexate (MTX, amethopterin)
Resistance to MTX can occur via 3 different mechanaisms:
1) Methotrexate permeation mutants (incl. MDR, increased efflux))
2) Altered DHFR with lower MTX binding affinity
3) Increased levels of P-glycoprotein efflux pump (MDR)
4) Overproduction of DHFR protein
MDR = multiple drug resistance
Glycine
Purine
nucleotides
(AMP and
GMP)
Thymidylic
acid
(TMP)
Gene amplification: dhfr
Historically:
• Methotrexate resistance
• MTX inhibits dihydrofolate reductase (DHFR)
• MTX-resistant cells have (in order of discovery):
• High DHFR enzyme activity
• High DHFR protein
• High protein synthetic rate
• High in vitro translatable mRNA
• High mRNA level (by hybridization)
• High DNA level.
Homogeneously staining, expanded chromosomal regions (HSRs)
HSRs are the location of the high number of dhfr genes.
Double minute chromosomes are an occasional alternative form.
Amplicons (distance between repeated genes) are large (300 KB).
(dhfr gene = ~ 25 kb)
HSRs can shrink, migrate.
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Reduction of folate to tetrahydrofolate
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Reduction of folate to tetrahydrofolate
MTX
DNA
RNA
FH4
IMP
serine
PurBios
DHFR
Precursors
CH
FH4
SHM
FH2
glycine
MTDH
Folate
TMP
TS
CH2=FH4
DHFR:
SHM:
TS:
PurBios:
MTDH:
DHFR
dUMP
Dihydrofolate reductase
Serine hydroxymethyltransferase
Thymidylate synthetase
Purine nucleotide biosynthetic pathway
Methylenetetrahydrofolate dehydrogenase
DNA
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Methotrexate:
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Gene amplification
HSR:
Homogenously
staining
region
Tritium grains from hybridized cDNA
Nunberg et al. PNAS 1978 75:5553-6.
(R.T. Schimke, Sci. Amer. 243:60-69, 1980)
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Gene amplification
“Homogeneously staining region”
FISH, here
FISH = fluorescent in situ hybridization
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+FISH
Original locus?
HSR  dmin upon DS break induced by a homing endonuclease (I-SceI).
HSR = homogeneously staining region
Dmin = double minute chromosomes
Restriction-type enzyme with a
very long recognition sequence (
~20 bp)
Arnaud Coquelle, Lorène Rozier, Bernard Dutrillaux and Michelle Debatisse
ONCOGENE, 31 October 2002, Volume 21, Number 50, Pages 7671-7679
Induction of multiple double-strand breaks within an hsr by meganuclease I-SceI expression
or fragile site activation leads to formation of double minutes and other chromosomal rearrangements
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Ampification models: over-replication, unequal sister chromatid
exchange, breakage and fusion (Tanaka et al. Mol. Cell. Biol. 2007 27:1993-2002
.).
Map dhfr amplicons: ~ 300 kb , but wide range (Nunberg et al., Proc Natl Acad
Sci U S A. 1978 Nov;75(11):5553-6; Looney et al., Mol Cell Biol. 1988. 8:5268-79)
Gene amplification is rare in normal cells p53- mutation allows it. (Livingstone
et al., Cell. 1992.70:923-35; Yin et al., Cell. 1992. 70:937-48).
In nature:
rDNA in oocytes; Drosophila chorion genes.
In medicine:
chemotherapy resistance (MDR, P-glycoprotein, efflux pump)
cancer (myc, ras)
In biotechnology:
high level recombinant protein production in mammalian cells
MDR = multiple drug resistance
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Gene amplification for high level recombinant protein production in mammalian cells.
Principal system = dhfr- CHO cells
Facilitated by the availability of DHFR-deficient mutant CHO cells
CHO dhfr- cells + vector with dhfr minigene + YFG
-GHT medium
Most cells die.
Transfectants live.
+ gradually increasing concentrations of MTX
Cells with gradually amplified dhfr transgenes survive.
YFG is co-amplified along with the dhfr minigene.
-GHT = without glycine, hypoxanthine (a purine source) and thymdine
DHFR-deficient cells require glycine, thymidine and a purine
and are resistant to tritiated deoxyuridine
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hypoxanthine
DNA
RNA
FH4
IMP
serine
PurBios
DHFR
Precursors
CH
FH4
SHM
DHFR
FH2
Folate
glycine
MTDH
TMP
TS
X
DNA 3H-DNA
X
CH2=FH4
dUMP
3H-dUMP
DHFR:
SHM:
TS:
PurBios:
MTDH:
TdR
Dihydrofolate reductase
Serine hydroxymethyltransferase
Thymidylate synthetase
Purine nucleotide biosynthetic pathway
Methylenetetrahydrofolate dehydrogenase
DHFR- cells selected by their resistance to radioactive 3H-deoxyuridine:
3H-dU  3H-dUMP  3H-TMP  3H-DNA  death from radioactive decay.
DHFR- cells require glycine, hypoxanthine and thymidine (GHT).
In GHT-free medium CHO dhfr- cells die,
but transfectants that have received a dhfr minigene, +/- YFG, survive.
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Some other amplifiable genes
ENZYME
ABBN
INHIBITOR
SELECTIVE MEDIUM
Adenosine deaminase
ADA
deoxycoformycin
MTX + adenosine
Ornithine decarboxylase
ODC
difluoromethyl-ornithine
Polyamine-free
Asparagine synthetase
AS
Ribonucleoside reductase
RR
hydroxyurea
Deoxynuceloside-free
Tri-functional pyrimidine
synthetic enzyme
CAD
PALA
Pyrimidine-free
Thymidylate synthetase
TS
FUdR
Thymidine-free
Dihydrofolate reductase
DHFR
MTX
Gly-, TdR-, purine-free
Glutamine synthetase
GS
Methionine sulfoximine
Gln-free
Asparagine-free
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A different major system for high level Mab production
NS0 cells:
Mouse myeloma cells, high IgG producers  IgG- variants = NS0
No endogenous IgG, but cell is a natural IgG secretor.
Lack glutamine synthetase (GS):
glutamate + NH3 + ATP  glutamine + ADP + Pi
Vector = MAb genes driven by strong promoters (H-chain, L-chain)
+ GS cDNA gene (Bebbington)
Select on glutamine-free medium
Inhibit GS with methionine sulfoximine (gln analog)
Select for GS overproducers
--->--> (gene amplification does not seem to be operating in this system of the
GS cDNA gene and linked Mab genes)
Proprietary (Lonza Biologics)
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Transfection strategies
1.
YFG (Your Favorite Gene) linked to a dhfr minigene on a single plasmid
A. ~Insures co-integration
B. ~Insures co-amplification
2.
YFG and dhfr on separate plasmids
A. Allows a high ratio of YFG to dhfr to start
B. Co-amplification not assured but commonly occurs.
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dhfr
DHFR-
YFG
CHO
cells
Select in
purine-free
medium
DHFR+
Increasing [MTX]
DHFR+++++
YFG +++++
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Co-amplification of genes on unlinked plasmids
DHFR
YFG
Transfection
Co-integration, usually
DHFR
YFG
Step-wise
Low MTX --> High MTX
Co-amplification
Wigler et al., PNAS, 1980 . 77: 3567-70 (Principle of co-amplification).
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Use a high ratio (e.g., 1000X) of YFG plasmid DNA to dhfr plasmid DNA
DHFR
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
Transfect with 10 ug of YFG and 10 ng of dhfr
Co-integration (with or without pre-ligation)
Multiple copies of YFG from the start
76(11): 5684–5688
Step-wise
Low MTX --> High MTX
Co-amplification
(Wigler et al, Cell, 1979:
DNA)
76:X174
5684–5688
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Transfection with both genes in one vector
and even in one transcription unit
Y.F.G.
DHFR
Y.F.G.
DHFR
Dicistronic mRNA
Y.F.G.
DHFR
Dicistronic mRNA
(ribosome
Poor translation initiation read-through)
Low DHFR
Supersensitive to MTX
Select initially in low MTX
More room for amplification
Kaufman, RJ, et al, NAR, 1993
Also, later, better dhfr translation
using an IRES,
Internal ribosome initiation site, used
mostly in viral but also in some cellular genes.
In theory, not an advantage.
Y.F.G.
IRES
DHFR
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Use a weak dhfr promoter to confer supersensitivity to MTX
DHFR
YFG
Transfection,
Co-integration, usually
DHFR
YFG
Stepwise
Very low MTX --> high MTX
Co-amplification
More room for amplification
Wigler et al., PNAS, 1980
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Use a high ratio (e.g., 1000X) of YFG plasmid DNA to dhfr plasmid DNA
and a poorly expressed dhfr minigene
DHFR
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
YFG
Transfect with 10 ug of YFG and 10 ng of dhfr
Co-integration
Multiple copies of YFG from the start
Stepwise
Very low MTX --> high MTX
Co-amplification
(Wigler et al, Cell, 1979:X174 DNA)
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Possible amplification protocol
Pool of transfectants
selected for growth
in purine-free medium
Check
selected
populations
for Ig
production
0
10
20
40
80
10
20
40
80
160 nM MTX
40
80
160
300
1000 nM MTX
etc.
Note: Process is lengthy and tedious.
nM MTX
in -GHT
medium
Finally clone several
for final stages
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Some marketed recombinant proteins
Erythropoietin (Epogen, Procrit) J&J, Amgen
Tissue plasminogen activator (TPA) Genentech
Growth Hormone (Genentech)
Insulin (Genentech)
Beta-interferon (Avonex) Biogen-IDEC
Alpha-interferon (IntronA) Schering-Plough
Neupogen (Amgen)
Etanercept – TNF receptor + IgG (Enbrel) Amgen
Monoclonal antibodies (mAbs): see next graphic
Top ten monoclonal antibodies in sales 2009-201
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