Download Tet-OFF

Expression in mammalian cells
Lab examples of cell lines:
HEK293 Human embyonic kidney (high transfection efficiency)
HeLa
Human cervical carcinoma (historical, low RNase)
CHO
Chinese hamster ovary (hardy, diploid DNA content, mutants)
Cos
Monkey cells with SV40 replication proteins (-> high transgene copies)
3T3
Mouse or human exhibiting ~regulated (normal-like) growth
+ various others, many differentiated to different degrees, e.g.:
BHK
Baby hamster kidey
HepG2 Human hepatoma
GH3
Rat pituitary cells
PC12
Mouse neuronal-like tumor cells
MCF7 Human breast cancer
HT1080 Human with near diploid karyotype
IPS
induced pluripotent stem cells
and:
Primary cells cultured with a limited lifetime (frozen stocks available)
E.g.,
MEF = mouse embryonic fibroblasts, HDF = Human diploid fibroblasts
Common in industry:
NS1
Mabs
Vero
vaccines
CHO
Mabs, other therapeutic proteins
PER6 Mabs, other therapeutic proteins
Mouse plasma cell tumor cells
African greem monkey cells
Chinese hamster ovary cells
Human retinal cells
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Mammalian cell expression
Generalized gene structure for mammalian expression:
polyA site
Mam.prom.
intron
5’UTR
Intron is
optional but
a good idea
cDNA gene
3’UTR
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Popular mammalian cell promoters
•
•
•
•
•
•
•
SV40 LargeT Ag: Simian Virus 40
RSV LTR: Rous sarcoma virus
MMTV: Mouse mammary tumor virus, glucocorticoid [Dex] inducible
HSV TK: Herpes simplex virus, low expression
Metallothionein: many sources, metal inducible, Cd++
CMV early: Cytomegalovirus, strong in most cell types
Engineered inducible / repressible:
tet-, ecdysone-, glucocorticoid- responsive (tet = tetracycline)
Tet-OFF
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Engineered regulated expression:
Tetracycline-reponsive promoters
Tet-OFF (add tet  shut off)
tTA = tet activator fusion protein: tetR
domain
VP16 tc’n
act’n domain (Herpes virus)
active
If no tet,
binds tet operator
(if tet not also bound)
Tet-OFF tetR
domain
VP16 tc’n
act’n domain
Tetracycline (tet), or,
better, doxicyclin (dox)
Tet bound,
allosteric
change in
conformation,
cannot bind
operator,
not active
tTA gene must be in cell (permanent transfection, integrated):
polyA site
CMV
prom.
tTA cDNA
(Bujold et al.)
Tet operator-repressor, original bacterial source state
tet
prom.
Tetracycline resistance gene
tet operator sequence
No doxicyclin:
tetR
protein
tet
prom.
inactive
no transcripton, RNA Pol blocked
Tetracycline resistance gene
tet operator sequence
tetR
protein
Doxicyclin present:
active
transcripton, no blockage
tet
prom.
your favorite gene
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Tet-OFF, exploits modulatable binding of the tet protein bytet
MIN. CMV prom.
polyA site
your favorite gene
Mutliple tet operator elements
No doxicyclin:
VP16 tc’n
tetR
domain act’n domain
active
Plenty of transcripton
MIN. CMV prom.
polyA site
your favorite gene
tetR
VP16 tc’n
domain act’n domain
Doxicyclin present:
MIN. CMV prom.
not active
little transcripton (2%?, bkgd)
polyA site
your favorite gene
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Tet-ON
Tetracycline-reponsive promoters
Tet-ON: add tet  turn on gene
Different fusion protein:
Does NOT bind tet operator
(if tet not bound)
tetR
VP16 tc’n
domain act’n domain
not active
tetR
VP16 tc’n
domain act’n domain
active
Tetracycline (tet), or,
better, doxicyclin (dox):
Now, can bind to operator seq.
polyA site
Full CMV prom.
tTA cDNA
tTA must be in cell (permanent transfection, integrated):
commercially available (293, CHO) or do-it-yourself
Tet-ON
polyA site
MIN. CMV prom.
your favorite gene
Mutliple tet operator elements
tetR
VP16 tc’n
domain act’n domain
Doxicyclin absent:
not active
little transcription (bkgd.)
polyA site
MIN. CMV prom.
your favorite gene
Add dox:
VP16 tc’n
doxicyclin tetR
domain act’n domain
active
active
Plenty of transcripton (> 50X)
MIN. CMV prom.
your favorite gene
polyA site
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Back to protein-protein interactions:
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Reporter
enzyme
F = reporter protein fragment
SW Michnick web site: http://michnick.bcm.umontreal.ca/research/images/pca_general_en.gif
Enzyme fragments
themselves do not
associate well enough
to reconstitute an
active enzyme
Dihydrofolate reductase (DHFR):
role in metabolism
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Folic acid
DHFR
(FH2)
DHFR
(FH4)
http://www.nature.com/onc/journal/v22/n47/images/1206946f1.gif
Clonal selection and in vivo quantitation of protein interactions with protein-fragment
complementation assays, I. Remy and S.W. Michnick PNAS 96, 394–5399, 1999
DHFR fragments
Rapamycin
promotes
the
association
of the 2
protein
domains
fMTX
Cell
growth
assay: CHO
DHFR- mutant
cells
Fluorescein – MTX
binding assay
IN PURINE-FREE MEDIUM
DHFR = dihydrofolate reductase
DHF=dihydrofolate = FH2
THF=tetrahydrofolate = FH4
fMTX=fluorescent methotrexate
FK506 = immunosuppressant drug
FKBP = FK506 binding protein
FRAP = FKBP–rapamycin binding protein
FRB= FKBP–rapamycin binding domain of FRAP
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FK506 recruits FKBP to bind to calcineurin and
inhibit its action as a specific phosphatase
a phosphatase
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No. of CHO colonies
Claim detection of
0.05 nM rapamycin
??
[rapamycin]
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Fluorescent
methotrexate
(fMTX) assay:
Wash in, wash out
CHO cells
(permanent transfection)
cos cells
(transient transfection)
Background association of
FKBP and FRB without rapamycin
(compare mixed input)
Leucine zipper protein
fragments instead of
rapamycin binding
proteins (positive contro)
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No. of cells
Fuorescence-activated flow cytometer
(FACS is this, plus more)
Allows quantitation of fluorescence
per cell
8-fold increase in
fluorescence per cell
Fluorescence intensity
Log of fluorescence intensity
Measure affinity for a drug in vivo
[rapamycin]
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Erythropoietin-erythropoietin receptor (dimer) interaction: Efficacy of a peptide mimetic
EPO
EPO bp2
EPO bp1
Erytropoietin (EPO) receptor
In vivo assay of drug effectiveness (EMP1)
(inexpensive substitute for erythropoietin?)
EMP1 = Erythropoietin mimetic peptide 1
Erythropoietin
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FACS =
Fluorescence-activated
cell sorter
Impart a charge
on the recognized cell
Can be used purely
anaytically
without the sorting
capability. Then
called “flow cytometry”,
or also called FACS
anyway.
Less than one cell or particle
per droplet. Thus the most
that most droplets contain is
one particle.
Charged plates attract droplets
containing a particle of the
opposite charge
Cells remain viable if
treated with care.
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Histogram-type display
No. of cells
No fluorescence (background autofluorescence)
Red stained
Usually a log scale
Having this much
fluorescence
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Scatter plot display
Amount of green fluorescence (log)
Analysis on 2 colors
One cell
You decide on
the positions of
of demarcations
Amount of red fluorescence (log)
Say, want high
reds but
low greens:
Instruct the
FACS to deflect
cells in this
quadrant only.
Collect and
grow or analyze
further.
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Beaming bead FACS analysis
Analysis of beads representing the
human genome using allele-specific
hybridization probes and the FACS
Both
signals
Red signal
A. Flow cytometry data: 2-D plots
where each point represents one
particle. Then contour lines plotted
around the point density. Here light
“forward” scattering (irrespective
of wavelength) is measured (FSC).
Instrument can be set to reject
data from 2-bead doublets that
scatter light more.
Green signal
Neither
signal
B-D. Amplified beads hybridized to
2 probes, one specific to the S allele of a certain
gene and one specific to the L allele. The beads
carry the amplified PCR products corresponding to
this region from 3 human individuals.
The blue points come from microspheres that
contained both types of PCR products from both
alleles, despite the high dilution.
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Biotechnology methods to study transcriptional regulation in cells
Mainly, use of reporter proteins whose cDNA sequence is linked to the promoter.
First, a synopsis of promoter structure:
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General model for transcriptional regulation in higher eukaryotes
Core transcriptional elements
TF… transcription factor
TBP: TATA binding protein
TAF: TBP associated protein
BRE: TFIIB response element
-35
INR: transcription initiator element
DPE: downstream promoter element
-28
GGGCGCC;
TATA(AT)AA(GA)
CCACGCC
YYAN(TA)YY
(AG)G(AT)(CT)(GAC)
Y = C or T (pyrimidine)
The transcription complex either recruits RNA Pol II or activates a bound RNA Pol II
For review see Smale and Katonga, Ann. Rev. Biochem. 72: 449-479 (2003)
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Many transcriptional enhancer elements often lie upstream of promoters,
allowing for many combinations of TF binding
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Got this far
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Put a DNA regulatory region upstream of a reporter gene to analyze its elements
Space for res. enz. to bind
PCR
Reporter
gene
Transfect
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Popular reporters to study promoter/enhancers
•
Beta-galactosidase (β-gal) – detection by several different assays
•
Chloramphenicol acetyl transferase (CAT) – detection, sensitive radioactive assay
•
Luciferase (firefly, Renilla [jellyfish]) – detection, easy dual, sensitive luminescent
assay
•
Green fluorescent protein (GFP, BFP, YFP)) – cytological, visible in living cells,
fusion proteins, FACS
•
•
Neomycin phosphotransferase (neo)–selectable drug resistance (G418R)
(similarly: resistance to hygromycin, puromycin, histidinol
•
Dihydrofolate reductase (DHFR) – selectable in dhfr- cells, amplifiable, fusion
proteins work
•
Suicide selection: Herpes simplex virus thymidine kinase (HSVTK)
FACS = fluorescence-activated cell sorter
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Gangcyclovir selection AGAINST the presence of enzyme activity
(compare to 5-fluoro-orotic acid (FOA) resistance in yeast, URA3-)
HSVTK
Gancilovir, ATP
Gangcylovir, ATP
Gancilovir-PO4
Mammalian TK
toxicity, death
(Ganciclovir itself is not toxic)
lox
lox
Use example: Site-directed recombination
Engineered chromosome:
Replacement plasmid:
WT protein of interest
HSVTK
CRE recombinase
(cassette excnahge)
Mut. protein of interest
gangcylovir
Mut. protein of interest
Select recombinants as HSVTK-, gancilovir-resistant
Testing for a cell-specific promoter: chloramphenicol acetyl transferase (CAT)
reporter assay
CAT cDNA is from a
prokaryotic source.
CAT is not found
in mammalian cells.
Therefore low backgrounds
A
Thin layer
chromatography (TLC)
B
diacetylated
14C-chloramphenicol
monoacetylated
Positive control
Negative control
unacetylated
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Reporter enzyme substrates for different purposes
Substrates for beta-galactosidase, for example:
•
ONPG (ortho-nitrophenyl-beta-galactoside) – spectrophotometric
measurement (420 nm – blue color – simplest)
•
X-gal (5-Bromo-4-chloro-3-indolyl-ß-D-galactoside) – blue precipitate - for
cytology or colony detection
•
Umbelliferyl–galactoside (-> umbelliferone, fluorescent, reading in a
fluorimeter allows more sensitive quantification than spectrophotometry)
•
Galacton-STAR or some such (-> chemiluminescent product = emission of
light, so lower background than fluorescence)
•
Lactose (glucose-beta-galactose disaccharide) – allows growth if
hydrolyzed; growth phenotype. For microbial cells usually.
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Mapping transcriptional
elements upstream of a
promoter:
Mapping with restriction
enzyme mediated deletions
Conclusion:
Light units of
luciferase in
hepatocytes
Footprinting: detects sites on DNA to which protein are bound
DNA + DNA-binding protein
Population of
molecules
Naked DNA
Population of
molecules
Partial DNase
missing
Gel
electrophoresis.
autoradiography
Footprint
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Note uneven cleavage
of naked DNA by DNase
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Protein-DNA binding: EMSA or gel shift
(EMSA = electrophoretic mobility shift assay)
1
2
3
4
5
competitor
(supershift)
(shift)
DNA element
(Even though the hexagon looks like a protein here)
U. Arizona
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Protein DNA complexes
migrate more slowly than
naked DNA
Gel shifts (EMSA
(competed only by specific probe)
(two molecules
of protein bound)
Supershift
(surpershifted
complex is not
competed by NONspecific probe)
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SELEX
for protein binding sites
(T7 RNA Pol from an embedded
T7Pol promoter
Systematic Evolution of
Ligands by Exponential
Enrichment
(huge number)
Synthetic, range usually 6 to 40-mers
(usually a protein)
;
by PCR
(re-iterate 3-10 times)
Binding to Protein,
e.g.
Separate using nitrocellulose binding,
gel electrophoresis, etc.
sequences
 consensus
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Practical capacity ($700):
1014 random sequences
(random ~21-mer = 421)
Binding to
protein of
interest
http://www.molmed.uniluebeck.de/T.%20Restle/
Bilder/SELEX.jpg
RT
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Binding site for a “puf “
protein, implicated in mRNA
degradation
PUM2, a novel murine puf protein, and its consensus RNA-binding site
.
White EK, Moore-Jarrett T, Ruley HE. RNA. 2001 Dec;7(12):1855-66
20-mer
Consensus:
Description
Nucleic acid degenerate base abbreviations
Cod
e
Intege
r
Base Name
Meanin
g
Complemen
t
A
1
Adenine
A
T
C
2
Cytosine
C
G
G
3
Guanine
G
C
T
4
Thymine
T
A
U
4
Uracil
U
A
R
5
(PuRine)
G|A
Y
Y
6
(PYrimidine)
T|C
R
K
7
(Keto)
G|T
M
M
8
(AMino)
A|C
K
S
9
Strong interaction (3 H bonds)
G|C
S
W
10
Weak interaction (2 H bonds)
A|T
W
B
11
Not-A (B follows A)
G|T|C
V
D
12
Not-C (D follows C)
G|A|T
H
H
13
Not-G (H follows G)
A|T|C
D
V
14
Not-T (or U) (V follows U)
G|A|C
B
N,X
15
ANy nucleotide
G|A|T|C
N
-
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Gap of indeterminate length
Gap
-