1Expression in mammalian cellsLab 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 hepatomaGH3 Rat pituitary cellsPC12 Mouse neuronal-like tumor cellsMCF7 Human breast cancerHT1080 Human with near diploid karyotypeIPS 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 Mouse plasma cell tumor cellsVero vaccines African greem monkey cellsCHO Mabs, other therapeutic proteins Chinese hamster ovary cellsPER6 Mabs, other therapeutic proteins Human retinal cells
2
Mammalian cell expression
Generalized gene structure for mammalian expression:
cDNA geneMam.prom.
polyA site
intron
5’UTR3’UTR
Intron is optional but a good idea
3
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)
4Engineered regulated expression:Tetracycline-reponsive promotersTet-OFF (add tet shut off)
tTA cDNA
tTA = tet activator fusion protein: tetRdomain
VP16 tc’nact’n domain (Herpes virus)
If no tet,binds tet operator(if tet not also bound)
tetRdomain
VP16 tc’nact’n domain
Tetracycline (tet), or,better, doxicyclin (dox)
active
CMV prom.
polyA sitetTA gene must be in cell (permanent transfection, integrated):
Tet-OFF
Tet-OFF
(Bujold et al.)
Tet bound, allosteric change in conformation,cannot bind operator, not active
5
tet operator sequence
tet prom.
Tetracycline resistance gene
your favorite gene
tetRprotein
activetranscripton, no blockage
Doxicyclin present:
inactiveno transcripton, RNA Pol blocked
No doxicyclin:
RNA pol
Tet operator-repressor, original bacterial source state
tet operator sequence
tet prom.
Tetracycline resistance gene
tetRprotein
RNA poltet prom.
RNA pol
6
MIN. CMV prom. your favorite gene
polyA site
Mutliple tet operator elements
MIN. CMV prom. your favorite gene
polyA site
tetRdomain
VP16 tc’nact’n domain
not activelittle transcripton (2%?, bkgd)
Doxicyclin present:
MIN. CMV prom. your favorite gene
polyA siteactivePlenty of transcripton
No doxicyclin:
tetRdomain
VP16 tc’nact’n domain
RNA po l
Tet-OFF, exploits modulatable binding of the tet protein bytet
7
Tetracycline-reponsive promotersTet-ON: add tet turn on gene
tTA cDNA
tetRdomain
VP16 tc’nact’n domain
tetRdomain
VP16 tc’nact’n domain
Tetracycline (tet), or,better, doxicyclin (dox):Now, can bind to operator seq.
active
not active
Full CMV prom.
polyA site
Different fusion protein: Does NOT bind tet operator(if tet not bound)
Tet-ON
tTA must be in cell (permanent transfection, integrated): commercially available (293, CHO) or do-it-yourself
8
MIN. CMV prom. your favorite gene
polyA site
Mutliple tet operator elements
MIN. CMV prom. your favorite gene
polyA site
active
Doxicyclin absent:
MIN. CMV prom. your favorite gene
polyA siteactivePlenty of transcripton (> 50X)
Add dox:
tetRdomain
VP16 tc’nact’n domain
RNA pol II
Tet-ON
tetRdomain
VP16 tc’nact’n domain
not active little transcription (bkgd.)
doxicyclin
9
SW Michnick web site: http://michnick.bcm.umontreal.ca/research/images/pca_general_en.gif
F = reporter protein fragment
Enzyme fragmentsthemselves do not associate well enough to reconstitute an active enzyme
Reporterenzyme
Back to protein-protein interactions:
10Folic acid
DHFR
DHFR
http://www.nature.com/onc/journal/v22/n47/images/1206946f1.gif
(FH4)
(FH2)
Dihydrofolate reductase (DHFR): role in metabolism
11
FK506 = immunosuppressant drugFKBP = FK506 binding proteinFRAP = FKBP–rapamycin binding proteinFRB= FKBP–rapamycin binding domain of FRAP
DHFR = dihydrofolate reductaseDHF=dihydrofolate = FH2
THF=tetrahydrofolate = FH4
fMTX=fluorescent methotrexate
fMTX
DHFR fragments
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
IN PURINE-FREE MEDIUM
Fluorescein – MTXbinding assay
Rapamycin promotes the association of the 2 protein domains
Cell growth assay: CHO
DHFR- mutantcells
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a phosphatase
FK506 recruits FKBP to bind to calcineurin and inhibit its action as a specific phosphatase
13
Claim detection of 0.05 nM rapamycin??
No.
of
CH
O c
olon
ies
[rapamycin]
14
Background association of FKBP and FRB without rapamycin
(compare mixed input)
Leucine zipper protein fragments instead ofrapamycin binding proteins (positive contro)
CHO cells(permanent transfection)
cos cells(transient transfection)
Fluorescent methotrexate (fMTX) assay:Wash in, wash out
15
8-fold increase in fluorescence per cell
Measure affinity for a drug in vivo
Fuorescence-activated flow cytometer(FACS is this, plus more)Allows quantitation of fluorescence per cell
No.
of
cells
Flu
ores
cenc
e in
tens
ity
Log of fluorescence intensity
[rapamycin]
16
EMP1 = Erythropoietin mimetic peptide 1
Erythropoietin-erythropoietin receptor (dimer) interaction: Efficacy of a peptide mimetic
Erythropoietin
In vivo assay of drug effectiveness (EMP1)(inexpensive substitute for erythropoietin?)
Erytropoietin (EPO) receptor
EPO bp1EPO bp2EPO
17
FACS = Fluorescence-activated cell sorter
Impart a charge on the recognized cell
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.
Can be used purely anaytically without the sorting capability. Thencalled “flow cytometry”, or also called FACS anyway.
18
No.
of
cells
Having this much fluorescence
Histogram-type display
No fluorescence (background autofluorescence)
Red stained
Usually a log scale
19
One cell
Amount of red fluorescence (log)
Am
ount
of
gre
en f
luor
esce
nce
(log)
Say, want high reds butlow greens:Instruct the FACS to deflect cells in this quadrant only. Collect and grow or analyze further.
Analysis on 2 colors
Scatter plot display
You decide on the positions of of demarcations
20
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.
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.
Green signalR
ed s
ign
al
Both signals
Neither signal
Analysis of beads representing the human genome using allele-specific hybridization probes and the FACS
Beaming bead FACS analysis
21
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
TF… transcription factorTBP: TATA binding proteinTAF: TBP associated proteinBRE: TFIIB response element
INR: transcription initiator elementDPE: downstream promoter element
The transcription complex either recruits RNA Pol II or activates a bound RNA Pol II
Core transcriptional elements
-28-35
For review see Smale and Katonga, Ann. Rev. Biochem. 72: 449-479 (2003)
GGGCGCC; CCACGCC
TATA(AT)AA(GA) YYAN(TA)YY
Y = C or T (pyrimidine)
(AG)G(AT)(CT)(GAC)
23
Many transcriptional enhancer elements often lie upstream of promoters,allowing for many combinations of TF binding
24
Got this far
25
Put a DNA regulatory region upstream of a reporter gene to analyze its elements
PCR
Space for res. enz. to bind
Reportergene
Transfect
26
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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HSVTKGancilovir, ATP Gancilovir-PO4
Mammalian TKGangcylovir, ATP
toxicity, death
Use example: Site-directed recombination
Engineered chromosome: WT protein of interest HSVTK
lox
lox
Replacement plasmid:
Mut. protein of interest
gangcylovir
Mut. protein of interest
Select recombinants as HSVTK-, gancilovir-resistant
Gangcyclovir selection AGAINST the presence of enzyme activity
(compare to 5-fluoro-orotic acid (FOA) resistance in yeast, URA3-)
CRE recombinase(cassette excnahge)
(Ganciclovir itself is not toxic)
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diacetylated
monoacetylated
Testing for a cell-specific promoter: chloramphenicol acetyl transferase (CAT) reporter assay
Thin layer chromatography (TLC)
CAT cDNA is from a prokaryotic source. CAT is not foundin mammalian cells.Therefore low backgrounds
A B
14C-chloramphenicol
unacetylatedPositive controlNegative control
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Reporter enzyme substrates for different purposes
• 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.
Substrates for beta-galactosidase, for example:
30
Mapping transcriptional elements upstream of a promoter:
Mapping with restrictionenzyme mediated deletions
Conclusion:
Light units of luciferase in hepatocytes
31Footprinting: detects sites on DNA to which protein are bound
Naked DNA DNA + DNA-binding proteinP
opul
atio
n of
mol
ecul
es
missing
Pop
ulat
ion
of m
olec
ules
Partial DNase
Gel electrophoresis.autoradiography
Footprint
32
Note uneven cleavage of naked DNA by DNase
33
(EMSA = electrophoretic mobility shift assay)
(shift)
(supershift)
1 2 3 4 5
DNA element
U. Arizona
Protein-DNA binding: EMSA or gel shift
(Even though the hexagon looks like a protein here)
competitor
34
(surpershifted complex is not competed by NON-specific probe)
(competed only by specific probe)
(two molecules of protein bound)
Protein DNA complexes migrate more slowly than naked DNA
Gel shifts (EMSA
Super-shift
35
SELEX
Binding to Protein,e.g.
sequences consensus
by PCR
Synthetic, range usually 6 to 40-mers (huge number)
Separate using nitrocellulose binding, gel electrophoresis, etc.
(re-iterate 3-10 times)
(usually a protein)
(T7 RNA Pol from an embedded T7Pol promoter
;
for protein binding sites
Systematic Evolution of Ligands by Exponential Enrichment
36
Binding to protein of interest
RThttp://www.molmed.uni-luebeck.de/T.%20Restle/Bilder/SELEX.jpg
Practical capacity ($700):
1014 random sequences
(random ~21-mer = 421)
37
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.
Consensus:
Binding site for a “puf “ protein, implicated in mRNA degradation
Code
Integer
Base Name Meaning
Complement
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
- 16 Gap of indeterminate length Gap -
Description
Nucleic acid degenerate base abbreviations20-mer