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

12

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:

22

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

27

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