1 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) CosMonkey cells with SV40 replication proteins (-> high transgene copies) 3T3Mouse or human exhibiting ~regulated (normal-like) growth + various others, many differentiated to different degrees, e.g.: BHKBaby hamster kidey HepG2Human hepatoma GH3Rat pituitary cells PC12Mouse neuronal-like tumor cells MCF7Human breast cancer HT1080 Human with near diploid karyotype IPSinduced 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: NS1MabsMouse plasma cell tumor cells Vero vaccines African greem monkey cells CHOMabs, other therapeutic proteinsChinese hamster ovary cells PER6Mabs, other therapeutic proteinsHuman retinal cells

2 Mammalian cell expression Generalized gene structure for mammalian expression: cDNA gene Mam.prom. polyA site intron 5’UTR 3’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)

4 Engineered regulated expression: Tetracycline-reponsive promoters Tet-OFF (add tet  shut off) tTA cDNA tTA = tet activator fusion protein: tetR domain VP16 tc’n act’n domain (Herpes virus) If no tet, binds tet operator (if tet not also bound) tetR domain VP16 tc’n act’n domain Tetracycline (tet), or, better, doxicyclin (dox) active CMV prom. polyA site tTA gene must be in cell (permanent transfection, integrated): 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 tetR protein active transcripton, no blockage Doxicyclin present: inactive no transcripton, RNA Pol blocked No doxicyclin: RNA pol Tet operator-repressor, original bacterial source state tet operator sequence tet prom. Tetracycline resistance gene tetR protein RNA pol tet prom. RNA pol

6 MIN. CMV prom. your favorite gene polyA site Mutliple tet operator elements MIN. CMV prom. your favorite gene polyA site tetR domain VP16 tc’n act’n domain not active little transcripton (2%?, bkgd) Doxicyclin present: MIN. CMV prom. your favorite gene polyA site active Plenty of transcripton No doxicyclin: tetR domain VP16 tc’n act’n domain RNA po l Tet-OFF, exploits modulatable binding of the tet protein bytet

7 Tetracycline-reponsive promoters Tet-ON: add tet  turn on gene tTA cDNA tetR domain VP16 tc’n act’n domain tetR domain VP16 tc’n act’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 site active Plenty of transcripton (> 50X) Add dox: tetR domain VP16 tc’n act’n domain RNA pol II Tet-ON tetR domain VP16 tc’n act’n domain not active little transcription (bkgd.) doxicyclin

9 SW Michnick web site: F = reporter protein fragment Enzyme fragments themselves do not associate well enough to reconstitute an active enzyme Reporter enzyme Back to protein-protein interactions:

10 Folic acid DHFR (FH 4 ) (FH 2 ) Dihydrofolate reductase (DHFR): role in metabolism

11 FK506 = immunosuppressant drug FKBP = FK506 binding protein FRAP = FKBP–rapamycin binding protein FRB= FKBP–rapamycin binding domain of FRAP DHFR = dihydrofolate reductase DHF=dihydrofolate = FH 2 THF=tetrahydrofolate = FH 4 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 – MTX binding assay Rapamycin promotes the association of the 2 protein domains Cell growth assay: CHO DHFR - mutant cells

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 CHO colonies [rapamycin]

14 Background association of FKBP and FRB without rapamycin (compare mixed input) Leucine zipper protein fragments instead of rapamycin 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 Competition with a molecule that binds only one 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 Fluorescence intensity 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 bp2 EPO

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. Then called “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) Amount of green 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. 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 signal Red signal 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 factor TBP: TATA binding protein TAF: TBP associated protein BRE: TFIIB response element INR: transcription initiator element DPE: downstream promoter element The transcription complex either recruits RNA Pol II or activates a bound RNA Pol II Core transcriptional elements For review see Smale and Katonga, Ann. Rev. Biochem. 72: (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 Reporter gene 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 HSVTK Gancilovir, ATPGancilovir-PO 4 Mammalian TK Gangcylovir, ATP toxicity, death Use example: Site-directed recombination Engineered chromosome: WT protein of interest HSVTK 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)

28 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 found in mammalian cells. Therefore low backgrounds A B 14 C-chloramphenicol unacetylated Positive control Negative control

29 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 restriction enzyme mediated deletions Conclusion: Light units of luciferase in hepatocytes

31 Footprinting: detects sites on DNA to which protein are bound Naked DNA DNA + DNA-binding protein Population of molecules missing Population of molecules Partial DNase Gel electrophoresis. autoradiography Footprint

32 Note uneven cleavage of naked DNA by DNase

33 (EMSA = electrophoretic mobility shift assay) (shift) (supershift) 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 RT luebeck.de/T.%20Restle/ Bilder/SELEX.jpg Practical capacity ($700): random sequences (random ~21-mer = 4 21 )

37 PUM2, a novel murine puf protein, and its consensus RNA-binding site White EK, Moore-Jarrett T, Ruley HE. RNA Dec;7(12): White EKMoore-Jarrett TRuley HE Consensus: Binding site for a “puf “ protein, implicated in mRNA degradation Cod e Intege r Base NameMeanin g Complemen t A1 Adenine AT C2 Cytosine CG G3 Guanine GC T4 Thymine TA U4 Uracil UA R5 (PuRine) G|AG|AY Y6 (PYrimidine) T|CT|CR K7 (Keto) G|TG|TM M8 (AMino) A|CA|CK S9 Strong interaction (3 H bonds) G|CG|CS W10 Weak interaction (2 H bonds) A|TA|TW B11 Not-A (B follows A) G|T|CG|T|CV D12 Not-C (D follows C) G|A|TG|A|TH H13 Not-G (H follows G) A|T|CA|T|CD V14 Not-T (or U) (V follows U) G|A|CG|A|CB N,X15 ANy nucleotide G|A|T|CG|A|T|CN -16 Gap of indeterminate length Gap- Description Nucleic acid degenerate base abbreviations 20-mer