cDNA Expression Cloning in Mammalian Cells

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit contains protocols for expression cloning in mammalian cells. Either calcium phosphate? or liposome?mediated transfection of mammalian cells, or virus infection and liposome?mediated transfection are used to screen pools derived from a cDNA library. cDNA pools are prepared for cloning from library?transformed E. coli grown in liquid culture medium or on antibiotic?containing selection plates. Results of screening assays for expression can be detected using autoradiography of dishes of cultured cells to identify clones, direct visualization of radiolabeled cells on emulsion?coated and developed chamber slides, detection and quantification of gene activity by a functional (transport) assay with scintillation counting, or detection using a filter?based assay for binding of radioligand to membranes or whole cells. The most critical step of any cDNA cloning project is the establishment of the screening protocol. Therefore, the bioassay for the gene product must be established prior to executing any of these protocols, including construction of the cDNA library.

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Basic Protocol 1: Expression of Cloned cDNA Transfected into Mammalian Cells
Basic Protocol 2: Recombinant Vaccinia Virus Infection and Liposome‐Mediated Transfection of Mammalian Cells
Support Protocol 1: Preparing cDNA Pools by Culture in Antibiotic Selection Medium
Support Protocol 2: Preparing cDNA Pools by Plating onto Antibiotic Selection Plates
Support Protocol 3: Using Autoradiography to Detect Gene Expression
Support Protocol 4: Using Nuclear Emulsion–Coated Chamber Slides to Detect Gene Expression
Support Protocol 5: Transport Activity Assay to Detect and Quantify Activity of Expressed Genes
Support Protocol 6: Radioligand Binding to Membranes or Whole Cells to Detect Gene Expression
Reagents and Solutions
Commentary
Figures
Tables

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Materials

Basic Protocol 1: Expression of Cloned cDNA Transfected into Mammalian Cells

Materials

LBA medium: LB medium ( appendix 2A ) containing appropriate antibiotics
DMSO or glycerol stocks of cDNA pools (see protocol 3 or protocol 42 )
TE buffer, pH 7.5 ( appendix 2A )
Phosphate‐buffered saline (PBS; appendix 2A )
Trypsinization solution: 0.25% (w/v) trypsin/0.02% (w/v) EDTA
Complete DMEM/10% FBS, complete DMEM/20% FBS, and complete DMEM without serum ( appendix 2A )
COS‐1, COS‐7, or HEK‐293 cells (ATCC# CRL 1650, CRL 1651, and CRL 1573, respectively)
250‐ml flasks, sterile
Shaking incubator, 37°C
Single‐chamber tissue culture slides, 6‐well tissue culture plates, or 10‐cm tissue culture dishes, sterile
Additional reagents and equipment for bioassay of transfected cells (see Chapter 7), isolation and purification of plasmid DNA, counting cells using a hemacytometer, DNA quantitating, calcium phosphate– or lipid‐mediated transfection, and transfection by electroporation (see CPMB UNITS , , , & , CPMB APPENDICES & , and appendix 1A in this manual)

NOTE: All solutions and equipment coming into contact with live cells must be sterile and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

Basic Protocol 2: Recombinant Vaccinia Virus Infection and Liposome‐Mediated Transfection of Mammalian Cells

Materials

CV‐1 or HeLa cells (ATCC #CCL 70 and CCL 2, respectively)
Complete DMEM/10% FBS, complete DMEM/20% FBS, and complete DMEM without serum ( appendix 2A )
Recombinant T7 (Fuerst et al., ) or SP6 (Usdin et al., ) vaccinia virus stock (available from B. Moss, NIH)
Purified DNA from cDNA pools (see protocol 1 , steps and )
LipofectACE (Life Technologies)
Bleach

10‐cm tissue culture dishes, single‐chamber culture slides, or 6‐well tissue culture plates (optional)
Bath sonicator with ice water
24‐well polystyrene tissue culture plates or polystyrene tubes

Additional reagents and equipment for preparing vaccinia virus and counting cells using a hemacytometer (see CPMB UNIT , CPMB APPENDIX , and appendix 1A in this manual)

Support Protocol 1: Preparing cDNA Pools by Culture in Antibiotic Selection Medium

Materials

DMSO or glycerol stock cDNA library from appropriate cells or tissue transformed into E. coli or packaged into a phage (e.g., λ_ZAP , Stratagene)
LBA medium: LB medium ( appendix 2A ) containing appropriate antibiotics
LBA plates: 10‐cm LB agar plates ( appendix 2A ) containing appropriate antibiotics
Glycerol stock buffer (see recipe )

50‐ml polypropylene conical tubes, sterile
Shaking incubator, 37°C
Beckman centrifuge and JS4.2 rotor (or equivalent)
Oven, 37°C

Support Protocol 2: Preparing cDNA Pools by Plating onto Antibiotic Selection Plates

Materials

DMSO or glycerol stock cDNA library from appropriate cells or tissue transformed into E. coli or packaged into a phage (e.g., λ_ZAP , Stratagene)
LBA plates: 10‐ and 15‐cm LB agar plates ( appendix 2A ) containing appropriate antibiotics
LBA medium: LB medium ( appendix 2A ) containing appropriate antibiotics
Glycerol stock buffer (see recipe )

50‐ml polypropylene centrifuge tubes, sterile
Beckman centrifuge and JS4.2 rotor (or equivalent)

Support Protocol 3: Using Autoradiography to Detect Gene Expression

Materials

Physiological buffer: HEPES‐buffered Krebs‐Ringer buffer (see recipe ) or phosphate‐buffered saline (PBS; appendix 2A )
Cultures of adherent transformed cells in 10‐cm tissue culture plates (see protocol 1 or protocol 2 )
Glogos luminescent stickers (Stratagene)
SDS/EDTA: 0.6% (w/v) SDS/10 mM EDTA
5 M NaCl
recipeTris‐buffered phenol ( appendix 2A )
20 mg/ml glycogen
100% ethanol
recipe3 M sodium acetate ( appendix 2A )
TE buffer, pH 7.5 ( appendix 2A )
Electroporation‐competent E. coli cells
LBA medium: LB medium ( appendix 2A ) supplemented with appropriate antibiotics
Double‐sided tape
X‐ray film: XAR or Beta‐max MR (Kodak)
Film cassettes, preferably large
Additional reagents and equipment for binding or transport assays for gene expression (e.g., see Chapter 7), electroporation of E. coli ( appendix 1E ), preparation of glycerol stock (see protocol 3 ), and transfection of COS cells (see protocol 1 )

Support Protocol 4: Using Nuclear Emulsion–Coated Chamber Slides to Detect Gene Expression

Materials

Physiological buffer: HEPES‐buffered Krebs‐Ringer (see recipe ) or phosphate‐buffered saline (PBS; appendix 2A )
Transformed cells plated on single‐chamber tissue culture slides (see protocol 1 or protocol 2 )
Radioligand fixative (see Table 4.8.1 )
PBS ( appendix 2A )
NTB‐2 nuclear track emulsion (Kodak or equivalent)
Dektol or D19 developer and fixer (Kodak)
Counterstain: e.g., 1% (w/v) Giemsa or 0.05% (w/v) nuclear fast red
Low‐viscosity Cytoseal (e.g., Fisher)
Slide rack

Peel‐away slide holder
Light‐tight darkroom facility with safelight
Water bath, 42°C
Glass Coplin jar
Light‐tight slide box
Humicaps (desiccant)
Black tape
Coverslips
Light microscope

Additional reagents and equipment for performing binding or transport assay (see Chapter 7)

Table 4.8.1 Materials Radioligand and Radiosubstrate Fixatives a Radioligand and Radiosubstrate Fixatives

Fixative
4% (w/v) formaldehyde/PBS b
Acetone
Methanol
1% (v/v) acrolein c /2.5% glutaraldehyde d /0.1 M cacodylate buffer, pH 6.4 e
1% (v/v) acrolein c /2.5% glutaraldehyde/PBS, pH 7.4 b
10% (v/v) acrolein c /0.1 M cacodylate buffer e
1% (v/v) glutaraldehyde d /PBS, pH 7.4 b
1% (v/v) glutaraldehyde d /0.1 M cacodylate buffer, pH 6.4 e
5% (v/v) glutaraldehyde/0.1 M cacodylate buffer, pH 6.4 e
1% (v/v) glutaraldehyde d /4% (w/v) formaldehyde/5% (v/v) saturated picric acid solution
1% (w/v) osmium tetroxide l /0.1 M cacodylate buffer, pH 6.4 e
1% (w/v) potassium permanganate/0.1 M cacodylate buffer, pH 6.4 e
3% (w/v) potassium permanganate/acetate buffer e

^a These solutions represent a range of possibilities as a starting point for determining what might work best for the radioligand of interest. Selection of the optimal fixative will depend on the chemical structure of the ligand or substrate in use. For instance, acrolein worked well for the neurotransmitter 5‐hydroxytryptamine since acrolein reacts with the compound's indole nitrogen. For further information concerning fixatives, consult McManus and Mowry ( ).

^b See recipe in appendix 2A .

^c CAUTION: Acrolein is tear gas and should only be used in a hood.

^d Electon microscopy grade, stored at 4°C.

^e See recipe in Reagents and Solutions .

Support Protocol 5: Transport Activity Assay to Detect and Quantify Activity of Expressed Genes

Materials

Transfected cells grown in 6‐well tissue culture plates (see protocol 1 or protocol 22 )
Uptake buffer (see recipe ), 37°C and ice cold
Radioligand working solution at 7‐fold desired final concentration (see unit 7.9 )
Inhibitor of transport diluted in uptake buffer to 7‐fold desired final concentration (see unit 7.9 )
0.5 N NaOH or 1% (w/v) SDS
Scintillation fluid
Micropipettor with polypropylene tips

Scintillation vials

Additional reagents and equipment for transport or binding assay and data interpretation (see Chapter 7)

Support Protocol 6: Radioligand Binding to Membranes or Whole Cells to Detect Gene Expression

Materials

Transfected cells (see protocol 1 or protocol 2 ) cultured in 35‐mm or 10‐cm tissue culture dishes or 6‐well plate
Phosphate‐buffered saline (PBS; appendix 2A )
Hypotonic buffer: e.g., 50 mM Tris⋅Cl, pH 7.6 ( appendix 2A )/5 mM MgCl₂
Binding buffer appropriate for assay
PBS containing 1 mM EGTA (for use with whole cells)
Radioligand (see unit 7.5 & )
Displacing drug
Physiological buffer: e.g., PBS, 1× Krebs‐Ringer (see recipe ), or uptake buffer (see recipe )
0.1% to 1% (v/v) polyethyleneimine (PEI) at neutral pH
Scintillation fluid
Cell scraper

15‐ml round‐bottomed centrifuge tubes (e.g., Falcon 2059 tubes)
Sonicator/homogenizer (e.g., Tissuemizer, Polytronic)
Sorvall centrifuge and SM‐24 rotor (or equivalent)
Glass tubes or microtiter plate
Glass‐fiber filters appropriate for isotope (e.g., Schleicher and Schuell, Whatman, or Inotech)
Cell harvester (e.g., Brandel or Inotech)

Additional reagents and equipment for determining protein concentration (see CPMB UNIT and appendix 1A in this manual)

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Figures

Figure 4.8.1 Flow chart for expression cloning in mammalian cells. Filter binding assays should be used when there are large numbers of very low‐complexity pools to be analyzed; autoradiography may be used for smaller numbers of high‐complexity pools.

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Videos

Literature Cited

Literature Cited
	Alsobrook, J.P. and Stevens, C.F. 1988. Cloning the calcium channel. Trends Neurosci. 11:1‐2.
	Blakely, R.D., Clark, J.A., Rudnick, G., and Amara, S.G. 1991. Vaccinia‐T7 RNA polymerase expression system: Evaluation for the expression cloning of plasma membrane transporters. Anal. Biochem. 194:302‐308.
	Borowsky, B. and Hoffman, B.J. 1995. Neurotransmitter transporters: Molecular biology, function, and regulation. Int. Rev. Neurobiol. 38:139‐199.
	Erickson, J.D., Eiden, L.E., and Hoffman, B.J. 1992. Expression cloning of a reserpine‐sensitive vesicular monoamine transporter. Proc. Natl. Acad. Sci. U.S.A. 89:10,993‐10,997.
	Evans, C.J., Keith, D.E. Jr., Morrison, H., Magendzo, K., and Edwards, R.H. 1992. Cloning of a delta opioid receptor by functional expression. Science 258:1952‐1955.
	Fuerst, T.R., Niles, E.G., Studier, F.W., and Moss, B. 1986. Eukaryotic transient‐expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 83:8122‐8126.
	Hayat, M.A. 1981. Fixation for Electron Microscopy. Academic Press, New York.
	Hediger, M.A., Coady, M.J., Ikeda, T.S., and Wright, E.M. 1987. Expression cloning and cDNA sequencing of the Na+/glucose co‐transporter. Nature 330:379‐381.
	Hoffman, B.J. 1994. Expression cloning of the serotonin transporter: A new way to study antidepressant drugs. Pharmacopsychiatry 27:16‐22.
	Hoffman, B.J., Mezey, E., and Brownstein, M.J. 1991. Cloning of a serotonin transporter affected by antidepressants. Science 254:579‐580.
	Julius, D., MacDermott, A.B., Axel, R., and Jessell, T.M. 1988. Molecular characterization of a functional cDNA encoding the serotonin 1c receptor. Science 241:558‐564.
	Kaupmann, K., Huggel, K., Heid, J., Flor, P.J., Bischoff, S., Mickel, S.J., McMaster, G., Angst, C., Bittiger, H., Froestl, W., and Bettler, B. 1997. Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors. Nature 386:239‐246.
	Liu, Y., Peter, D., Roghani, A., Schuldiner, S., Prive, G.G., Eisenberg, D., Brecha, N., and Edwards, R.H. 1992. A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter. Cell 70:539‐551.
	Lubbert, H., Hoffman, B.J., Snutch, T.P., van Dyke, T., Levine, A.J., Hartig, P.R., Lester, H.A., and Davidson, N. 1987. cDNA cloning of a serotonin 5‐HT1C receptor by electrophysiological assays of mRNA injected Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 84:4332‐4336.
	McManus, J.F.A. and Mowry, R.W. 1960. Staining Methods, Histologic and Histochemical. Harper and Row, New York.
	Morel, A., O'Carroll, A.M., Brownstein, M.J., and Lolait, S.J. 1992. Molecular cloning and expression of a rat V1a arginine vasopressin receptor. Nature 356:523‐526.
	Murphy, T.J., Alexander, R.W., Griendling, K.K., Runge, M.S., and Bernstein, K.E. 1991. Isolation of a cDNA encoding the vascular type‐1 angiotensin II receptor. Nature 351:233‐236.
	Pacholczyk, T., Blakely, R.D., and Amara, S.G. 1991. Expression cloning of a cocaine‐ and antidepressant‐sensitive human noradrenaline transporter. Nature 350:350‐354.
	Seeburg, P.H., Wisden, W., Verdoorn, T.A., Pritchett, D.B., Werner, P., Herb, A., Luddens, H., Sprengel, R., and Sakmann, B. 1990. The GABAA receptor family: Molecular and functional diversity. Cold Spring Harbor Symp. Quant. Biol. 55:29‐40.
	Snutch, T.P. 1988. The use of Xenopus oocytes to probe synaptic communication. Trends Neurosci. 11:250‐256.
	Usdin, T.B., Brownstein, M.J., Moss, B., and Isaacs, S.N. 1993. SP6 RNA polymerase containing vaccinia virus for rapid expression of cloned genes in tissue culture. Biotechniques 14:222‐224.
	Yamauchi, A., Uchida, S., Kwon, H.M., Preston, A.S., Robey, R.B., Garcia‐Perez, A., Burg, M.B., and Handler, J.S. 1992. Cloning of a Na+‐ and Cl− dependent betaine transporter that is regulated by hypertonicity. J. Biol. Chem. 267:649‐652.
Key References
	Hoffman et al., 1991. See above.
	Use of emulsion autoradiography to clone the serotonin transporter by calcium phosphate transfection into COS cells and transport assays.
	Pacholcyzk et al, 1991. See above.
	Use of film autoradiography to clone the norepinephrine transporter by DEAE‐dextran transfection into COS cells, transport assays, and Hirt recovery of plasmid DNA.
	Kaupmann et al., 1997. See above.
	Use of film autoradiography to clone GABAB receptors by DEAE‐dextran transfection into COS cells starting with small pools of cDNAs.
	Murphy et al., 1991. See above.
	Use of binding assays to clone the angiotensin II receptor starting with very small (1000‐member) pools of cDNAs.

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cDNA Expression Cloning in Mammalian Cells

Abstract

Table of Contents

Materials

Basic Protocol 1: Expression of Cloned cDNA Transfected into Mammalian Cells

Basic Protocol 2: Recombinant Vaccinia Virus Infection and Liposome‐Mediated Transfection of Mammalian Cells

Support Protocol 1: Preparing cDNA Pools by Culture in Antibiotic Selection Medium

Support Protocol 2: Preparing cDNA Pools by Plating onto Antibiotic Selection Plates

Support Protocol 3: Using Autoradiography to Detect Gene Expression

Support Protocol 4: Using Nuclear Emulsion–Coated Chamber Slides to Detect Gene Expression

Support Protocol 5: Transport Activity Assay to Detect and Quantify Activity of Expressed Genes

Support Protocol 6: Radioligand Binding to Membranes or Whole Cells to Detect Gene Expression

Figures

Videos

Literature Cited