Expression Cloning of Neural Genes Using Xenopus laevis Oocytes

Jim Boulter1, Christopher Boyer2

1 University of California at Los Angeles, Los Angeles, California, 2 The Salk Institute, La Jolla, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.3
DOI:  10.1002/0471142301.NS0403s00
Online Posting Date:  May, 2001
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Abstract

Expression cloning requires a representative cDNA or genomic DNA library and a host organism in which the cloned genes can be transcribed and/or translated. It likewise requires a method to detect the expressed protein using, for example, the inherent biological activity of the gene or antibodies specific for the gene product. Most successful expression cloning strategies have employed cDNA libraries constructed in plasmid or bacteriophage lambda vectors and Xenopus oocytes or cultured mammalian cells as hosts. This unit presents several protocols designed for expression cloning paradigms that rely on electrophysiological recordings from Xenopus laevis oocytes.

     
 
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Table of Contents

  • Basic Protocol 1: Preparing Bacteriophage λ Template DNA for In Vitro Transcription
  • Alternate Protocol 1: Preparing Plasmid DNA Template for In Vitro Transcription
  • Basic Protocol 2: In Vitro Transcription of Sublibrary DNA
  • Basic Protocol 3: Injection of Xenopus laevis Oocytes with In Vitro–Transcribed cRNA
  • Support Protocol 1: Preparation of Xenopus laevis Oocytes for cRNA Injection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparing Bacteriophage λ Template DNA for In Vitro Transcription

  Materials
  • 15 × 150–mm NZYM agarose plates (see recipe)
  • Escherichia coli strain BB4 (Stratagene)
  • NZYM medium (see recipe) containing 0.2% (w/v) filter‐sterilized maltose
  • Bacterial resuspension buffer (see recipe)
  • Bacteriophage λ library stock
  • NZYM agar plates (see recipe)
  • NZYM soft agarose medium (see recipe)
  • Phage dilution buffer (see recipe)
  • 10 mg/ml DNase I
  • 10 mg/ml pancreatic RNase
  • Cesium chloride step gradient solutions (see recipe)
  • 20% (w/v) sucrose in phage dilution buffer (see recipe)
  • Phage DNA extraction buffer (see recipe)
  • TE buffer ( appendix 2A)
  • Restriction endonuclease and buffer
  • 25:24:1 (v/v/v) Tris⋅Cl‐buffered phenol/chloroform/isoamyl alcohol, pH 8.0 ( appendix 2A)
  • Ether
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 75% and 100% ethanol
  • 15 × 150–mm and 15 × 100–mm plastic petri dishes, sterile
  • Sorvall RC‐2B centrifuge and SS‐34 rotor or equivalent
  • 50‐ml polypropylene screw‐cap centrifuge tubes, sterile
  • 17 × 100–mm polystyrene tubes, sterile
  • Rubber spatula, sterile
  • Sorvall RT6000B centrifuge and H‐1000B rotor or equivalent
  • 30‐ml centrifuge tubes
  • SW 28 centrifuge tubes (Beckman), clear
  • Beckman L80 centrifuge and SW 28 rotor or equivalent
  • 5‐ml disposable plastic syringes
  • 18‐G needles
  • Dialysis membrane (Spectra/Por2, 12,000 to 14,000 MWCO)
  • RNase‐free aerosol‐resistant micropipet tips
  • 1.7‐ml microcentrifuge tubes, RNase‐free and silanized
  • Additional reagents and equipment for spectrophotometric determination of DNA concentration, and restriction endonuclease digestion of DNA (CPMB APPENDIX , unit 3.1, and appendix 1A in this manual)

Alternate Protocol 1: Preparing Plasmid DNA Template for In Vitro Transcription

  Materials
  • 15 × 150–mm LB agarose plates containing 50 µg/ml ampicillin ( appendix 2A)
  • Plasmid library glycerol stock
  • LB medium ( appendix 2A) with and without 50 µg/ml ampicillin
  • 15 × 100–mm LB agar plates containing 50 µg/ml ampicillin ( appendix 2A)
  • 70% ethanol
  • 2× bacterial freezing medium (see recipe)
  • ∼2‐mm‐diameter L‐shaped‐glass rod
  • 17 × 100–mm polystyrene centrifuge tubes, sterile (Falcon)
  • 2‐ml sterile cryotube (Nunc)
  • Sorvall RC2‐B centrifuge and SS‐34 rotor or equivalent
  • Additional reagents and equipment for purifying plasmid DNA and spectrophotometric determination of DNA concentration (CPMB UNIT , CPMB APPENDIX , and appendix 1A in this manual)

Basic Protocol 2: In Vitro Transcription of Sublibrary DNA

  Materials
  • 5× RNA transcription buffer (see recipe)
  • 0.1 M dithiothreitol (DTT)
  • 10 mM ATP
  • 10 mM CTP
  • 10 mM GTP
  • 10 mM UTP
  • 10 mM m7G(5′)ppp(5′)G (methyl‐diguanosine triphosphate, RNA “cap” structure; Ambion)
  • RNase‐free water (see recipe)
  • 1 U/µl RNase inhibitor (e.g., Prime RNase Inhibitor, 5 Prime → 3 Prime), stored at −20°C
  • 10 mCi/ml [α‐32P]UTP (800 Ci/mmol)
  • Linearized λ DNA template (see protocol 1) or linearized plasmid sublibrary DNA (see protocol 2 2)
  • 200 U/µl T3 RNA polymerase (Ambion), stored at −20°C
  • 10 M lithium chloride
  • 0.5 M sodium phosphate (Na 2HPO 4)
  • 75% and 95% ethanol
  • 1.7‐ml microcentrifuge tubes, RNase‐free and silanized
  • 2.4–cm diameter DE81 filters (Whatman)
  • Scintillation fluid and vials

Basic Protocol 3: Injection of Xenopus laevis Oocytes with In Vitro–Transcribed cRNA

  Materials
  • Barth's solution containing calcium( recipe)
  • Sigmacote
  • Fluorinert (ISCO) or light mineral oil (Sigma)
  • In vitro–transcribed cRNA (see protocol 3)
  • Collagenase‐treated Xenopus laevis oocytes (see protocol 5)
  • 2.5‐cm metal cork borer
  • 2.5‐cm‐diameter nylon monofilament mesh circle (Nitex Nylon, 500 µm mesh; Tetko)
  • Fine‐toothed hacksaw
  • 2.5‐cm‐diameter polyvinyl chloride (PVC) tubing
  • 18‐G blunt‐end needle
  • 1‐ and 3‐ml plastic disposable syringes
  • Epoxy cement
  • 8‐in. glass capillary pipets (Drummond), sterile
  • 170° to 180°C drying oven
  • Electrophysiology pipet puller (e.g., Sutter Instruments or equivalent)
  • Injection micropipet holder: a sterile, dust‐free, static‐free, RNase‐free 15 × 100–mm petri dish containing a 0.3 × 0.3 × 10–cm strip of dental wax or clay
  • Digital microdispenser (Drummond)
  • Micromanipulator (Brinkman or equivalent)
  • Dissecting microscope and fiber optic light source
  • 3‐in. 26‐G needles
  • 1.7‐ml microcentrifuge tubes, RNase‐free and silanized
  • 15 × 60–mm plastic petri dishes, sterile
  • Wide‐bore (∼3‐mm), blunt‐end, fire‐polished Pasteur pipet
  • Additional reagents and equipment for silanizing glassware (see CPMB APPENDIX and appendix 1A in this manual)

Support Protocol 1: Preparation of Xenopus laevis Oocytes for cRNA Injection

  Materials
  • Adult, female Xenopus laevis (>2 years old, ≥ 10 cm in length)
  • Tricaine anesthetic (see recipe)
  • Barth's solution with and without calcium and antibiotics (see recipe)
  • Collagenase solution (see recipe)
  • Blunt‐end forceps
  • Towels or absorbent pads
  • Large‐ and small‐tooth forceps, sterile
  • Small, straight scissors, sterile
  • 15 × 60–mm and 15 × 100–mm plastic petri dishes, sterile
  • Absorbable surgical sutures (e.g., cutting FS‐2 chromic gut, Ethicon)
  • 50‐ml, conical, screw‐cap centrifuge tubes, sterile
  • Orbital shaker or platform rocker
  • Dissecting microscope with fiber optic light source
  • Wide‐bore (∼3‐mm), fire‐polished Pasteur pipets
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.
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Figures

Videos

Literature Cited

   Ballivet, M., Nef, P., Couturier, S., Rungger, D., Bader, C.R., Bertrand, D., and Cooper, E. 1988. Electrophysiology of a chick neural nicotinia acetylcholine receptor expressed in Xenopus oocytes after cDNA injection. Neuron 1:847‐852.
   Bertran, J., Werner, A., Chillaron, J., Nunes, V., Biber, J., Testar, X., Zorzano, A., Estivill, X., Murer, H., and Palacin, M. 1993. Expression cloning of a human renal cDNA that induces high‐affinity transport of L‐cystine shared with dibasic amino acids in Xenopus oocytes. J. Biol. Chem. 268:14842‐14849.
   Bertrand, D., Ballivet, M., and Rungger, D. 1990. Activation and blocking of neuronal nicotinic acetylcholine receptor reconstituted in Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 87:1993‐1997.
   Bertrand, D., Cooper, E., Valera, S., Rungger, D., and Ballivet, M. 1991. Electrophysiology of neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes following nuclear injection of genes or cDNAs. Methods Neurosci. 4:174‐193.
   Boyle, M.B. and Kaczmarek, L.K. 1991. Electrophysiological expression of ion channels in Xenopus oocytes. Methods Neurosci. 4:157‐173.
   Brake, A.J., Wagenbach, M.J., and Julius, D. 1994. New structural motif for ligand‐gated ion channels defined by an ionotropic ATP receptor. Nature 371:519‐523.
   Brunden, M.N., Huff, R.M., Vidmar, T.J., and Cooper, M.M. 1990. Planning the purification process of active cDNA in expression cloning strategies. J. Theor. Biol.. 144:145‐154.
   Chao, M.V., Bothwell, M.A., Ross, A.H., Koprowski, H., Lanahan, A.A., Buck, C.R., Sehgal, A. 1986. Gene transfer and molecular cloning of the human NGF receptor. Science 232:518‐521.
   Costa, A.C., Patrick, J.W., and Dani, J.A. 1994. Improved technique for studying ion channels expressed in Xenopus oocytes, including fast perfusion. Biophys. J. 67:395‐401.
   Dascal, N. 1987. The use of Xenopus oocytes for the study of ion channels. CRC Crit. Rev. Biochem. 22:317‐387.
   Dumont, J.N. 1972. Oogenesis in Xenopus laevis (Daudin) 1. Stages of oocyte development in laboratory‐maintained animals. J. Morph. 136:153‐180.
   Etheridge, A.L. and Richter, S.M.A. 1978. Xenopus laevis: Rearing and breeding the African clawed frog. Nasco, Fort Atkinson, Wisc.
   Frech, G.C., van Dongen, A.M., Schuster, G., Brown, A.M., and Joho, R.H. 1989. A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning. Nature 340:642‐645.
   Gundersen, C.B., Miledi, R., and Parker, I. 1984. Messenger RNA from human brain induces drug‐ and voltage‐operated channels in Xenopus oocytes. Nature 308:421‐424.
   Gurdon, J.B., Lane, C.D., Woodland, H.R., and Marbaix, G. 1971. Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature 233:177‐182.
   Gurdon, J.B. and Wickens, M.P. 1983. The use of Xenopus oocytes for the expression of cloned genes. Methods Enzymol. 101:370‐386.
   Hollmann, M., O'Shea‐Greenfield, A., Rogers, S.W., and Heinemann, S. 1989. Cloning by functional expression of a member of the glutamate receptor family. Nature 342:643‐648.
   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.
   Kushner, L., Lerma, J., Bennett, M.V.L., and Zukin, S.R. 1991. Using the Xenopus oocyte system for expression and cloning of neuroreceptors and channels. Methods Neurosci. 1:157‐173.
   Lam, A., Kloss, J., Fuller, F., Cordell, B., and Ponte, P.A. 1992. Expression cloning of neurotrophic factors using Xenopus oocytes. J. Neurosci. Res. 32:43‐50.
   Leonard, J.P. and Snutch, T.P. 1991. The expression of neurotransmitter receptors and ion channels in Xenopus oocytes. In Molecular Neurobiology: A Practical Approach (J. Chad and H. Wheal, eds.) pp 162‐182. IRL Press, Oxford.
   Lustig, K.D., Shiau, A.K., Brake, A.J., and Julius, D. 1993. Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc. Natl. Acad. Sci. U.S.A. 90:5113‐5117.
   MacKinnon, R., Reinhart, P.H., and White, M.M. 1988. Charybdotoxin block of shaker K+ channels suggests that different types of K+ channels share common structural features. Neuron 1:997‐1001.
   Methfessel, C., Witzemann, V., Takahashi, T., Mishina, M., Numa, S., and Sakmann, B. 1986. Patch clamp measurements on Xenopus laevis oocytes: Currents through endogenous channels and implanted acetylcholine receptor and sodium channels. Pflügers Arch.Eur. J. Physiol. 407:577‐588.
   Perez‐Reyes, E., Kim, H.S., Lacerda, A.E., Horne, W., Wei, X.Y., Rampe, D., Campbell, K.P., Brown, A.M., and Birnbaumer, L. 1989. Induction of calcium currents by the expression of the alpha 1‐subunit of the dihydropyridine receptor from skeletal muscle. Nature 340:233‐236.
   Pfaff, J.L., Tamkun, M.M., and Taylor, W.L. 1990. pOEV: A Xenopus oocyte protein expression vector. Anal. Biochem. 188:192‐199.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Shih, C. and Weinberg, R.A. 1982. Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell 29:161‐169.
   Short, J.M., Fernandez, J.M., Sorge, J.A., and Huse, W.D. 1988. Lambda ZAP: A bacteriophage lambda expression vector with in vivo excision properties. Nucl. Acids Res. 16:7583‐7600.
   Snutch, T.P. 1988. The use of Xenopus oocytes to probe synaptic communication. Trends Neurosci. 11:250‐256.
   Soreq, H. 1985. The biosynthesis of biologically active proteins in mRNA‐injected Xenopus oocytes. CRC Crit. Rev. Biochem. 18:199‐235.
   Straub, R.E., Oron, Y., and Gershengorn, M.C. 1991. Expression of mammalian plasma membrane receptors in Xenopus oocytes: Studies of thyrotropin‐releasing hormone action. Methods Neurosci. 1:46‐61.
   Tanaka, K. 1993. Expression cloning of a rat glutamate transporter. Neurosci. Res. 16:149‐153.
   Tate, S.S., Yan, N., and Udenfriend, S. 1992. Expression cloning of a Na+‐independent neutral amino acid transporter from rat kidney. Proc. Natl. Acad. Sci. U.S.A. 89:1‐5.
   Thornhill, W.B. and Levinson, S.R. 1987. Biosynthesis of electroplax sodium channels in Electrophorus electrocytes and Xenopus oocytes. Biochemistry 26:4381‐43883.
   Yi, J.R., Lu, S., Fernandez‐Checa, J., and Kaplowitz, N. 1995. Expression cloning of the cDNA for a polypeptide associated with rat hepatic sinusoidal reduced glutathione transport: Characteristics and comparison with the canalicular transporter. Proc. Natl. Acad. Sci. U.S.A. 92:1495‐1499.
Key References
   Dumont, 1972. See above.
  This is the definitive paper on Xenopus laevis oogenesis: it describes aspects of oocyte development with an excellent presentation of the anatomical and histological characteristics of each stage of oocyte development and includes some very useful photographs of oocytes during each stage of development.
   Leonard and Snutch 1991. See above.
  A complete and thorough discussion of how to utilize Xenopus oocytes for electrophysiological analysis of ion channels and receptors. The text covers RNA synthesis, size‐fractionation of poly(A)+ RNA and RNA purification methods, the preparation and injection of oocytes, and detailed account of electrophysiological recording techniques including two‐electrode voltage clamp, patch clamping, and single‐channel recording.
   Kushner et al., 1991. See above.
  Includes a compendium of ion channels and receptors which are expressed in oocytes after injection of tissue mRNA or in vitro transcribed cRNA methods for the preparation of RNA, in vitro synthesis of RNA from cloned genes, preparation and injection of oocytes, and a brief discussion of cloning strategies utilizing oocytes.
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