手机版 发布询价单
厂商登录 新闻快讯 网站地图
全站
发布询价单
 热门搜索词： 移液工作站 液相色谱 原子吸收 标准物质 直读光谱仪 扫描电镜 微波消解 粒度仪 ICP-MS
导购
实验方法> 生物信息学技术> 数据库>Expression Cloning of Neural Genes Using Xenopus laevis Oocytes

Expression Cloning of Neural Genes Using Xenopus laevis Oocytes

关键词： expression cloning来源： 互联网
  • Abstract
  • Table of Contents
  • Materials
  • Literature Cited

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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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 m7 G(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‐32 P]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 2 HPO 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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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. Clonin
推荐方法
分析测试百科网| 快捷面板| 站点地图| 律师声明| 关于我们| About Us| 广告服务| 改善您的体验| 友情链接| 联系我们
 移动版： 资讯   Webinar   仪器谱

Copyright ©2007 ANTPedia, All Rights Reserved

京ICP备07018254号 京公网安备1101085018 电信与信息服务业务经营许可证：京ICP证110310号