Protein Knockouts in Living Eukaryotes Using deGradFP and Green Fluorescent Protein Fusion Targets

Emmanuel Caussinus1, Oguz Kanca1, Markus Affolter1

1 University of Basel, Basel
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 30.2
DOI:  10.1002/0471140864.ps3002s73
Online Posting Date:  September, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

ABSTRACT

This unit describes deGradFP (degrade Green Fluorescent Protein), an easy‐to‐implement protein knockout method applicable in any eukaryotic genetic system. Depleting a protein in order to study its function in a living organism is usually achieved at the gene level (genetic mutations) or at the RNA level (RNA interference and morpholinos). However, any system that acts upstream of the proteic level depends on the turnover rate of the existing target protein, which can be extremely slow. In contrast, deGradFP is a fast method that directly depletes GFP fusion proteins. In particular, deGradFP is able to counteract maternal effects in embryos and causes early and fast onset loss‐of‐function phenotypes of maternally contributed proteins. Curr. Protoc. Protein Sci. 73:30.2.1‐30.2.13. ‐ 2013 by John Wiley & Sons, Inc.

Keywords: GFP; nanobodies; F‐Box; Drosophila

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Protein Knockout in Drosophila Embryos
  • Alternate Protocol 1: Protein Knockouts in Drosophila Larva
  • Support Protocol 1: How to Know a Particular Protein of Interest is Targetable by deGradFP
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Protein Knockout in Drosophila Embryos

  Materials
  • Fly line carrying a functional GFP fusion gene in a genetic background where no wild‐type product of that gene (not labeled with GFP) is produced
  • Fly line carrying a UAS_NSlmb‐vhhGFP4 transgene
  • Fly line carrying a Gal4 driver
  • Apple juice agar plates (see recipe)
  • Baker's yeast powder
  • 3% (w/v) sodium hypochlorite (prepared fresh each day)
  • Embryo glue (see recipe)
  • Mineral oil (PCR grade)
  • Basic fly husbandry material (fly vials, fly food, binocular microscope, CO 2 fly station)
  • Fine nylon mesh
  • 50‐ml conical polypropylene centrifuge tube (e.g., BD Falcon) with the base cut off and a hole ∼1 cm in diameter in the center of the cap
  • 22‐mm square no. 1 coverslip
  • BioFolie 25 (Sartorius)
  • 2‐mm‐thick plastic or metallic microscope slides each with a hole ∼1 cm in diameter
  • Double‐sided tape
  • Confocal microscope

Alternate Protocol 1: Protein Knockouts in Drosophila Larva

  Materials
  • Fly line carrying a functional GFP fusion gene in a genetic background where no wild‐type product of that gene (not labeled with GFP) is produced
  • Fly line carrying a UAS_NSlmb‐vhhGFP4 transgene
  • Fly line carrying a Gal4 driver
  • Fly line carrying a tubP_Gal80ts transgene
  • Baker's yeast powder
  • 30% (v/v) glycerol
  • Phosphate buffered saline (PBS; APPENDIX 2E)
  • 4% (w/v) methanol‐free formaldehyde in PBS
  • Vectashield or Vectashield with DAPI if a nuclear counterstain is required (Vector Laboratories)
  • Basic fly husbandry material (fly vials, fly food, binocular microscope, CO 2 fly station)
  • 29°C incubator
  • Fluorescence stereo microscope
  • Microscope slides
  • 22‐mm square no. 1 coverslip
  • Nail polish
  • Confocal microscope

Support Protocol 1: How to Know a Particular Protein of Interest is Targetable by deGradFP

  Additional Materials (also see protocol 1Basic Protocol)
  • Fly line carrying a functional GFP fusion gene: in contrast to the protocol 1Basic Protocol and protocol 2Alternate Protocol, the genetic background of this line is not important and can be wild type in respect to the gene of interest.
  • Fly line carrying a UAS_NSlmb‐vhhGFP4 transgene, a UAS_mCherry‐NLS transgene, and an engrailed‐Gal4 (enGal4) driver used to express, in Drosophila embryos, both NSlmb‐vhhGFP4 and mCherry‐NLS in a simple pattern: posterior compartment of each segment of the epidermis from stages 6 to 17 (Tabata et al., )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Asakawa, K. and Kawakami, K. 2009. The tol2‐mediated Gal4‐UAS method for gene and enhancer trapping in zebrafish. Methods 49:275–281.
  Ashburner, M. 1990. Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  Banaszynski, L.A., Chen, L.C., Maynard‐Smith, L.A., Ooi, A.G. and Wandless, T.J. 2006. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126:995–1004.
  Berdougo, E., Terret, M.E., and Jallepalli, P.V. 2009. Functional dissection of mitotic regulators through gene targeting in human somatic cells. Methods Mol. Biol. 545:21–37.
  Brand, A.H. and Perrimon, N. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415.
  Buszczak, M., Paterno, S., Lighthouse, D., Bachman, J., Planck, J., Owen, S., Skora, A.D., Nystul, T.G., Ohlstein, B., Allen, A., Wilhelm, J.E., Murphy, T.D., Levis, R.W., Matunis, E., Srivali, N., Hoskins, R.A., and Spradling, A.C. 2007. The carnegie protein trap library: A versatile tool for Drosophila developmental studies. Genetics 175:1505–1531.
  Carette, J.E., Guimaraes, C.P., Varadarajan M., Park, A.S., Wuethrich, I., Godarova, A., Kotecki, M., Cochran, B.H., Spooner, E., Ploegh, H.L., and Brummelkamp, T.R. 2009. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326:1231–1235.
  Caussinus, E., Kanca, O., and Affolter, M. 2011. Fluorescent fusion protein knockout mediated by anti‐GFP nanobody. Nat. Struct. Mol. Biol. 19:117–121.
  Ciechanover, A. 1998. The ubiquitin‐proteasome pathway: On protein death and cell life. EMBO J. 17:7151–7160.
  Dohmen, R.J., Wu, P., and Varshavsky, A. 1994. Heat‐inducible degron: A method for constructing temperature‐sensitive mutants. Science 263:1273–1276.
  Ekker, S.C. 2000. Morphants: A new systematic vertebrate functional genomics approach. Yeast 17:302–306.
  Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double‐stranded RNA in C. elegans. Nature 391:806–811.
  Greenspan, R.J. 2004. Fly pushing: the theory and practice of Drosophila genetics second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  Harder, B., Schomburg, A., Pflanz, R., Küstner, K., Gerlach, N., and Schuh, R. 2008. Tev protease‐mediated cleavage in Drosophila as a tool to analyze protein functions in living organisms. Biotechniques 44:765–772.
  Hartenstein, V. 1993. Atlas of Drosophila Development. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  Haruki, H., Nishikawa, J., and Laemmli, U.K. 2008. The anchor‐away technique: Rapid, conditional establishment of yeast mutant phenotypes. Mol. Cell 31:925–932.
  Inoue, T., Heo, W.D., Grimley, J.S., Wandless, T.J. and Meyer, T. 2005. An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nat. Methods 2:415–418.
  Jiang, J. and Struhl, G. 1998. Regulation of the hedgehog and wingless signalling pathways by the F‐box/WD40‐repeat protein Slimb. Nature 391:493–496.
  Kawakami, K., Abe, G., Asada, T., Asakawa, K., Fukuda, R., Ito, A., Lal, P., Mouri, N., Muto, A., Suster, M.L., Takakubo, H., Urasaki, A., Wada, H., and Yoshida M. 2010. Ztrap: Zebrafish gene trap and enhancer trap database. BMC Dev. Biol. 10:105.
  Kelso, R.J., Buszczak, M., Quiñones, A.T., Castiblanco, C., Mazzalupo, S., and Cooley, L. 2004. Flytrap, a database documenting a GFP protein‐trap insertion screen in Drosophila melanogaster. Nucleic Acids Res. 32:D418–D420.
  Mavrakis, M., Rikhy, R., Lilly, M. and Lippincott‐Schwartz, J. 2008. Fluorescence imaging techniques for studying Drosophila embryo development. Curr. Protoc. Cell Biol. 39:4.18.1–4.18.43.
  McGuire, S.E., Le, P.T., Osborn, A.J., Matsumoto, K., and Davis, R.L. 2003. Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302:1765–1768.
  Morin, X., Daneman, R., Zavortink, M., and Chia, W. 2001. A protein trap strategy to detect gfp‐tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 98:15050–15055.
  Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T., and Kanemaki, M. 2009. An auxin‐based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6:917–922.
  Pauli, A., Althoff, F., Oliveira, R.A., Heidmann, S., Schuldiner, O., Lehner, C.F., Dickson, B.J., and Nasmyth, K. 2008. Cell‐type‐specific TEV protease cleavage reveals cohesin functions in Drosophila neurons. Dev. Cell 14:239–251.
  Pastrana, E. 2012. Degrading proteins with intracellular antibodies. Nat. Methods 9:228.
  Pina, C. and Pignoni, F. 2012. Tubby‐RFP balancers for developmental analysis: FM7c 2xTb‐RFP, CyO 2xTb‐RFP, and TM3 2xTb‐RFP. Genesis 50:119–123.
  Quiñones‐Coello, A.T., Petrella, L.N., Ayers, K., Melillo, A., Mazzalupo, S., Hudson, A.M., Wang, S., Castiblanco, C., Buszczak, M., Hoskins, R.A., and Cooley, L. 2007. Exploring strategies for protein trapping in Drosophila. Genetics 175:1089–1104.
  Rothbauer, U., Zolghadr, K., Muyldermans, S., Schepers, A., Cardoso, M.C., and Leonhardt, H. 2008. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol. Cell Proteomics 7:282–289.
  Royou, A., Field, C., Sisson, J.C., Sullivan, W., and Karess, R. 2004. Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos. Mol. Biol. Cell 15:838–850.
  Saerens, D., Pellis, M., Loris, R., Pardon, E., Dumoulin, M., Matagne, A., Wyns, L., Muyldermans, S., and Conrath, K. 2005. Identification of a universal vhh framework to graft non‐canonical antigen‐binding loops of camel single‐domain antibodies. J. Mol. Biol. 352:597–607.
  Sakamoto, K.M., Kim, K.B., Kumagai, A., Mercurio, F., Crews, C.M., and Deshaies, R.J. 2001. Protacs: Chimeric molecules that target proteins to the SKP1‐Cullin‐F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. U.S.A. 98:8554–8559.
  Schornack, S., Fuchs, R., Huitema, E., Rothbauer, U., Lipka, V., and Kamoun, S. 2009. Protein mislocalization in plant cells using a GFP‐binding chromobody. Plant J. 60:744–754.
  Tabata, T., Eaton, S., and Kornberg, T.B. 1992. The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. Genes Dev. 6:2635–2645.
  Taxis, C., Stier, G., Spadaccini, R., and Knop, M. 2009. Efficient protein depletion by genetically controlled deprotection of a dormant N‐degron. Mol. Syst. Biol. 5:267.
  Varshavsky, A. 2012. The ubiquitin system, an immense realm. Ann. Rev. Biochem. 81: 167–176.
  Zhang, J., Zheng, N., and Zhou, P. 2003. Exploring the functional complexity of cellular proteins by protein knockout. Proc. Natl. Acad. Sci. U.S.A. 100:14127–14132.
  Zhou, P., Bogacki, R., McReynolds, L., and Howley, P.M. 2000. Harnessing the ubiquitination machinery to target the degradation of specific cellular proteins. Mol. Cell 6:751–756.
Key Reference
  Caussinus et al., 2011. See above.
  The original article about deGradFP.
Internet Resources
  http://flytrap.med.yale.edu
  Flytrap is a database of GFP protein trap Drosophila lines generated in the Chia, Cooley, and Spradling labs.
  http://www.flyprot.org/
  Flyprot is a database of Venus protein trap Drosophila lines generated in the Cambridge protein trap project.
  http://flybase.org
  Flybase is the leading database and Web portal for genetic and genomic information on Drosophila melanogaster and related fly species.
  http://kawakami.lab.nig.ac.jp/ztrap/
  A gene trap and enhancer trap database dedicated to Danio rerio.
  http://www.addgene.org/
  Addgene is a non‐profit organization that operates a plasmid repository for the research community.
  http://flystocks.bio.indiana.edu/
  The Bloomington Drosophila Stock Center (at Indiana University) collects, maintains, and distributes Drosophila melanogaster strains for research.
  http://www.sdbonline.org/fly/atlas/00atlas.htm
  Online version of the Atlas of Drosophila Development by Volker Hartenstein ().
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library