In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP

D. A. Stark1, P. M. Kulesa1

1 Stowers Institute for Medical Research, Kansas City
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.8
DOI:  10.1002/0471143030.cb1208s28
Online Posting Date:  October, 2005
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Abstract

Selective marking of a single cell within a living embryo is often difficult due to the inaccuracy and invasiveness of standard techniques. This unit describes a minimally invasive optical protocol that uses 405‐nm laser light to photoactivate a variant of green fluorescent protein (PAGFP). This method takes advantage of the accessibility of the chick embryo to inject PAGFP into a region of interest and uses electroporation to deliver the construct into cells. This unit describes in detail how single and small groups of cells (n<10) that express PAGFP can be made visually distinguishable from the host population using the photoactivation process. Included is a means to maximize the fluorescence increase due to photoactivated GFP signal and to reduce photobleaching. Briefly outlined are previously developed chick culture and time‐lapse imaging techniques to allow for the subsequent monitoring of photoactivated cell migratory behaviors. The technique has the potential to be a less‐invasive, accurate tool for in vivo studies that involve following cell lineage and cell migration.

Keywords: photoactivation; GFP; chick; embryo; cell labeling; lineage

     
 
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Table of Contents

  • Basic Protocol 1: Photoactivation of GFP in Single Cells in Chick Embryos
  • Alternate Protocol 1: In Ovo Photoactivation
  • Support Protocol 1: Imaging Using High‐Magnification Acquisition
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Photoactivation of GFP in Single Cells in Chick Embryos

  Materials
  • Fertilized white Leghorn chick eggs incubated at 37°C
  • India ink (drawing ink, Pelikan)
  • Howard Ringer's solution (J.A. Webster), sterile
  • 10 mg/ml Fast Green FCF (Fisher) in Howard Ringer's solution, optional
  • PAGFP construct (available from J. Lippincott‐Schwartz; )
  • H2B‐mRFP construct (optional; available from P. Kulesa; )
  • 70% ethanol
  • High‐vacuum silicone grease (Dow Corning)
  • B27 supplement (Invitrogen)
  • Neural basal medium (Invitrogen)
  • L‐glutamine (Sigma‐Aldrich)
  • H 2O, sterile
  • Human fibronectin (Invitrogen), diluted to 20 µg/ml in phosphate butter
  • Egg incubator (G.Q.F. Manufacturing Co., Model 1550)
  • 15‐ml tubes
  • Glass capillary tubes; borosilicate with filament (Sutter Instrument)
  • Glass needle puller (Sutter Instrument, Model P‐87)
  • Micropipet loader
  • 20‐µl pipettor
  • Picospritzer (Picospritzer III, Parker Hannifin Corporation)
  • Dumont no. 5 forceps and iris scissors (Fine Science Tools)
  • Electroporator (BTX)
  • Platinum electrodes (A.M. Systems)
  • 18‐ and 25‐G needles (Becton Dickinson)
  • 1‐ and 5‐ml syringes (VWR)
  • Tape (e.g., Scotch)
  • Plastic transfer pipet, sterile
  • Tungsten needle (A.M. Systems)
  • Micromanipulator (World Precision Instruments)
  • 6‐well culture plates
  • Soldering tool (Weller)
  • Sandpaper, medium grade (3M)
  • Glass cover slips, 22‐mm round (VWR)
  • Culture insert (Millipore)
  • Petri dishes (Falcon)
  • Fluorescence stereo microscope with halogen light source and appropriate filters (LP‐DAPI, TRITC; Leica)
  • Whatman no.1 filter paper
  • Dissecting scissors
  • Stereo dissecting microscope
  • Confocal inverted laser scanning microscope with 488‐ and 405‐nm laser lines (Zeiss LSM5 PASCAL) and cell tracking software (optional)
  • Parafilm
  • Cardboard (1/4‐in. thick)
  • Velcro (sticky back tape)
  • Insulation for cardboard box (5/16‐in. thick; Reflectix)
  • Clear plastic packaging tape
  • Chick incubator heater (Model 115‐20, Lyon)

Alternate Protocol 1: In Ovo Photoactivation

  • Beeswax (Eastman Kodak)
  • Microinjected and electroporated eggs (see protocol 1)
  • 3.8‐cm × 7.5‐cm × 15‐µm Teflon membrane, high‐sensitivity, oxygen permeable (Fisher)
  • ∼2.2‐cm i.d. × 2.6‐cm o.d. × 0.5‐cm high acrylic ring (constructed in machine shop)
  • 2.4‐cm i.d. × 2.1‐cm o.d. rubber O ring (constructed in machine shop)
  • Upright, laser scanning confocal microscope with 488‐nm and 405‐nm laser excitation, equipped with a long working distance 10× Plan Neofluar objective (NA = 0.3)

Support Protocol 1: Imaging Using High‐Magnification Acquisition

  • Embryos on culture inserts (see protocol 1)
  • Glass‐bottomed petri dishes (Mat Tek)
  • Glass microscope slides (VWR)
  • Coverslips, 22‐mm
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Figures

Videos

Literature Cited

Literature Cited
   Ando, H., Furuta, T., Tsien, R.Y., and Okamoto, H. 2001. Photo‐mediated gene activation using caged RNA/DNA in zebrafish embryos. Nat. Genet. 28:317‐325.
   Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802‐805.
   Chudakov, D.M., Verkhusha, V.V., Staroverov, D.B., Souslova, E.A., Lukyanov, S., and Lukyanov, K.A. 2004. Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22:1435‐1440.
   Fraser, S.E. 1996 Iontophoretic dye labeling of embryonic cells. Methods Cell Biol. 51:147‐160.
   Hamburger, V. and Hamilton, H.L. 1951. A series of normal stages in the development of the chick embryo. J. Embryol. Exper. Morph. 88:49‐92.
   Itasaki, N., Bel‐Vialar, S., and Krumlauf, R. 1999. Shocking developments in chick embryology: Electroporation and in ovo gene expression. Nat. Cell Biol. 1:E203‐E207.
   Patterson, G.H. and Lippincott‐Schwartz, J. 2002. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873‐1877.
   Patterson, G.H. and Lippincott‐Schwartz, J. 2003. Development and use of fluorescent protein markers in living cells. Science 300:87‐91.
   Sawin, K.E. and Nurse, P. 1997. Photoactivation of green fluorescent protein. Curr. Biol. 7:R606‐R607.
   Stern, C.D. and Fraser, S.E. 2001. Tracing the lineage of tracing cell lineage. Nat. Cell Biol. 3:E216‐E218.
   Yokoe, H. and Meyer, T. 1996. Spatial dynamics of GFP‐tagged proteins investigated by local fluorescence enhancement. Nat. Biotechnol. 14:1252‐1256.
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