Culturing Ovarian Somatic and Germline Stem Cells of Drosophila

Yuzo Niki1

1 Ibaraki University, Ibaraki, Japan
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2E.1
DOI:  10.1002/9780470151808.sc02e01s10
Online Posting Date:  September, 2009
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Abstract

This unit describes how to collect, culture, and establish stable cell lines of ovarian somatic and germline stem cells of Drosophila. We also describe a protocol for culturing embryonic cells that overexpress growth factors, which serve as a source for conditioned medium. Curr. Protoc. Stem Cell Biol. 10:2E.1.1‐2E.1.9. © 2009 by John Wiley & Sons, Inc.

Keywords: Drosophila; somatic and germline stem cells; isolation; expansion

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Culture of Ovarian Somatic Stem Cells and Germline Stem Cells
  • Support Protocol 1: Preparation of Fly Extract
  • Support Protocol 2: Preparation of Conditioned Medium from Embryos Overexpressing Growth Factors
  • Support Protocol 3: Fixation and Staining of Cultured Drosophila Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Culture of Ovarian Somatic Stem Cells and Germline Stem Cells

  Materials
  • 20‐ to 30‐day‐old female flies of w1118; P[w+ hsp‐70 bam+] 11‐d bamΔ86 ry e/bamΔ86 P[ovo‐lacZ] or w1118; P[w+ hsp‐70 bam+] 11‐d bamΔ86 ry e/bamΔ86 P[ovo‐lacZ) P[vasa‐egfp] genotype (Fig. A,B)
  • 70% (v/v) ethanol
  • Drosophila phosphate‐buffered saline (PBS; Robb, ), sterile
  • Culture medium (see recipe)
  • Penicillin/streptomycin (see reciperecipes), optional
  • Distilled water
  • DMSO
  • Liquid nitrogen
  • 15‐ml conical tubes
  • Single concave depression glass slides
  • Forceps
  • Sterilized tungsten needles
  • 96‐well tissue culture plates
  • Sealed container (e.g., Tupperware)
  • Phase‐contrast and fluorescent microscope
  • 200‐µl pipet tips
  • Cryotubes (Nunc)
  • −20° and −80°C freezers
  • 1.5‐ml microcentrifuge tubes

Support Protocol 1: Preparation of Fly Extract

  Materials
  • Young adult flies, 2 to 3 days after emergence
  • 70% (v/v) ethanol
  • Drosophila phosphate‐buffered saline (PBS; Robb, ), sterile
  • M3 (BF) medium (see recipe; also see Cross and Sang, ), 25°C
  • Culture medium (see recipe)
  • 15‐ml conical tubes
  • Dounce homogenate
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge

Support Protocol 2: Preparation of Conditioned Medium from Embryos Overexpressing Growth Factors

  Materials
  • actinGal 4 Drosophila lines (National Institute of Genetics, Mishima, Japan)
  • UASdpp, wingless, or hedgehog Drosophila lines (Bloomington fly stock center)
  • 35‐mm culture dish filled with 10% agar pasted with “microwaved” yeast (see recipe)
  • Saponated cresol solution (Japanese Pharmacopoeia)
  • 2.5% sodium hypochlorite (NaOCl)
  • 0.1% Triton X100/PBS (PBT)
  • Culture medium (see recipe)
  • 15‐ml conical tube with bottom cut and stainless steel mesh attached
  • 1.5‐ml microcentrifuge tubes
  • Microhomogenizer
  • 100‐ to 150‐µm nylon mesh
  • 48‐ or 96‐well culture plates
  • 15‐ml conical tubes
  • 0.22‐µm syringe filter

Support Protocol 3: Fixation and Staining of Cultured Drosophila Cells

  Materials
  • Cultures of Drosophila cells
  • Ice
  • 4% (w/v) paraformaldehyde in PBS
  • 0.1% Triton X100 in PBS (PBT)
  • Primary antibodies
    • Rabbit anti‐Vasa antibody (S. Kobayashi, National Institute for Basic Biology, and A. Nakamura, Riken Center for Developmental Biology) at a 1:250 dilution
    • Rabbit anti‐Spectrin antibody (R. Dubreuil, University of Illinois‐Chicago) at a 1:1000 dilution
    • Mouse anti‐Fusome antibody (F. Maruo, Tsukuba University) at a 1:3 dilution
    • Mouse anti‐GFP antibody (Molecular Probes)
    • Mouse anti‐Drosophila Dpp antibody (R&D systems, Lot number FRW02) at 1:100 to 1:200 dilutions
    • Mouse anti‐Fasciclin III (FasIII) antibody at a 1:50 dilution (Developmental Studies Hybridoma Bank, Iowa University)
  • Secondary antibodies:
    • FITC‐ or TRITC‐conjugated goat anti‐rabbit IgG or Alexa Fluor 546 (Molecular Probes) at 1:100 or 1:200–1:500 dilution in 5% (w/v) BSA
    • FITC‐ or TRITC‐conjugated goat anti‐mouse IgG or Alexa Fluor 488 (Molecular Probes) at 1:100 or 1:200–1:500 dilution in 5% (w/v) BSA
  • Hoescht 33258 or 33342
  • 50% glycerol in PBS or Aqua Poly‐Mount (Polysciences)
  • Appropriate microscopes
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Figures

Videos

Literature Cited

Literature Cited
   Cross, D.P. and Sang, J.H. 1978. Cell culture of individual Drosophila embryos. I. Development of wild‐type cultures. J. Embryol. Exp. Morphol. 45:161‐172.
   Currie, D.A., Milner, M.J., and Evans, C.W. 1988. The growth and differentiation in vitro leg and wing imaginal disc cells from Drosophila melanogaster. Development 102:805‐814.
   Fuller, M.T. and Spradling, A.C. 2007. Male and female Drosophila germline stem cells: Two versions of immortality. Science 316:402‐404.
   Kanatsu‐Shinohara, M., Ogonuki, N., Inoue, K., Miki, H., Ogura, A., Toyokuni, S., and Shinohara, T. 2003. Long‐term proliferation in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 69:612‐616.
   Lau, N.C., Robine, N., Martin, R., Chung, W., Niki, Y., Berezikov, E., and Lai, E.C. 2009. Abundant primary piRNAs, endo‐siRNAs and microRNAs in a Drosophila ovary cell line. Genome Res. 2009 Jul 14. [Epub ahead of print].
   Matsui, Y., Zsebo, K., and Hogan, B.L. 1992. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70:841‐847.
   Niki, Y. 2008. In vitro approach of germline stem cells in fly and mouse. In Stem Cell Applications in Disease and Health (W.B. Burnsides and R.H. Ellsley, eds.) pp. 127‐149. Nova Science Publishers, New York.
   Niki, Y. and Mahowald, A.P. 2003. Ovarian cystocytes can repopulate the embryonic germline and produce functional gametes. Proc. Natl. Acad. Sci. U.S.A. 100:14042‐14045.
   Niki, Y., Yamaguchi, F., and Mahowald, A.P. 2006. Establishment of stable cell lines of Drosophila germ‐line stem cells. Proc. Natl. Acad. Sci. U.S.A. 103:16325‐16330.
   Robb, J.A. 1969. Maintenance of imaginal discs of Drosophila melanogaster in chemically defined media. J. Cell Biol. 41:876‐885.
   Ueda, R., Ui‐Tei, K., Roberts, J., and Cherbas, L. 2007. Standard protocol for establishing cell lines from Drosophila embryos. CGB Technical Report 2007‐04.
   Ui, K., Ueda, R., and Miyake, T. 1987. Cell lines from imaginal discs of Drosophila melanogaster. In Vitro Cell. Dev. Biol. 23:707‐711.
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