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Contributor Information

  • Name Matthew Holley
  • Institute University of Sheffield

Tool Details

  • Tool name: GATA3eGFP reporter cell line
  • Alternate names: US/VOT- GATA3eGFP (Univ Sheffield/Ventral Otocyst – gata3-enhanced green fluorescent protein)
  • Tool type: Cell Lines
  • Organism: Mouse
  • Tissue: Embryo
  • Cancer type: Not specific
  • Disease: Development, immune system responses, adipogenesis, tumor malignancy, hearing loss, Hypoparathyroidism, Hearing Loss and Renal Anomaly (HDR) Syndrome
  • Morphology: Inner ear otocyst at embryonic day E10
  • Model: Transgeneic
  • Model description: The mice were derived from a cross between two different transgenic mouse lines. One mouse line stably expressed the H-2Kb-tsA58 transgene in which a temperature-sensitive variant of the SV40 large T-antigen was expressed under the control of an inducible promoter driven by gamma-interferon. The second, GATA3eGFP BAC-transgenic mouse line expressed eGFP under the control of the GATA3 promoter.
  • Conditional: Yes
  • Conditional description: Conditionally immortal, see production details
  • Description: The expression of eGFP correlates with that of endogenous gata3 in V2c interneurons within the spinal cord from embryonic days E10.5-11.5 [Panayi et al., 2010]. We found that in freshly dissected or unfixed, cryosectioned tissue it correlated with endogenous expression of gata3 in many other tissues throughout embryonic development. At E10.5 these included the midline dorsal aorta, mesonephric ridge, endothelial cells around the region of the neural lumen, the lens of the eye and specific neurons of the central nervous system. At E12.5 they included the vomeronasal organ and neurons in the mesencephalon, pons and the diencephalon. Images of the whole head clearly revealed expression in the olfactory region, lens, midbrain, hindbrain and spinal cord. A similar pattern was observed at E14.5, including ventral spinal cord, trigeminal ganglion, thyroid glands and tongue. At E16.5, eGFP was observed in the diencephalon, pons, spinal cord, lens, tongue, thymus, carotid arteries, olfactory region and whisker follicles.
  • Research area: Regulation of gata3 in development, cancer, immune system, deafness and in the adult function of central and peripheral nervous systems and organs including the kidneys, hair follicles and visual system.
  • Production details: Animal studies were licensed under the UK Home Office, Animals (Scientific Procedures) Act 1986, and had prior approval of The University of Sheffield Ethical Review Committee. Tissue was harvested from animals culled by a Schedule 1 method. GATA3eGFP mice were a kind gift of Dr Stavros Malos from the Cyprus Institute of Neurology and Genetics and were generated by Bacterial Artificial Chromosome (BAC) recombination [Panayi et al., 2010]. For characterization of embryos, homozygous GATA3eGFP mice were interbred and checked daily for vaginal plugs. The time of earliest detection of plugs was considered to be embryonic day E0.5 and embryos were culled for inner ear tissue at E10.5, E12.5, E14.5 and E16.5. Transgene expression was screened by shining ultraviolet light into the eyes or onto fresh, unfixed slices of tissue from ear-clips, which revealed gata3 expression in the hair follicles [Kurek 2007]. ImmortomiceĹ  (Charles River) were bred with C57BL/6 mice to generate mice heterozygous for the T-antigen. These were crossed with homozygous GATA3eGFP mice to yield embryos heterozygous for both GATA3eGFP and T-antigen. Embryos were culled at E10.5 for derivation of conditionally immortal reporter cell lines for GATA3 and cultures were screened to confirm expression of eGFP and T-antigen by fluorescence and immunofluorescence microscopy, respectively. Removal and dissection of otocysts and the subsequent primary culture and cloning of otic epithelial cells was as previously described (Lawoko et al 2004), except that the media for cloning included Ultraculture (BiowhittakerĹ˝ Lonza), GlutaMAX™ (Gibco, LifeTechnologies), 5% fetal calf serum (Invitrogen), 100U/ml murine .–interferon (PeproTech), 20nM p160ROCK inhibitor (Tocris) to enhance cell survival and a mixture of penicillin and streptomycin (Gibco). At least 3 rounds of dilution cloning were carried out to ensure clonality.

  • For Research Use Only

Target Details

Application Details

Handling

  • Growth medium: Cell culture depends on the experiment and these cells can be cultured under different conditions [eg Milo et al. 2009; Lawoko et al. 2004; Rivolta & Holley, 2002; Lawlor et al., 1999]. Under immortalising conditions they will continue to proliferate so the temperature and level of g-interferon should be managed for optimal growth. The cells can be cultured both in serum and serum free conditions. In serum they can be cultured in MEM (Invitrogen-GIBCO, Paisley, UK) and 10% fetal bovine serum (FBS) (Invitrogen-GIBCO, Paisley, UK) under immortalising conditions (33C with g-interferon (Peprotech Ltd, London UK) and under differentiating conditions (39C without g-interferon). In serum free media for neural conditions cells can be cultured in Neurobasal Medium (NM) (Invitrogen-GIBCO, Paisley, UK) with 1% L-Glutamine (Invitrogen-GIBCO, Paisley, UK) and 2.5% FBS under immortalizing condition, and then 2% B27 supplement (Invitrogen-GIBCO, Paisley, UK) under differentiating conditions. Under epithelial conditions cells can be cultured in Ultraculture Medium (Cambrex-BioWhittaker, Europe) with 1% L-Glutamine. Flasks and dishes can be coated with poly-D-lysine (Signa-Aldrich, UK) with fibronectin (Sigma-Aldrich, UK) added to the medium just before plating.

Documentation

  • Available on request

References

  •   PMID: 1711218
  •   PMID: 17151017
  •   PMID: 11135239
  •   PMID: 10531448
  •   PMID: 11793341
  •   PMID: 15499550
  •   PMID: 19774072
  •   PMID: 20844123
  •   PMID: 12100886
  •   PMID: 12382283