National Avian Research Facility
National Avian Research Facility

Roslin Green (Cytoplasmic GFP)

Ubiquitous cell expression of cytoplasmic Green Fluorescent Protein (eGFP)

GFP chick and wild type chick
Cytoplasmic GFP chick on right

Summary

The Roslin Green (Cytoplasmic GFP) chicken line is a powerful tool for developmental biology, facilitating fate mapping, cell lineage and tissue grafting. GFP is expressed ubiquitously in early embryos and extra embryonic tissues. Roslin Green has been used to understand the embryonic origin of adult tissues and structures, including limb development1,2, heart muscle3, brain structures4 and neurons5. The Roslin Green line has also provided insights into the regulatory mechanisms that control embryogenesis6–8. In addition, Roslin Green eggs have been used to show that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone9,10.

 

Utility

Embryos from this line have been used as donors in transplantation experiments for cell lineage analysis11, by following the methods originally developed by Le Douarin et al. for transplanting quail cells into chick embryo hosts12. Chick-chick transplantation avoids issues that may result from differences between quail and chick and has the major advantage that the graft can be imaged immediately after transplantation and visualised in vivo. McGrew et al. (2008) have also shown that serial transplantation is possible13. Furthermore, embryos can undergo grafting and be taken through embryonic development14, and from hatch to sexual maturity to analyse long-term development of grafted cells.

The distribution of expression in older embryos and birds appears to be widespread but has not been systematically characterised. Confounding issues can arise during visualisation because of autofluorescence of tissues, which need to be distinguished from GFP when imaging.

 

Line origin

The Roslin Green line is well-established, generated at the Roslin Institute by Professor Helen Sang’s group, and was originally described in McGrew et al. (2008). The Roslin Green line was industrially funded, and generated by using an EIAV lentiviral vector to deliver a transgene consisting of the CAGGs enhancer/promoter developed by Niwa et al. (1991), driving expression of eGFP (CMV enhancer/chicken beta-actin promoter)15.

 

Cytoplasmic - GFP
From McGrew et al. (2008). GFP fluorescence in CAG-GFP transgenic chickens. (A) The provirus is flanked by self-inactivating LTRs (ΔLTR), and contains the virus packaging site (ψ) and a neomycin resistance open reading frame 5’ to the CAG-eGFP transgene. (B-E) GFP fluorescence in transgenic embryos at (B) new laid egg stage, (C) stage 4 HH, (D) stage 15 HH and (E) 5 days (transgenic on left). (F,G) Transverse sections of (F) stage 15 HH GFP+ and control embryos, and (G) day 5 GFP+ and control embryos. (H) Stage 13 HH embryos hybridised for GFP (transgenic on left). (I) Flow cytometric analysis of GFP fluorescence (stage 11 HH embryos). Black line, non-transgenic embryo; blue and green lines, transgenic lines 158 and 205, respectively. Scale bars: 0.5 mm.

 

For publications please reference McGrew, M. J. et al. Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity. Development 135, 2289–2299 (2008).

Publications

  1. Dunn, I. C. et al. The chicken polydactyly (Po) locus causes allelic imbalance and ectopic expression of Shh during limb development. Dev. Dyn. 240, 1163–1172 (2011).
  2. Fisher, M., Downie, H., Welten, M., Delgado, I. & Bain, A. Comparative analysis of 3D expression patterns of transcription factor genes and digit fate maps in the developing chick wing. PLoS One 6, 18661 (2011).
  3. Camp, E., Dietrich, S. & Münsterberg, A. Fate mapping identifies the origin of SHF/AHF progenitors in the chick primitive streak. PLoS One 7, (2012).
  4. Pearson, C. A. et al. FGF-dependent midline-derived progenitor cells in hypothalamic infundibular development. Development 138, 2613–2624 (2011).
  5. Sabado, V., Barraud, P., Baker, C. V. H. & Streit, A. Specification of GnRH-1 neurons by antagonistic FGF and retinoic acid signaling. Dev. Biol. 362, 254–262 (2012).
  6. Anderson, C. et al. A strategy to discover new organizers identifies a putative heart organizer. Nat. Commun. 7, 1–9 (2016).
  7. Towers, M., Signolet, J., Sherman, A., Sang, H. & Tickle, C. Insights into bird wing evolution and digit specification from polarizing region fate maps. Nat. Commun. 2, 1–7 (2011).
  8. Tschopp, P. et al. A relative shift in cloacal location repositions external genitalia in amniote evolution. Nature 516, 391–394 (2014).
  9. Moreno-Jiménez, I. et al. Remodelling of human bone on the chorioallantoic membrane of the chicken egg: De novo bone formation and resorption. J. Tissue Eng. Regen. Med. 12, 1877–1890 (2018).
  10. Moreno-Jiménez, I. et al. The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: A refinement animal model for tissue engineering. Sci. Rep. 6, 1–12 (2016).
  11. Davey, M. G., Balic, A., Rainger, J., Sang, H. M. & McGrew, M. J. Illuminating the chicken model through genetic modification. Int. J. Dev. Biol. 62, 257–264 (2018).
  12. Balaban, E., Teillet, M. A. & Douarin, N. L. E. Application of the quail-chick chimera system to the study of brain development and behavior. Science (80-. ). 241, 1339–1342 (1988).
  13. McGrew, M. J. et al. Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity. Development 135, 2289–2299 (2008).
  14. Zhao, D. et al. Somatic sex identity is cell autonomous in the chicken. Nature 464, 237–242 (2010).
  15. Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).