National Avian Research Facility
National Avian Research Facility

Membrane GFP

Ubiquitous membrane bound expression of Green Fluorescent Protein (eGFP)

Summary & Utility

Membrane GFP is expressed ubiquitously in the cell membranes of all cells in the embryonic and extra embryonic tissues, allowing detailed characterization of cell behaviours. The Membrane GFP line is a powerful tool for tracking cell movements, cell boundaries, cell lineage and cell shape changes in vivo (with live imaging) and in fixed tissues. Membrane GFP has provided insight into primitive steak formation1, early embryonic development more broadly2–4, and the process of eye development during embryogenesis5.

 

Line origin

The Membrane GFP (memGFP) line was generated at Roslin Institute by Professor Helen Sang’s group, funded by BBSRC. Briefly, a lentiviral expression vector for ubiquitous expression of Myr-EGFP was generated containing the amino-terminal myristoylation/palmitoylation sequence MGCGCSSHPED. The CAG promoter/enhancer (CMV-IE enhancer fused to the chicken β-actin promoter/1st intron) was fused to the Myr-EGFP lentiviral expression vector. Viral titres were used to generate chicks with transgene insertion. Southern blotting was used to confirm each transgenic chick had a single integrated copy of the Myr-GFP expression vector. A transgenic line was established from one of these chicks. All fertile eggs from this line are hemizygous for Myr-EGFP.

 

MemGFP chick optic fissure
MemGFP used to reveal optic fissure fusion dynamics in the chick eye. A) Whole mounted image of memGFP chicken embryo (HH ~ st26); inset - dissected eye showing optic fissure margin and the ventral region (arrows). B) Confocal optical section brightfield image of chick optic fissure at point depicted by arrows in A. C) Fluorescent image of same memGFP chick optic fissure used to distinguish fusion point.

 

For publications please reference; Rozbicki, E, et al. "Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation." Nature cell biology 17(4), 397-408 (2015).

 

Publications

  1. Rozbicki, E. et al. Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation. Nat. Cell Biol. 17, 397–408 (2015).
  2. Firmino, J., Rocancourt, D., Saadaoui, M., Moreau, C. & Gros, J. Cell division drives epithelial cell rearrangements during gastrulation in chick. Dev. Cell 36, 249–261 (2016).
  3. Ferro, V., Chuai, M., McGloin, D. & Weijer, C. J. Measurement of junctional tension in epithelial cells at the onset of primitive streak formation in the chick embryo via non-destructive optical manipulation. Development 147, (2020).
  4. Wood, T. R. et al. Neuromesodermal progenitors separate the axial stem zones while producing few single- and dual-fated descendants. BioRxiv (2019). doi:10.1101/622571
  5. Hardy, H. et al. Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential mediator of epithelial fusion. Elife 8, (2019).