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.
The Membrane GFP (memGFP) line was generated at Roslin Institute by Professor Helen Sang's group, and funded by BBSRC (grant number: BB/E011276/1). 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. For welfare reasons, only heterozygous birds are maintained for this insertion, resulting in the provision of fertile eggs that are 50% memGFP (hemizygous for Myr-EGFP) and 50% wildtype.
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).
- 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).
- 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).
- 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).
- 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
- Hardy, H. et al. Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential mediator of epithelial fusion. Elife 8, (2019).