In vivo imaging and differential localization of lipid‐modified GFP‐variant fusions in embryonic stem cells and mice

Abstract

SummaryThe visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice. Here we report the generation of two strains of mice, each expressing a spectrally distinct lipid-modified GFP-variant fluorescent protein fusion. The CAG::GFP-GPI strain exhibited widespread expression of a glycosylphosphatidylinositol-tagged green fluorescent protein (GFP) fusion, while the CAG::myr-Venus strain exhibited widespread expression of a myristoyl-Venus yellow fluorescent protein fusion. Imaging of live transgenic embryonic stem (ES) cells, either live or fixed embryos and postnatal tissues demonstrated that glycosylphosphatidyl inositol-and myristoyl-tagged GFP-variant fusion proteins are targeted to and serve as markers of the plasma membrane. Moreover, our data suggest that these two lipid-modified protein fusions are dynamically targeted both to overlapping as well as distinct lipid-enriched compartments within cells. These transgenic strains not only represent high-contrast reporters of cell morphology and plasma membrane dynamics, but also may be used as in vivo sensors of lipid localization. Furthermore, combining these reporters with the study of mouse mutants will be a step forward in understanding the inter-and intracellular behaviors underlying morphogenesis in both normal and mutant contexts.

Adult Neurogenesis Requires Smad4-Mediated Bone Morphogenic Protein Signaling in Stem Cells

“…For transplantation experiments Myristoyl (myr)-Venus animals were used. In this mouse line, Venus protein is fused with Myristoyl protein, which is a lipid-modified protein present in plasma membrane of all cells (Rhee et al, 2006). SEZ of 6-week-old myr-Venus animals were dissected and prepared for transplantation as described previously (Seidenfaden et al, 2006;Berninger et al, 2007).…”

Regulatory Mechanisms that Mediate Tenascin C-Dependent Inhibition of Oligodendrocyte Precursor Differentiation

“…OPCs were collected by centrifugation for 5 min at 200 ϫ g. For each experimental condition, 4 -5 ϫ 10 6 cells were transfected with the Amaxa Rat Oligodendrocyte Nucleofection Kit (Lonza) according to the manufac-turer's instructions. For the knockdown experiments, 200 pmol siRNA duplexes against Cntn1, Sam68, or control RNA were cotransfected with 0.5 g pMaxGFP (Lonza) or pCX-myrVenus (Rhee et al, 2006) using electroporation program O-17. This represents a 1:5 ratio of green fluorescent protein (GFP) DNA to siRNA (in g).…”

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour <em>In Vitro</em>

“…Please click here to view a larger version of this figure. 19,22,27,30,[36][37][38][39][40] have been characterized for the formation of aggregates and, although subtle differences between cell lines are expected and have been observed, all the above lines show similar dynamics in terms of elongation and morphology. In terms of gene expression, the expression pattern is specific to the gene expressed, but within each cell line, the expression pattern is generally consistent over a number of passages.…”