Imaging endoplasmic reticulum calcium with a fluorescent biosensor in transgenic mice.
The use of biosynthetic fluorescent sensors is an important new approach for imaging Ca(2+) in cells. Genetically encoded indicators based on green fluorescent protein, calmodulin, and fluorescence resonance energy transfer (FRET) have been utilized to measure Ca(2+) in nonmammalian transgenic organisms and provide information about the organization and regulation of Ca(2+) signaling events in vivo. However, expression of biosynthetic FRET-based Ca(2+) indicators in transgenic mammals has proven to be problematic. Here, we report transgenic expression of an endoplasmic reticulum (ER) Ca(2+) biosensor in mouse pancreas. We targeted expression of a yellow cameleon3.3er (YC3.3er) transgene with mouse insulin I promoter. YC3.3er protein expression was limited to pancreatic beta-cells within islets of Langerhans and absent in the exocrine pancreas and other tissues. Animals developed and matured normally; sensor expression was unaffected by age. Glucose tolerance in transgenic mice was also unaffected, indicating the transgenic biosensor did not impair endocrine pancreas function. ER Ca(2+) responses after administration of thapsigargin, carbachol, and glucose were measured in individual beta-cells of intact islets using confocal microscopy and confirmed the function of the biosensor. We conclude that controlling transgene transcription with a cell-specific promoter permits transgenic expression of FRET-based Ca(2+) sensors in mammals and that this approach will facilitate real-time optical imaging of signal transduction events in living tissues.
Hara
Manami
M
Department of Medicine MC1027, The University of Chicago, 5841 South Maryland Ave., Chicago, IL 60637, USA.
Bindokas
Vytautas
V
Lopez
James P
JP
Kaihara
Kelly
K
Landa
Luis R
LR
Harbeck
Mark
M
Roe
Michael W
MW
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis*; D003952:Diagnostic Imaging / Q000379:methods*; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D007328:Insulin / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D010179:Pancreas / Q000378:metabolism; D011401:Promoter Regions, Genetic
D007328:Insulin; D002118:Calcium