Transgenic mice expressing a pH and Cl- sensing yellow-fluorescent protein under the control of a potassium channel promoter.
During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca2+, Cl-, or transmembrane voltage have been developed. These sensors are based on variants of green-fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow-fluorescent protein (EYFP), a yellow-green pH- and Cl- sensitive variant of GFP, under control of the Kv3.1 K+ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl- concentration using imaging techniques and conditions analogous to those used with conventional ion-sensitive dyes. We conclude that transgenic mice expressing GFP-derived sensors under the control of cell-type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.
Metzger
Friedrich
F
Laboratory for Neuronal Circuit Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
Repunte-Canonigo
Vez
V
Matsushita
Shinichi
S
Akemann
Walther
W
Diez-Garcia
Javier
J
Ho
Chi Shun
CS
Iwasato
Takuji
T
Grandes
Pedro
P
Itohara
Shigeyoshi
S
Joho
Rolf H
RH
Knöpfel
Thomas
T
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis*; D001921:Brain / Q000033:anatomy & histology; D001923:Brain Chemistry / Q000235:genetics*; D002712:Chlorides / Q000378:metabolism*; D018755:GABA Agonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000096:biosynthesis*; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D009118:Muscimol / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics*; D024642:Potassium Channels, Voltage-Gated; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051670:Shaw Potassium Channels
D001426:Bacterial Proteins; D002712:Chlorides; D018755:GABA Agonists; C490839:Kcnc1 protein, mouse; D008164:Luminescent Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D051670:Shaw Potassium Channels; C054562:yellow fluorescent protein, Bacteria; D009118:Muscimol; D018698:Glutamic Acid