Analysis of cerebellar Purkinje cells using EAAT4 glutamate transporter promoter reporter in mice generated via bacterial artificial chromosome-mediated transgenesis.
The EAAT4 glutamate transporter helps regulate excitatory neurotransmission and prevents glutamate-mediated excitotoxicity in the cerebellum. Immunohistochemistry and in situ hybridization have previously defined a cerebellar cell population expressing this protein. These methods, however, are not well suited for evaluating the dynamic regulation of the transporter and its gene-especially in living tissues. To better study EAAT4 expression and regulation, we generated bacterial artificial chromosome (BAC) promoter eGFP reporter transgenic mice. Histological analysis of the transgenic mice revealed that the EAAT4 promoter is active predominantly in Purkinje cells, but can also be modestly detected in other neurons early postnatally. EAAT4 promoter activity was not present in non-neuronal cells. Cerebellar organotypic slice cultures prepared from BAC transgenic mice provided a unique reagent to study transporter and Purkinje cell expression and regulation in living tissue. The correlation of promoter activity to protein expression makes the EAAT4 BAC promoter reporter a valuable tool to study regulation of EAAT4 expression.
Gincel
Dan
D
Department of Neurology, Johns Hopkins University, Baltimore, MD 21287, USA.
Regan
Melissa R
MR
Jin
Lin
L
Watkins
Andrew M
AM
Bergles
Dwight E
DE
Rothstein
Jeffrey D
JD
D000818:Animals; D002525:Cerebellar Cortex / Q000166:cytology / Q000378:metabolism*; D002876:Chromosomes, Bacterial / Q000235:genetics; D050581:Excitatory Amino Acid Transporter 4 / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008967:Molecular Biology / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics; D011689:Purkinje Cells / Q000166:cytology / Q000378:metabolism*; D009435:Synaptic Transmission / Q000235:genetics*
D050581:Excitatory Amino Acid Transporter 4; C493390:Slc1a6 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid