Mouse gα15 pcmv6plasmid

In order
to generate a tetracycline (Tet) inducible rat mGlu₃ stable
cell line to be used for a calcium mobilizaion assay, TREx293 cells
(Invitrogen) were transfected with mouse G_α15-pCMV6
plasmid (Origene) using Fugene6 (Promega). The cells were selected
for G_α15 expression with 1 mg/mL G418 in the presence
of 10 μg/mL blasticidins to maintain Tet repressor expression.
Two weeks after the selection, polyclonal TREx293 G_α15 cells were obtained. The entire coding sequence of rat mGlu₃ was amplified by polymerase chain reaction (PCR) and cloned
into the Tet-inducible expression plasmid pcDNA5/TO (Invitrogen).
Rat mGlu₃-pcDNA5/TO was transfected into TREx293 G_α15 cells and selected for mGlu₃ expression
with 200 μg/mL hygromycin in the presence of G418 and blasticidins.
The resulting polyclonal TREx293 mGlu₃ G_α15 cells were plated for monoclonal selection, and positive monoclones
were identified in the calcium mobilization assay. Cells were maintained
in growth medium containing DMEM, 10% Tet-tested FBS (Atlanta Biogicals),
20 mM HEPES, 2 mM l-glutamine, antibiotic/antimycotic, nonessential
amino acids, 500 μg/mL G418, 100 μg/mL hygromycin, and
5 μg/mL blasticidin S at 37 °C in the presence of 5% CO₂.

In order to generate a rat mGlu₂ stable cell line to
be used for a calcium mobilization assay, HEK293A cells (ATCC) were
transfected with mouse G_α15-pCMV6 plasmid (Origene)
using Fugene6 (Promega). The cells were selected for G_α15 expression with 1 mg/mL G418. Two weeks after the selection, polyclonal
HEK293A G_α15 cells were obtained. The entire coding
sequence of rat mGlu₂ was amplified by PCR and cloned into
the expression plasmid pIRESpuro3 (Invitrogen). Rat mGlu₂-pIRESpuro3 was transfected into HEK293A G_α15 cells
and selected for mGlu₂ expression with 0.6 μg/mL
puromycin in the presence of G418. The resulting polyclonal HEK293A
mGlu₂ G_α15 cells were then utilized for
calcium mobilization assays. Cells were maintained in growth medium
containing DMEM, 10% FBS, 20 mM HEPES, 2 mM l-glutamine,
antibiotic/antimycotic, nonessential amino acids, 700 μg/mL
G418, and 0.6 μg/mL puromycin at 37 °C in the presence
of 5% CO₂.