Preparation of Stable HEK293 Cell Lines expressing
GABAARs
To test the target selectivity of α5-SOP002, a stable cell line of
HEK293 cells expressing α5β2γ2 subunits of the GABAAR
was developed using the previously established method based on
antibiotic selection (Brown et al., 2016). HEK293 cells (2 × 106) were
transfected using Lipofectamine LTX (catalog no. 15338-100, Invitrogen)
with the α5 pcDNA3.1(+) construct, incorporating the G418 disulfate
(Neomycin) resistance gene and β2 pcDNA3.1(+) construct, incorporating
the Zeocin resistance gene. Cells were subsequently plated at the ratios
of 1:3, 1:5, 1:7, 1:10, 1:15, and 1:20, and selected with G418
(Neomycin; catalog no. G5013, Sigma) and Zeocin (catalog no. R25001
Gibco) antibiotics (both at 800 μg/ml) until colonies were formed. After
7 days, ∼5–20 single colonies were selected and gradually scaled up.
The clone expressing the highest level of GABAAR α5 and
β2 subunits, as well as the previously established α2β2-HEK293 (Brown et
al., 2016) stable cell line were further transfected with the γ2
pcDNA3.1(+) construct, incorporating the Hygromycin
resistance gene, in order to produce triple cell lines. Expression of
all three subunits was characterised by immunoblotting and
immunocytochemistry. The α1β2γ2-HEK293 was characterised previously
(Fuchs et al., 2013).