Knockdown of TauT Expression Impairs Human Embryonic Kidney 293 Cell Development - Advances in Experimental Medicine and Biology - page 288

(Invitrogen) supplemented with 10% fetal bovine serum (Gibco), 50 units/ml peni-
cillin, and 50 mg/ml streptomycin. The 293 cells were maintained in a humidified
37°C incubator with 5% CO 2 , fed every 3 days with a complete medium, and sub-
cultured when confluence was reached.

28.2.2 Generation of TauT-De fi cient 293 Cell Line

A TauT knockdown cell line was created by the stable transfection of a SureSilencing
shRNA plasmid DNA (5¢ -ggcatcaagttctatctgtat-3 ¢ and 5¢ -ggaatctcattcgatgcatac-3 ¢ )
into 293 cells. Cells (3 × 10 4 ) were seeded in a 6-well plate with 2 ml of growth medium
and cultured for 24 h. To facilitate the transfection process, 1 mg of TauT shRNA plas-
mid was added (using the shRNA plasmid as a negative control) to separate 50 m l
aliquots of Opti-MEM I Reduced-Serum Medium (Gibco). Lipofectamine2000 (1 m l)
was added to the 50 ml Opti-MEM solution and incubated for 10 min at room tempera-
ture. Fifty ml Lipofectamine2000 mixture was combined with 50 ml of each shRNA
mix and incubated for another 20 min at room temperature, then added to the cells,
and incubated at 37°C in a CO 2 incubator for 24-96 h. For colony selection, G418
(3 mg/ml) was added to the cells. The medium was changed every 2-3 days for up to
2 weeks or until drug-resistant clones appeared in the transfected and selected plates.
The concentration of G418 used for colony selection was determined using mock 293
cells. The drug-resistant clones were confirmed by an immunohistochemistry analysis
of TauT expression and taurine uptake assay.

28.2.3 Measurement of Taurine Transport

Taurine transport studies were performed on confluent monolayers 3 days after
seeding cells. The cells were rinsed with Earle's balanced salt solution (EBSS) at
37°C. Uptake was initiated by the addition of an uptake buffer (2 mM KCl, 1 mM
MgCl 2 , 96 mM NaCl, 1.8 mM CaCl 2 , 5 mM Hepes, pH 7.6) to which 50 m M unla-
belled taurine and 0.5 m Ci/ml 14 C-taurine (Perkin Elmer, Boston, MA) were added.
After a 30-min period of incubation at room temperature, uptake was terminated by
the removal of uptake buffer followed by three rapid washes with cold EBSS. Cells
were dissolved in 1% SDS in 0.2N NaOH and radioactivity counted in a Packard
2000-CA Liquid Scintillation Analyzer.

28.2.4 Construction of the Reporter Gene

The promoter region of TauT was identified in previous studies (Han et al. 2000a ) ,
and a p53-binding consensus site was found in the TauT promoter sequence, located