Knockdown of TauT Expression Impairs Human Embryonic Kidney 293 Cell Development - Advances in Experimental Medicine and Biology - page 289

at −663 to −695. In this study, ~1.1 kb of the TauT promoter region DNA was used
as the template for PCR (GenBank TM /EBI accession number AR151716), and the
PCR fragment was cloned into the promoter-less luciferase vector pGL3-Basic
(Promega, Madison, WI) to generate the plasmid p963 for use in transfections and
luciferase assays. The conditions used are 30 cycles of 1 min of denaturation at 94°C,
1 min of annealing at 58°C, and 1 min of elongation at 72°C. The sense primer
(5¢ -GGGGTACCTTACTGAAGGTCACACAGC-3 ¢) designed for PCR contained a
unique site for KpnI, and the antisense primer (5¢ -AAGATCTTGGCACGG
GAGTTCA-3¢) contained a unique site for BglII. PCR products were digested with
KpnI and BglII and religated into KpnI and BglII sites of pGL3-Basic to generate
plasmids containing segments of the TauT promoter sequence extending from the
+48 nucleotide corresponding to the transcriptional start site. The constructs were
verified by DNA sequencing. The p53-binding site deletion (del pGL-563) and p53
mutation (mt pGL-963) constructs were generated from the p963 plasmid by using
sense primers 5¢ -GGGGTACCGAGTTGGGGAGGGA-3 ¢ and 5¢ -GGGGTACCAG
ATGAGGAAACCCCCACACAGAAGGTCTGGGGCTTGCCTGATGTCA-3¢ ,
respectively. The antisense primer used for these constructs was the same as
described above.

28.2.5 Transient Transfection

Plasmid DNA was introduced into cultured 293 cells using cationic liposomes
(Lipofectamine). Transfection was carried out for 16-18 h, and then cells were washed
twice with phosphate-buffered saline and incubated in fresh medium for 24-48 h
before harvesting. pGL-control, which contains a luciferase gene driven by the SV40
early region promoter/enhancer, and empty pGL-Basic vectors were used as positive
and negative controls, respectively. To standardize the transfection efficiency, 0.1 m g
of pRL-CMV vector (pRL Renilla reniformis luciferase control reporter vector;
Promega) was cotransfected in all experiments. Cells were harvested 48 h after trans-
fection and lysed in 200 ml of reporter lysis buffer (Promega). A luciferase assay was
performed using a dual luciferase assay kit (Promega), and activity was measured
with an Optocomp one luminometer (MGM Instruments, Inc., Hamden, CT). Promoter
activity (mean ± SD of four samples in relative light units) of each construct is repre-
sented by relative light output normalized to pRL-CMV control. Graphs represent
typical results of four separate experiments. The concentration of protein in the cell
extracts was determined using the Bradford method (Bio-Rad, Hercules, CA).

28.2.6 Western Blot Analysis

Cells were lysed in 50 ml M-PER mammalian protein extraction reagent (Pierce, Inc.,
Rockford, IL) supplemented with a protease inhibitor cocktail for use with mammalian
cell and tissue extracts (Sigma). The lysates were cleared by centrifugation at 14,000 × g