Name 3F 4F 5F 9F 8F 7F 6F 11F HHL-685Sac HHL-306Nhe HHL-79Kpn HHL-36Kpn HHL+13Xba HHL-10Kfw HHL-10Krev HHL-14Kfw HHL-14Krev HHL-22Kfw HHL-22Krev CATREV2d T3primere BMC Genomics 2007, 8:99 http://www.biomedcentral.com/1471-2164/8/99 Table 2: Oligonucleotides used to generate serial 5'-deletions of the rat and human HL promoter region. species and orientationa positionb 5'→3' sequencec r r r r r r r r h h h h h h h h h h h - - F F F F F F F F F F F F R F R F R F R R R -1697/-1675 -1048/-1030 -754/-736 -211/-196 -86/-71 -75/-58 -39/-22 -23/-8 -685/-667 -306/-291 -79/-55 -36/-20 +13/-6 -10457/-10437 -9956/-9975 -14202/-14183 -13524/-13549 -21929/-21909 -21543/-21562 cggaagc TTA GCA GAC AGC GAT TGG C cggaag CTT GCC TCC TCC TGA GTG C cggaagc TTG TCC AGG GCG TCC ATA C cggaagctt AGC TTG GCT CAA AAG G cggaagctt GTG TTC AAA TAC TGG G cggaagct TGG GTA ACA TGT TTT AGG cggaag CTT CCA CAA CTA AAT ACC cggaagctt CCA AGA AGC ATT CTG G ccgagctc TGG TCG CCT TTT CCC TAC C gcatgctagc GAA GCC ACC TAC CCC G ggggtacc TAA CAT GTT GAG AGG ggggtac CAA AGT ATC TAA TAG GC gctctaga CTT GGT AAT TTC TGA AGC C gtcgac GAA GGA TCA GGT GAG GGA TGG ggatcc GCT TCA AGG GCA ATG AAA GC gtcgac GAC TTG GGG ATA TCC ACA TC ggatc CTG GTA AAA GGA CAT GAA CAA TAT GG gtcgac GAT GGG AAA TGG ACC TAC AGC ggatcc GAT GAG GAC TGA TTC TCA GC gca act gac tga aat gcc tc att aac cct cac taa ag a: r and h: rat and human, respectively; F and R: forward and reverse orientation. b: numbering of the rat and human sequence according to [35] and Ensembl e!42 [26], respectively. c: rat and human HL specific parts of the primer sequences are given in capitals. d: oligonucleotide specific for pCAT-Basic. e: oligonucleotide specific for pBluescript. genomic DNA was isolated from a buffy coat, and the -14 kb, -22 kb and -10 kb elements were PCR amplified using specific primers (Table 2). The PCR products were cloned into the pGEM T-easy vector (ProMega, Madison, WI, USA). After digestion of the plasmids with BamHI and SalI, the inserts were cloned into hHL-685Luc in either sense or anti-sense orientation. All clones were verified by DNA sequencing using the Thermo-sequenase dye terminator kit (Amersham, UK) and the ABI 377 sequencer (Applied Biosystems, Foster City, CA, USA). Promoter reporter assays HepG2 hepatoma cells and HeLa cells were cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (ICN, Costa Mesa, CA, USA) supplemented with 10% (v/v) fetal calf serum (Gibco, Breda, Netherlands) and penicillin/streptomycin. Transfection of HepG2 cells with CAT-reporter constructs was performed by the cal- cium-phosphate co-precipitation method. At 24 h before transfection, the cells were plated in 6-wells plates at 20– 30 % confluence. At 3 h before transfection, the medium was refreshed. Cells were co-transfected with 2.5 μg/well of the CAT reporter test plasmid and 0.2 μg/well of control RSV β-galactosidase expression plasmid (Promega) [36]. Parallel transfections with SV40-CAT-Control and empty pCAT-Basic plasmids were used as controls. Fourty-eight hours post-transfection, cell lysates were prepared. CAT and β-galactosidase were determined by ELISA (Roche). Promoter activity was expressed as pg CAT/ng β-galactosi- dase to correct for differences in cell number and transfec- tion efficiency. Transfections of HepG2 and HeLa cells with the luci- ferase-reporter constructs were performed in 24-wells plates with Lipofectamine Plus (Invitrogen, Groningen, Netherlands) using 0.4 μg of the luciferase-reporter con- struct and 20 ng of pRL-CMV (Promega) per well [37]. Cell extracts were prepared at 48 h post-transfection. The luciferase activity in the cell extracts was determined with the FireLight kit (Perkin-Elmer, Boston MA, USA) and the Packard Top Count NXT luminometer. Data were normal- ized for the Renilla activity measured in the same sample. Statistics Experimental data are expressed as mean ± SD. Differ- ences were tested for statistical significance by paired Stu- dent's t-test. List of abbreviations CAT, chloramphenicol acetyltransferase; HDL, high-den- sity lipoprotein; HL, hepatic lipase; HNF, hepatocyte Page 12 of 13 (page number not for citation purposes)