4.2. Plasmids

The Car9 promoter construct was generated by insertion of −191/+47 Car9 genomic region amplified by PCR upstream of the firefly luciferase gene into pGL3-Basic luciferase reporter vector (Promega, Madison WI, USA). The generation of human CA9 promoter construct pGL3-CA9 was described previously [47]. pRL-TK Renilla vector served for the control of transfection efficiency.

HRE-, SP1-, and AP1-binding sites in the pGL3-Car9 promoter construct were mutated using certain mutagenic oligonucleotides and their reverse counterparts (summarized in Table 1) through PCR-based in vitro mutagenesis. The mutated fragments were amplified in the second round of PCR using pGL3-basic specific primers, digested with SacI and XhoI, and inserted into pGL3-basic. A deletion construct lacking the PR4 region was prepared by the amplification of the construct containing −191/+47 fragment in pGL3 by inverse PCR, using sense and antisense primers from the downstream and upstream PRs, respectively. The resulting PCR products were gel-purified, phosphorylated, and ligated. The resulting mutations and deletions were verified by sequencing.

Sequences of the oligonucleotides used in this study. Sequences of the strands are written in the 5′ -> 3′ direction. Mutations against the wild-type sequence are indicated in bold.

The eukaryotic expression plasmids pSG5C-Car9 and pSG5C-CA9 were described previously [18,48]. The generation of mCA IX-expressing NIH3T3 and MDCK cells was described previously [18].