Sfold (http://sfold.wadsworth.org/cgi-bin/index.pl ) and siDESIGN center (http://www.thermoscientificbio.com/design-center/ ) were used to identify siRNAs targeting canine hepsin and prostasin. Selected oligos (sense sequence) are listed in Table 2 . Oligonucleotides for shRNA strategy were designed combining the siRNA sequence (sense) followed by a loop (sequence 5’-3’: TTCAAGAGA), the reverse complement of the siRNA sequence (antisense) and RNA polymerase III terminator (sequence 5’-3’: TTTTTTGGAA). Oligos were phosphorylated, annealed and inserted in pENTR/pTER+, a gift from Eric Campeau (plasmid # 17453, Addgene, Cambridge, MA) (Campeau et al., 2009 (link)). MDCK cells stably expressing uromodulin were co-transfected with pENTR/pTER+ vector and EGFP-expressing vector pcDNA3x(+)MyEGFP (Life Technologies). Cells were collected 24 hr after transfection and sorted using MoFlo sorter (Beckman Coulter, Brea, CA). Recovered EGFP-positive cells were maintained in culture for 48 hr.10.7554/eLife.08887.027
Oligonucleotide sequences used to generate shRNAs.
| Target gene | Target position from ATG codon | siRNA oligo (5’-3’) |
|---|---|---|
| HPN_siRNA#1 | 122-140 | CCTTCCTACTCAAGAGTGA |
| HPN_siRNA#2 | 1195-1213 | TGGATCTTCCAGGCCATAA |
| PRSS8_siRNA#1 | 258-276 | GAAGGAAGACTATGAGGTA |
| PRSS8_siRNA#2 | 593-611 | TGTACAACATCAACGCTAA |
| Control siRNA | NA | TAGTGAGATTCGTTAAGAT |
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