Establishment of lentiviral cell lines and transient transfections
shRNA constructs targeting the coil-coiled domain 3 (sh1) and 5 (sh2) of the canine RPGRIP1L mRNA sequences (sh1: 5′-GGATAGAATTAATGATTTA-3′, sh2: 5′-GCAAGATTATGAACTCAAA-3′) were designed and cloned into the lentiviral pSICOR vector, lentiviruses were produced in HEK293T cells, and MDCK type II cells were infected, as described previously (Delous et al., 2009). MDCK control and RPGRIP1L-KD cells were transduced with human full-length NPHP4-V5 C-terminal (Burcklé et al., 2011) and human full-length RPGRIP1L-Myc C-terminal cDNAs both cloned in pRRL.SIN.cPPT.PGK/WPRE vector (Zufferey et al., 1998). HEK293T cell transfections were performed as described previously (Delous et al., 2009). inv-Flag was provided by I. Drummond (Harvard Medical School, Charlestown, MA); inv-GFP was provided by A. Benmerah (INSERM U1016, Paris, France); Dvl2-Myc was provided by S. Sokol (Mount Sinai School of Medicine, New York, NY). In cotransfection experiments with partial Rpgrip1l-Flag N-terminal (Rpgrip1l-Flag, Rpgrip1l-T615P-Flag and Rpgrip1l-T677I-Flag were in pCMV3-Flag described previously in Delous et al. [2007]) and Dvl2-Myc, or with inv-Flag and NPHP4-V5, 55–72% of the cells were cotransfected.