Retro x tet on 3g inducible expression system

Gene transfer experiments were performed using the pCX4 series of retroviral vectors.^{41 (link)} pCX4 vectors were transfected into PLT cells using FuGENE (Roche) and the culture supernatant used as the source of virus. KD of ARHGEF5 was performed using lentiviral vectors. Lentiviruses were generated from PLT cells using the MISSION Lentiviral packaging mix (Sigma-Aldrich) and FuGENE. siRNA and cDNA were transiently transfected using RNAimax and Lipofectamine 3000 (Thermo Fisher Scientific), respectively. The active form of Src (SrcF572) was introduced into mouse embryonic fibroblasts and MCF10A cells using the Retro-X Tet-On 3G Inducible Expression System (Clontech, Mountain view, CA, USA).

For rescuing the CEP44 conversion phenotype, a cell line expressing a siRNA-resistant construct was made from RPE1 wild type cells. Firstly, the Retro-X™ Tet-On® 3G Inducible Expression System (Clontech) was introduced in RPE1 wt cells. siRNA-resistant constructs of CEP44 were the integrated in RPE1 Tet3G cells under the TRE3G promoter via Retrovirus infection (Clontech) (Supplementary Table 3).
CEP44^−/− cell lines were generated via electroporation of RPE1 p53^−/− (Prof. Dr. Bryan Tsou) with pX458 cloned with the sequence for the sgRNA (Supplementary Table 3). Two days after electroporation GFP positive cells were FACS-sorted and the pool of cells obtained from the sorting was tested with antibodies against CEP44. In the knockout pool the signal of CEP44 vanished in 50% of the cells. Single clones were then tested by indirect immunofluorescence (IF), immunoblotting and chromosomal DNA sequencing to select cells in which both CEP44 alleles were successfully knocked out. Two CEP44^−/− genetically different clones were used for experiments. Clone #2 had a heterozygote CEP44 genotype (2 nt deletion on one copy and 5 nt deletion on the other copy), generating a non-sense frame shift. Clone #7 is homozygote with 2 nt deletion on both gene copies (Supplementary Fig. 3).

Clones of H1299 cells inducibly expressing miR-146a in the presence of doxycycline were generated using the Retro-X Tet-On 3G inducible expression system (Clontech). The pRetroX Tet 3G vector was used to produce retroviruses according to the manufacturer's suggested protocol. A preliminary cell line stably expressing the Tet transactivator protein (H1299-Tet) was created by transducing H1299 cells with these retroviruses and selecting with 500 μg/mL G418. A 616 bp fragment including the full miR-146a genomic sequence and flanking regions was cloned into the pRetroX TRE 3G vector using the BamHI and EcoRI sites. TRE-empty and TRE-miR-146a retroviruses were produced and used to transduce H1299-Tet cells, followed by selection with 1 μg/mL puromycin. Cells surviving both G418 and puromycin selection were diluted and plated in 96-well plates such that there was approximately one cell per well. Single clones were grown up and analyzed individually. H1299 Tet/TRE-empty and H1299 Tet/TRE-miR-146a cells were cultured, where indicated, in 1 μg/mL doxycycline. All cells were cultured in RPMI supplemented with 10% Tet System Approved FBS (Clontech) and 4 mM L-glutamine.