Jos Jansen

32 Chapter 2 Cloning Studies CCDC115 Wild-Type Sequence in pLIB-GSKBrd for Transfection in Skin-Derived Fibroblasts A retroviral pLIB construct was purchased from Clontech, and a PGK-Blasticidin resistant cassette was introduced to create a pLIB-PGKBsr vector. Human CCDC115 was then cloned into this vector. Skin fibroblasts from individual F1-II4 were transfected with either pLIB2-pgkBsr construct (empty vector) or pLIB2- CCDC115 -PGKBsr construct. pcDNA3.1-CCDC115-V5-His for Transfection in HeLa cells CCDC115 cDNAwasobtainedfromhealthycontrolfibroblastswiththeTranscriptor First Strand cDNA Synthesis Kit (Roche) and primers spanning the whole cDNA (see Table S1 for primer sequences). cDNA was sequenced and cloned into the mammalian expression vector pcDNA3.1_V5_HisTOPO-TA (LifeTechnologies). The construct was checked via Sanger sequencing and transformed in competent E. coli. DNA was extracted with the Birnboim method and checked for correct placement of the CCDC115 strand.(20) Plasmid purification was done with the Plasmid Midi Kit (QIAGEN) according to the manual. Transfection was done with FuGENE HD Transfection Reagent (Promega) on coverslips coated with poly-LLysine (Sigma) and incubated overnight in DMEM (GIBCO) with 30%–50% confluency for immunofluorescence studies. Immunofluorescence 48 hr. after transfection of HeLa cells with pcDNA3.1- CCDC115 -V5-His, cells were fixed with 3.7% paraformaldehyde (PFA) for 12 min, washed three times in PBS, and permeabilized with 0.1% Triton in 3% BSA/13 PBS at 4°C for 10 min. After washing in PBS, cells were blocked for 30 min with 3% BSA/13 PBS solution. Primary antibodies were diluted in 3% BSA/13 PBS. Cells were incubated with primary antibody in a wet environment for 1 hr. at room temperature (RT) and, after washing with PBS, incubated for 1.5 hr. with secondary antibodies in 3% BSA/13 PBS. After washing the cells three times with PBS and once with distilled water, they were mounted on a slide with Prolong Gold antifade with DAPI (Life Technologies) and left to dry at RT for at least 24 hr. Under identical settings, the cells were visualized with a confocal Leica SP8 (Leica Microsystems) with 603 water immersion and 1.2 NA objective. Picture processing was done with ImageJ