primary cell culture: Passages 3 to 8 treatment: Reference control cell type: Foreskin fibroblasts
The Open Reading Frame (ORF) sequences of transgenes were isolated from reverse transcribed adult human testis total RNA (Clontech). Isolated PCR products were cloned into the lentiviral backbone pLenti7.3-V5 (Life Technologies) using the Gateway system (Life Technologies). Lentiviral packaging was generated by transient co-transfection using Lipofectamine 2000 (Life Technologies) of the 293-T packing cell line (CRL-11268, ATCC). The collected viral supernatants supplemented with 8µg/mL polybrene (Sigma Aldrich) were used in a dilution 1:2 with conventional medium for transduction. Remaining viral supernatants were washed off 24h later and GC-M was added the following day.
Briefly, conventional growth medium, DMEM/F12 supplemented with 10% FBS (Life Technologies) and 2mM L-glutamine (Millipore) was used to grow human foreskin fibroblasts (hFSK (46,XY), CRL-2429, ATTC). Induced Germ-like Cells (iGC) were obtained, cultured and maintained in germ cell medium (GC-M), consistent on DMEM/F12 supplemented with 20% KnockOut Serum Replacement (all from Life Technologies), 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol (Millipore), and supplemented with 1000U/ml human recombinant Leukemia Inhibitor Factor (LIF, Sigma Aldrich), 20ng/mL recombinant human Glial-Derived Neurotrophic Factor (GDNF, Peprotech), 5µM Forskolin (Sigma Aldrich), 10 ng/ml basic Fibroblast Growth Factor (bFGF, Life Technologies), 5ng/mL Stem Cell Factor (SCF, Peprotech) and 2µM retinoic acid (Sigma Aldrich) at 37°C, 5% CO2. All the experiments were performed at least in triplicate using low passages (up to passage 8).
Total RNA was extracted with the RNAeasy mini kit (Qiagen) according to manufacturer´s instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4845A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Hybridized microarrays were scanned in an Axon 4100A scanner (Molecular Devices, Sunnyvale, CA), and the data were extracted with the GenePix Pro 6.0 software (Molecular Devices) (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
Sample name: 251485079647_3-4 Gene expression 14 days post-transduction Replicate 3
Gene expression scanned values were preprocessed, normalized, and statistically analyzed. The half-background median intensity values were subtracted from the average intensity of each spot, and were normalized between arrays using the quantile method implemented in the Bioconductor (www.bioconductor.org) LIMMA package run under the R software (www.r-project.org). Probe sets belonging to the same gene were merged by median and transformed to the logarithmic scale (log2) giving rise to a final matrix of 25,512 genes by sample. The microarray analysis was performed using Babelomics software version 4.3 (babelomics.bioinfo.cipf.es) . Exploratory analysis of whole gene expression data was performed to check the behavior of samples using Principal Component Analysis. Functional enrichment for differential expressed genes was performed with FatiGO  a widely used SEA implementation, which is included in the Babelomics  web-based package using the Gene Ontology (GO)  and Reactome curated databases . The significant over-represented terms related to human genome comparison (adjusted p-value< 0.05) were considered. Results were validated by qPCR for three of the top up-regulated and down-regulated genes (data not shown). we applied the LIMMA test for deep screening analysis between groups (two-class comparisons), and compared the expression patterns from treated samples with i12F/i6F versus MOCK controls, obtaining adjusted p-values and fold changes for each comparison (FC higher than 2; adjusted p-value of less than 0.05); we corrected the p values using the false discovery rate accounting for multiple testing effects. Gene expression data fold change from significant genes was used to compare with RT-qPCR values (data not shown).