RT-qPCR and shRNA-Lentivirus

Primers used for RT-qPCR were selected in order to include at least one intron in the amplicon, no match with other murine genes by standard blast, no genomic amplification and for identical amplification efficiency. Results shown in Fig. 3b were obtained from samples of 50 sorted cells, using the single cell RT-qPCR techniques we described previously [23]. Oligonucleotide sequences are shown in Additional file 3: Materials.

For the production of lentivirus vectors we used the pLV-HTM plasmid (kindly provided by Dr. Trono Didier, Geneva University) which is a self-inactivation third generation HIV1-derived vector [24]. The annealed oligonucleotides coding for shRNA were ligated into ClaI and MluI double-restricted plasmid by standard cloning procedures, using restriction enzymes and T4 DNA ligase (New England BioLabs Inc). In these conditions, the production of siRNA is under the control of H1 (RNA polymerase type III) promoter, and the reporter gene, the green fluorescent protein (EGFP) is expressed under the control of eukaryotic EF-1α promoter. All plasmids were verified by sequence analysis. Lentivirus particles were produced by transient transfection of 293 T cells according standard protocols. Briefly, subconfluent 293 T cells were co-transfected with 20 μg of plasmid vector, 15 μg of pCMVR8.74 and 5 μg of pMD.G envelope (VSVG) by calcium phosphate method. After overnight incubation, medium was changed for a fresh one and supernatants were harvested at 48 and 72 h. Supernatants were further concentrated (100X) by ultracentrifugation, titrated and stored at −80 °C until use. Viral titers expressed as TU/ml were determined by assessing transduction of 293 T cells with serial dilutions of virion preparations. Batches with titers ≥10⁸ TU/ml were used.