To explore whether endogenous IMP1 regulated the production of retroviral vectors, the expression level of IMP1 was downregulated by RNA interference in the producer cells and the production of MLV-luc vector was assayed.
The apoptosis of the cells infected with miR-494-3p miR-494-3p miR-494-3pegatinhibitorol vector was assayed by staining with Annexin V-PE (BD BiorcienegativeA) and detecontrol flow cytometry.
Six colonies, harboring bait and prey vectors, were assayed for β-galactosidase activity.
Wild type and corresponding mutant NBS1 and NBS4 harboring reporter vectors were assayed for reporter gene activity by transfecting HEK293 cells in the presence of NKX3.1 expressing pcDNA-NKX3.1-HA expression vector or control pcDNA.
The tetracycline-inducible piggyBac vectors were assayed using a slightly different method in which Sf9 cells were co-transfected with the vector of interest in the presence of a helper plasmid, pBSK.Hr5IE1.rtTAM2.SV40-1.1k.ITR-3xP3-EGFP, which encodes the mutant repressor needed to induce transcription in the presence of tetracycline.
In vivo function of each of the immunosuppressant vectors was assayed by co-injection with an enhanced green fluorescent protein (EGFP -expressing first-gEGFP -expressingiral vector (AdEGFP) into the tibialis anterior muscle of C57BL/10 mice.
Colonies were picked on passage 4 or 5. Presence or absence of transgene vectors was assayed by genomic PCR for the CAG promoter and TaqMan Copy Number Assays against the blasticidin resistance gene with RNase P and TERT reference assays.
As with the level of Ser-5 phosphorylated CTD, the level of Ser-2 phosphorylated CTD was not altered when empty vector controls were assayed in repressing or non-repressing conditions (Figure 4B, bottom panel, lanes 1 4).
After 48 h of transfection with MYOGENIN containing the S160/164A mutations or empty vector, RH30 cells were assayed for their capacity to form colonies as previously described.
