Figure 2.

Insulated SIN-lentivirus vector for regulated Rlx expression (Ins-SIN-LV-Rlx). (a) Scheme of integrated provirus. The Rlx gene is under the control of a tTR-KRAB system.⁵¹ tRT-KRAB bound to tet-operator sequences represses promoters in the vicinity of 3–4 kb. Addition of Dox releases this repression. The vector also contains a central polypurine tract (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRC). A 0.4-kb cHS4 insulator element⁵² is inserted into the 398-bp U3 promoter/enhancer deletion (U3δ). Upon proviral integration into host genome, the U3 region containing the cHS4 is copied over to the 5′ LTR. (b) BT474-M1 cells were transduced with VSV-G-pseudotyped Ins-SIN-LV-Rlx at an MOI of 1. Twenty-four hours after transduction, cells were subjected to limited dilution in 96-well plates. Individual colonies were expanded and treated with Dox for 24 hours. Then, mRNA was isolated and subjected to qRT-PCR for GAPDH and Rlx mRNA. The powered δCt values represent Rlx mRNA levels compared to GAPDH mRNA levels. The right column shows the induction factor upon Dox addition. Dox, doxycycline; LTR, long-terminal repeat; MOI, multiplicity of infection; qRT-PCR, quantitative reverse transcription; Rlx, relaxin; SIN, self-inactivating.