Figure 2.

Design and validation of transgenic mice that overexpress or inhibit miR-21. A, Murine primary mir-21 and miR-21 sponge (MSP) sequence was inserted into ROSA26 targeting construct 3′ to PGK-neomycin cassette containing a trimer of the SV40 polyadenylation sequence (3xpA) flanked by loxP sites. The transgene is driven by a CAG promoter with a splice acceptor site (SA). This design allows for controlled expression of primary miR-21 or MSP via recombination of loxP sites by Cre-recombinase protein. (A, AscI; E, EcoRV; P, PacI; R, EcoRI; S, SalI; Sa, SacII; X, XbaI.) See Material and Methods for transgenic mouse generation. B, MSP transcript contains seven repeats of sequence complimentary to mature miR-21. Base pair mismatch minimizes enzymatic degradation of MSP-miR-21 pair. C, Functional inhibition of mature miR-21 by MSP was tested in HEK 293T cells. Increasing amounts of pCMV-d2eGFP-miR21 (M) compared withpCMV-d2eGFP-CXCR4 (C) sequestered miR-21, allowing for increased expression of pCMV-Luc-miR21-B (firefly luciferase). (1C:0M—pCMV-d2eGFP-CXCR4 plasmid only, 0.5C:0.5M—equal amounts of pCMV-d2eGFP-CXCR4 and pCMV-d2eGFP-miR21. 0C:1M—pCMV-d2EGFP-miR21 only.) n = 3, **p < 0.01 by ANOVA, compared with 1C:0M. D, E, Southern blot analyses for 5′ integration of ROSA26 targeting construct for miR-21 and MSP mice, respectively. Five clones and WT are shown for ROSA-miR21. Four clones are shown for MSP. F, ROSA-miR21 ES cells confirmed ROSA26 integration by Southern blot and PCR exhibited ∼8-fold mature miR-21 expression when infected with Ad-Cre compared with Ad-GFP. G, Similarly, confirmed ROSA-MSP ES cells expressed MSP RNA in the presence of Cre (+C) and when RNA was processed by reverse transcriptase (+R). GAPDH served as control for qPCR. Two clones outlined by dashed lines are shown.