Inducible miRNA Expression

Cellutron has developed a novel inducible miRNA expression system with Cre-Loxp technology and provided the ability to regulate miRNA expression. This system contains two expression vectors with a strong CAG promoter for high-efficient gene expression. pEGFP/RFP-miRNA-BL is a miRNA expression vector with EGFP and 3X termination sequences flanked by two loxP sites, followed by a co-cistronic sequence for RFP and miRNA expression.  In addition, there is a blasticidin (BL) expression cassette (for drug selection in the mammalian cells) cloned at the downstream of the miRNA expression cassette. pCreER-IRES-Puro is used for co-transfection with the miRNA expression vector.  It expresses a 4-hydroxytamoxifen (4-OHT)-activated form of Cre recombinase and also confers puromycin-resistance for drug selection.

After co-transfection the system initially expresses only EGFP, a reporter protein for monitoring the transfection efficiency. 4-OHT can be added in vitro and in vivo to activate the conditionally active form of Cre recombinase, leading to high-level and regulated expression of miRNAs.

The inducible miRNA expression allows you to:

1. Transfect with high efficiency in dividing and non-diving cells, including adult and embryonic stem cells and iPS cells.

2. Conditionally express miRNAs of your choice in vivo and in vitro.

3. Generate stable cell lines for long-term miRNA expression.

- 4OHT	+ 4OHT
This system expressed EGFP initially and, after 4-OHT treatment, a conditionally active form of Cre recombinase was activated and catalyzed the site-specific recombination by crossover between the two loxP sites, resulting in excision of "floxed" EGFP and the termination sequence and allowing RFP and miRNA to be expressed.

                              RT-PCR Analysis for Inducible miRNA Expression in Cells

Cells were cotransfected with pEGFP/RFP-miR-1-BL and pCreER-IRES-Puro and treated with 4-OHT 48 hours after transfection.

4OHT (4-hydroxytamoxifen) Treatment for miRNA Expression   For in vivo injection, 4OHT (Sigma, St. Louis, MO) is dissolved in ethanol at a concentration of 20 mg/ml and then diluted with 9 volumes of corn oil (Sigma). Diluted 4OHT (2 mg/ml) is i.p. injected (500 µl per animal) into the animals with a 26-gage needle.   For cultured cells, 4OHT diluted in ethanol (0.39 mg/ml) is directly added to the cell culture media (final concentration: 1-2 µM = 0.39-0.78 µg/ml).