Analyzing miRNA Targets with Luciferase Reporters
The pmirGLO Vector (Cat. # E1330) has a multiple cloning site downstream of the luc2 stop codon for insertion of the 3′ UTR of interest.
- MicroRNAs (miRNAs) are short RNAs that interact with targets in the 3′ UTR of transcripts and result in either mRNA degradation or inhibition of translation.
- Observing miRNA-mediated effects requires a reporter under the control of a weaker promoter so that subtle changes in gene expression can be observed.
- The pmirGLO Dual-Luciferase miRNA Target Expression Vector contains a 3′ UTR cloned downstream of the luc2 firefly luciferase gene under the control of the human phosphoglycerate kinase (PGK) promoter.
- The psiCHECK™-2 Vector, designed for target knockdown experiments, has been used to analyze miRNA targets.
- 3′ UTR of interest is cloned into the MCS of psiCHECK™-2 Vector.
Renilla luciferase under control of the strong SV40 promoter is the reporter.
- Strong promoter may mask subtle changes in gene expression.
Investigating Effect of miR200 Family of miRNAs on WAVE3-Dependent Cancer Cell Invasion
WAVE3 is a cytoskeleton remodeling enzyme which is upregulated in advanced breast cancer. The full 3´ UTR and each of the three seed sequences were cloned in to the pmirGLO Vector and transfected into HEK293, cancer cells of epithelial origin and cancer cells of mesenchymal origin and the effect of miR200b on WAVE3 expression was evaluated.
Sossey-Alaloui, K., Bialkowska, K. and Plow, E.F. (2009) J. Biol. Chem. 284, 33019–29.
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