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miTarget™ 3′ UTR miRNA Target Clones

Introduction

GeneCopoeia offers genome-wide human, mouse and rat microRNA (miRNA) 3' UTR target clones in mammalian expression vectors. miRNA 3' UTR target clones can be used for miRNA target identification and functional validation of predicted targets, or to study the regulatory effect of miRNAs on target genes.  

3' UTR sequences were obtained from public domain gene sequence databases and inserted downstream of a secreted Gaussia luciferase (GLuc) reporter gene, which is expressed under control of the SV40 promoter in mammalian cells. A chimeric mRNA is transcribed consisting of the GLuc and a 3' UTR target sequence. Thus, the study of mRNA-miRNA target interaction can be easily performed with a live cell assay for GLuc using only 10 μl of the cell culture medium.

Besides using GLuc as the miRNA 3' UTR target reporter, a secreted Alkaline Phosphatase (SEAP) reporter driven by a CMV promoter is also cloned into the same vector (pEZX-MT05) and serves as the internal control. The dual-reporter vector system enables transfection normalization for accurate across-sample comparison.

In addition to the GLuc/SEAP dual-reporter vector system, a firefly/Renilla Duo-Luciferase reporter vector (pEZX-MT06) is also available. 

mirna target with luciferase reporter

Figure 1. How 3’UTR clone works

 

mirna target with luciferase reporter

Figure 2. Vector backbone of miTarget miRNA 3' UTR target clones

 

 

 

 

 

Figure 3. The inhibitory effect of miRNA on a 3' UTR target sequence expression clone 

LIN28 is the known target gene for miR-125a. HEK 293 cells were transfected with LIN28 3' UTR target sequence expression plasmid alone (left bar) or co-transfected with miR-125a precursor expression plasmid (right bar). Both the GLuc activity and an internal control SEAP activity were determined 24 hours post-transfection. The activity ratio of GLuc to SEAP was set to 1 for the single transfection sample with LIN28 3'-UTR target sequence expression clone (left bar), against which the activity of the co-transfection sample was compared to. The result shows that miR-125a suppressed the luciferase activity from the GLuc-LIN28-3'-UTR clone by more than 70% (right bar).
   

 

 

 

 

 

Advantages

Live cell assays

  • Naturally secreted GLuc reporter
  • No lysis of the cells is necessary
  • Save samples, reduce variations, and simplify experiments

Dual-reporter vector system

  • Enables transfection normalization for accurate across-sample comparison

High-throughput compatible

  • Group study or high sample number compatible

High sensitivity

  • GLuc is 1000-fold more sensitive than firefly or Renilla luciferase

Convenience

  • All 3' UTR target clones are ready to use