A Novel Transgenic Mouse Line for Tracing MicroRNA-155-5p Activity In Vivo.

Jianhua Zhang,Tanyu Hu,Krung Phiwpan,Bhargavi M Boruah,Jie Guo,Wei Zhang,Xuyu Zhou,Sunil K Ahuja

doi:10.1371/journal.pone.0128198

Abstract

MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity in vivo by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level.

Highlights

MicroRNA-155 is processed from the non-protein coding transcript of the BIC (B cell Integration Cluster) gene located on chromosome 21 in human and chromosome 16 in mice [1, 2]. miR-155, like other microRNAs, is transcribed by RNA polymerase II to generate primary transcripts that is processed in the nucleus to generate miRNA precursors
The results showed that the miR-155-5p target was responsible for overall miR-155-5p interaction leading to decreased luciferase signals compared to HEK293T cells transfected with miR-146a (Fig 1A)
To further determine sensitivity of the miR-155-5p-OFF system in response to expression of miR-155-5p, HEK293T cells were transfected with pDTR.BFP-155TN1 along with varying amounts of synthetic precursor miR-155 ranging from 0 to 40 nM (Fig 1C)

Summary

Introduction

MicroRNA-155 (miR-155) is processed from the non-protein coding transcript of the BIC (B cell Integration Cluster) gene located on chromosome 21 in human and chromosome 16 in mice [1, 2]. miR-155, like other microRNAs (miRNAs), is transcribed by RNA polymerase II to generate primary transcripts (pri-miR-155) that is processed in the nucleus to generate miRNA precursors (pre-miR-155). Based on the stability of the 5’ end, one strand (passenger miR-155) of the miRNA duplex is released and degraded while the other strand (guide strand or mature miR-155), is retained and loaded into the RNA-induced silencing complex (RISC) which binds to target mRNAs as well as regulates gene expression by either repressing protein translation or inducing mRNA degradation. Both arms of pre-miR-155 can develop into mature miR-155-5p or miR-155-3p based on the selection of either 5’ or 3’ strand respectively [4]. The expression level of miR-155-5p is reported to be ~20–200 fold higher than that of miR-155-3p [5].

Methods

Results

Conclusion