SINEUP

Code Description Price Qty
GE02 SINEUP expression kit £750.00

Overview

SINEUP™ can be used to up-regulate protein expression for any target mRNA. The gene encoding the protein can be endogenous or non-endogenous, and in any mammalian cell. SINEUP™ increases the efficiency of translation but does not affect mRNA levels. Therefore, SINEUP™ only works in cells where a gene is already being expressed. This targeting mechanism and lack of activity in cells where a gene is quiescent provide a very high degree of cell specificity, even in mixed cell populations.

SINEUP™ utilizes a plasmid construct, pDUAL, which can be transiently or stably transfected into cells. The SINEUP™ construct contains a H1 promoter which drives the expression of the SINEUP™ sequence. This is non-coding and contains two elements separated by a short spacer. The first of these two elements is the binding domain (BD), a short sequence (around 40 nucleotides) complementary to the target gene. The second element is an effector domain (ED) which contains sequences which (similar to IRES elements) recruits ribosomes to the target mRNA.

Importantly, SINEUP™ is not a gene editing technology, and the target gene operon is completely unaltered. Increases in protein levels are typically 1.5-fold, but increases as much as 10-fold have been observed. Although relatively small, these effects are sufficient to generate measurable phenotypic effects. The hundreds of haploinsufficiency disorders and syndromes associated with trisomy illustrate the long-term effects of subtle changes in expression levels.

Product Data

 

Targeting of Uchl1 protein expression using a SINEUP. 

1. A SINEUP which specifically attached to the Uchl1 gene was designed. 

2. Cultured cells were either transfected with an empty vector (empty) or with a SINEUP targetting Uchl1 mRNA  (ASUchl1). The western blot (top panel) shows expression levels of Uchl1 in the two cell cultures.  mRNA levels of Uchl1 and ASUchl1 are shown in the bottom panel. Note that the levels of Uchl1 mRNA are unchanged although the levels of protein have increased significantly. This is a key characteristic of SINEUP.

3. Uchl1 protein levels can be titrated by increasing the amount of ASUchl1 constructs.

 

SINEUP™ Key characteristics

  • Up-regulates proteins up to 10-fold
  • Simple mechanism
  • Rapid effect -works with transient transfection
  • No need to create stable cell lines
  • Cost-effective
  • Target endogenous or exogenous genes
  • Target locus is unaffected (not a gene-editing technology)
  • mRNA levels are unaltered
  • Works across a range of cell lines 

 

SINEUP™ Kit contents

Each kit contains sufficient material to generate 3 SINEUPs and perform control experiments. The supplied plasmids may be propagated to generate further material.

  • pDUAL/ED.  This construct contains the SINEUP Effector Domain (ED), upstream of which the Binding Domain (BD) for any target gene can be cloned in via specific restriction sites. Once the BD has been inserted, the complete SINEUP™ will be downstream of the H1 promoter.
  • pDUAL/eGFP. This construct expresses eGFP downstream of a CMV promoter
  • pDUAL/eGFP_GFP-BD/ED. In this construct, eGFP is expressed downstream of a CMV promoter and also eGFP-BD, followed by the SINEUP™ ED downstream of an H1 promoter.
  • User Guide

 

 

References

Synthetic long non-coding RNAs [SINEUPs] rescue defective gene expression in vivo.
Indrieri A, Grimaldi C, Zucchelli S, Tammaro R, Gustincich S, Franco B. (2016) Sci Rep. 6: 27315

SINEUPs: A new class of natural and synthetic antisense long non-coding RNAs that activate translation.
Zucchelli S, Cotella D, Takahashi H, Carrieri C, Cimatti L, Fasolo F, Jones MH, Sblattero D, Sanges R, Santoro C, Persichetti F, Carninci P, Gustincich S. (2015)
RNA Biol.;12(8):771-9.

Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat.
Carrieri C, Cimatti L, Biagioli M, Beugnet A, Zucchelli S, Fedele S, Pesce E, Ferrer I, Collavin L, Santoro C, Forrest AR, Carninci P, Biffo S, Stupka E, Gustincich S. (2012)
Nature. 491:454-7.