Specifications

The psiTEST™ System comprises two components:

- psiTEST™ plasmid featuring the SEAP reporter gene and the GFP::zeo fusion gene.
- QUANTI-Blue™, a SEAP detection medium

The psiTEST™ plasmid can be used to clone fragments of genes or entire genes, the optimum size being 1.5 kb, although genes up to 3.5 kb can be targeted.
The psiTEST™ plasmid is selectable with Zeocin in E. coli and mammalian cells. Transfection efficicency can be monitored by assessing GFP expression.

Documents

• TDS psiTEST System : 1 kit (ksitest)
• Sequence psiTEST System : 1 kit (ksitest)

Contents

The psiTEST™ System includes the following components:

- 20 μg of psiTEST-mcs
- 20 μg of psiTEST control (psiTEST-hp53)
- 20 μg of psiRNA positive control (psiRNA-hp53)
- 20 μg of psiRNA negative control (psiRNA-Luc)
- 2 pouches of QUANTI-Blue™
- 4 pouches of Fast-Media® Zeo (2 TB, 2 Agar)

psiTEST-hp53, psiRNA-hp53 and psiRNA-Luc are control plasmids. psiTEST-hp53 contains a fusion between the SEAP and human p53 genes. psiRNA-hp53 produces an shRNA that functionally silences the human p53 resulting in the absence of phosphatase activity. psiRNA-Luc expresses a shRNA insert that will not affect the expression of the phosphatase reporter gene.

Description

The psiTEST™ system is based on a transcriptional fusion between a reporter gene and your target gene. The reporter gene is an optimized alkaline phosphatase gene engineered to be secreted (SEAP). The efficacy of an siRNA or shRNA to induce RNAi on your target gene is determined by measuring the expression levels of the SEAP reporter gene (see figure). Since SEAP is a secreted protein, the expression level can be evaluated from supernatants therefore allowing to perform kinetics of the silencing process.

The psiTEST™ System includes a cloning vector and a SEAP detection medium:

- the psiTEST™ plasmid features the SEAP reporter gene and the GFP::zeo fusion gene. The SEAP gene is cloned upstream of a multiple cloning site (MCS) that contains many common restriction sites compatible with many other restriction sites. The zeo moiety of GFP::zeo gene allows to amplify the plasmid in E. coli and select stable mammalian clones while the GFP moiety enables the monitoring of transfection efficiency.
The psiTEST™ plasmid can be used to clone fragments of genes or entire genes, the optimum size being 1.5 kb, although genes up to 3.5 kb can be targeted.

- QUANTI-Blue™ is a SEAP detection medium that turns purple/blue in the presence of phosphatase activity. Functional siRNAs or shRNAs will induce the degradation of the SEAP mRNA resulting in the reduction or absence of SEAP activity.
Silencing efficiencies of the siRNAs or shRNAs can be observed visually and unlike fluorescent reporters can also be easily quantified using  a microplate reader or spectrophotometer.

Details

Procedure

1- Clone your target gene (or a fragment) into the psiTEST plasmid.
2- Cotransfect mammalian cells with recombinant psiTEST and siRNA or shRNA-producing plasmid, such as psiRNA.
3- Incubate at 37°C with 5% CO2 for 72 hours.
4- Collect supernatant and add to a QUANTI-Blue™-containing well.
5- Incubate at 37°C for 30 min to 3 hours.
6- Determine the functionality of your siRNA ou shRNA by observing the coloration of QUANTI-Blue visually and/or with a spectrophotometer at 620-655 nm.

Screening of functional siRNA or shRNAs with the psiTEST system