Inducible Expression Vector System

 


Cellutron has developed a novel inducible expression system with Cre-Loxp technology and provided the ability to regulate transgene expression. This system contains two expression vectors with a strong CAG promoter for high-efficient gene expression. pEGFP/RFP-miRNA-BL and pEGFP/RFP-T2A-GOI-BL are expression vectors with EGFP and 3X termination sequences flanked by two loxP sites, followed by a co-cistronic sequence for expression of RFP and miRNA or protein-coding transgenes.  In addition, there is a blasticidin (BL) expression cassette (for drug selection in the mammalian cells) cloned at the downstream of the expression cassette for the miRNA and protein-coding transgene. pCreER-IRES-Puro is needed for co-transfection with the inducible expression vector.  It expresses a 4-hydroxytamoxifen (4-OHT)-activated form of Cre recombinase and also confers puromycin-resistance for drug selection.

 

After co-transfection the system initially expresses only EGFP, a reporter protein for conveniently monitoring the transfection efficiency. 4-OHT can be added in vitro and in vivo to activate the conditionally active form of Cre recombinase, leading to high-level and regulated expression of miRNAs or protein-coding transgenes.

 

The inducible expression system allows you to:

 

1. Transfect with high efficiency in dividing and non-diving cells, including adult and embryonic stem cells and iPS cells.

 

2. Conditionally express miRNAs or protein-coding transgenes in vivo and in vitro.

 

3. Generate stable cell lines for long-term expression of miRNAs or protein-coding transgenes.

 

 

inducible mirna

 

pCrer-IRES-Puro expression vector is used for co-transfection

 

 

- 4OHT

+ 4OHT

- 4OHT + 4OHT

 

This system expressed EGFP initially and, after 4-OHT treatment, a conditionally active form of Cre recombinase was activated and catalyzed the site-specific recombination by crossover between the two loxP sites, resulting in excision of "floxed" EGFP and the termination sequence and allowing expression of RFP and miRNA or protein-coding transgene.

 

 

                              RT-PCR Analysis for Inducible miRNA Expression in Cells

 

rt-pcr analysis

Cells were cotransfected with pEGFP/RFP-miR-1-BL
and pCreER-IRES-Puro and treated with 4-OHT 48 hours after transfection.

 

 

4OHT (4-hydroxytamoxifen) Treatment for miRNA Expression

 

For in vivo injection, 4OHT (Sigma, St. Louis, MO) is dissolved in ethanol at a concentration of 20 mg/ml and then diluted with 9 volumes of corn oil (Sigma). Diluted 4OHT (2 mg/ml) is i.p. injected (500 µl per animal) into the animals with a 26-gage needle.

 

For cultured cells, 4OHT diluted in ethanol (0.39 mg/ml) is directly added to the cell culture media (final concentration: 1-2 µM = 0.39-0.78 µg/ml).

 

 

*GOI = Gene of Interest

 Cat #  

 Product  Qty Price  
  v-0924   pCAG-CreER-IRES-Puro   5ug   $ 698   

  v-0919

  pCAG-LoxP-EGFP-LoxP-RFP-BL (Control)   5ug   $ 650  
  v-0922   pCAG-LoxP-EGFP-LoxP-RFP-T2A-GOI-BL   5ug

  $ 850

 
  v-0923   pCAG-LoxP-EGFP-LoxP-RFP-miRNA-BL   5ug

  $ 850

 
  v-0937   pCAG-LoxP-Stop-LoxP-RFP-BL (Control)   5ug   $ 650  
  v-0938   pCAG-LoxP-Stop-LoxP-RFP-miRNA-BL   5ug   $ 850  
  v-0939   pCAG-LoxP-Stop-LoxP-GOI-BL   5ug   $ 850  
  v-0940   pCAG-LoxP-Neo-LoxP-RFP (Control)   5ug   $ 650  
  v-0941   PCAG-LoxP-Neo-LoxP-miRNA   5ug   $ 850  
  v-0942   pCAG-LoxP-Neo-LoxP-GOI   5ug   $ 850  

 

 

Fax: 1-800-994-6482

 

customerservice@cellutron.com

Techsupport@cellutron.com

 

 

 

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Minicircle expression vectors

 

miRNA expression vectors   

 

 

 

Terms and conditions

 

 

 

 

 

All Cellutron products should be used only in accordance with labels on the products and other products literature made available.
All Cellutron products are labeled “For Research Use Only”. Products are not approved for human or veterinary use or for application to humans.

 

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