Takara Bicistronic IRES vectors

IRES-containing bicistronic vectors allow the simultaneous expression of two proteins separately, but from the same RNA transcript (Jackson et al. 1990; Jang et al. 1988). Click the more button for detailed information.

IRES-containing bicistronic vectors allow the simultaneous expression of two proteins separately, but from the same RNA transcript (Jackson et al. 1990; Jang et al. 1988). Click the more button for detailed information.

How does an IRES work?

The IRES of the encephalomyocarditis virus (EMCV) permits the translation of two open reading frames from one messenger RNA. Although translation initiation of eukaryotic mRNAs occurs almost exclusively at the 5` cap, the IRES allows ribosomes to bind and initiate translation at a second, internal location. Thus, two proteins are expressed simultaneously from the same bicistronic mRNA transcript.

Our pIRES vectors contain two multiple cloning sites flanking the central IRES for you to clone and express two genes of interest.

pIRES vectors containing antibiotic selection markers

The pIRES selection vectors pIRESpuro3, pIREShyg3, pIRESneo3, and pIRESbleo3 each contain a multiple cloning site located upstream of the IRES from which a selection marker is expressed. Because your gene of interest and a selection marker are translated from a single RNA, you can be certain that nearly 100% of the colonies that are resistant to puromycin, hygromycin, G418, or bleomycin also express your protein of interest.

All IRES selection vectors contain a partially disabled IRES which reduces the efficiency of translation initiation for the selection marker relative to that of the cloned gene, and allows preferential selection of cells expressing high levels of your protein of interest (Rees et al. 1996).

An IRES combined with a fluorescent protein

Similarly, fluorescent protein-containing IRES vectors allow coexpression of your target protein and a fluorescent protein. Successfully transfected target cells are easily identified by fluorescence microscopy or flow cytometry. The are several options for different fluorescent proteins, which include mCherry, ZsGreen1, and tdTomato (pLVX-IRES Vectors), and AcGFP1, DsRed2, and DsRed-Express (pIRES2 Vectors).

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Overview

• Express your protein of interest and an antibiotic resistance marker or fluorescent protein from a single mRNA
• Quickly identify cells expressing your protein of interest at high levels
• Bicistronic expression allows faster, more stable clone selection
• Choice of vectors enables screening via antibiotic selection, fluorescence microscopy, or flow cytometry
  • pLVX vectors are lentiviral vectors—a lentiviral packaging system is required to make lentiviral particles from these constructs.

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Applications

• Rapidly select stable clones that show high-level expression of your target protein
• Identify transfected cells by antibiotic selection, fluorescence microscopy, or flow cytometry

References

Jackson, R. J. et al., The novel mechanism of initiation of picornavirus RNA translation Trends Biochem. Sci. 15:477–483 (1990).

Jang, S. K. et al., A segment of the 5` nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J. Virol. 62:2636–2643 (1988).

Rees, S. et al., Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein. BioTechniques 20:102–110 (1996).

Additional product information

Please see the product`s Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.