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Bicistronic or Internal Ribosome Entry Site (IRES) Element — Efficient co-expression of two genes in a single vector

Introduction

The IRES element is exactly what's needed for certain experiments when a single-gene vector falls short. IRES enables the coordinated co-expression of two genes with the same vector. For example, you can monitor the delivery of one gene by using a second gene with a fluorescent tag or express a protein of interest and simultaneously biotinylate it with the same vector. An IRES element is especially useful for gene delivery when working with stem and primary cells and when the following tasks are pursued:

  • Monitor gene delivery efficiency
  • In vivo biotinylation
  • Stable transfection

 

OmicsLink ORF cDNA clones with IRES element

 

Technology

The IRES technology serves a dual-purpose. It allows the coordinated and efficient expression of two genes using the same promoter in a single vector (see figures 1 and 2). Virtually any combination of genes is possible. GeneCopoeia has already developed many constructs.

 

Combined with OmicsLink expression-ready clones

For a variety of applications, GeneCopoeia has incorporated the IRES element into several mammalian and lentiviral vectors using a wide choice of reporter genes including the following:

  • eGFP
  • mCherry
  • SUMO
  • biotin ligase
  • neomycin
  • luciferase

 

More than 20,000 human and 15,000 mouse genes are available in CMV promoter driven bicistronic (IRES) expression vectors.

Choice of co-expressing any reporter gene with IRES element

 

Advantages

  • Minimal effect on biological activities of target proteins when co-expressed with reporter/ assaying genes.
  • Efficient co-transfection due to coordinated co-expression of two genes from a single expression vector.

 

 

 

 

 

OmicsLink ORF cDNA clones with IRES element

 

 

 

 

Figure1. OmicsLink™ ORF cDNA expression clones with IRES element in mammalian and lentiviral expression vector systems.

Applications and Data

Applications

  • Monitor gene delivery efficiency by co-expressing proteins with reporter genes including eGFP, mCherry or Luciferase.
  • In vivo biotinylation of expressed proteins with AviTag by co-expressing with biotin ligase.
  • Establish stable transfected cell lines for efficient selection by co-expressing with selection marker genes like Neomycin.

 

Rigorous quality control and assurance process

All ORF cDNA clones in OmicsLink collection were generated from sequence validated full-length cDNA clones or high-quality human tissue cDNA libraries to construct expression-ready clones.

Once the clone is constructed, GeneCopoeia follows additional quality control processes to ensure the right clone is delivered.

  • All ORF cDNA are fully sequenced.
  • PCR amplification and size validation
  • Enzyme digestion check of the integrity of whole plasmid

How it works

Schematic of protein expression with IRES technology

Specific biotinylation of AviTag eGFP by E. coli biotin ligase in 293 cells

Figure 2. Specific biotinylation of AviTag eGFP by E. coli biotin ligase in 293 cells

Vectors

List of expression vectors with IRES technology

Vector Promoter Host Cell Selection Marker Tag Protease Site
pReceiver-Lv165 EF1a Lentiviral N/A IRES2-eGFP N/A
pReceiver-Lv166 EF1a Lentiviral N/A IRES2-mcherry N/A
pReceiver-Lv201 CMV Lentiviral Puromycin SV40-eGFP-IRES-puromycin N/A
pReceiver-Lv202 CMV Lentiviral Puromycin C-Myc-SV40-eGFP-IRES-puromycin N/A
pReceiver-Lv203 CMV Lentiviral Puromycin C-Flag-SV40-eGFP-IRES-puromycin N/A
pReceiver-Lv204 CMV Lentiviral Puromycin C-Myc-SV40-mCherry-IRES-puromycin N/A
pReceiver-Lv205 CMV Lentiviral Puromycin IRES2-eGFP-IRES-puromycin N/A
pReceiver-Lv206 CMV Lentiviral Puromycin SV40-mCherry-IRES-puromycin N/A
pReceiver-Lv207 CMV Lentiviral Hygromycin SV40-eGFP-IRES-hygromycin N/A
pReceiver-Lv211 CMV Lentiviral Hygromycin C-Halotag-IRES-hygromycin N/A
pReceiver-Lv213 CMV Lentiviral Puromycin IRES2-mCherry-IRES-puromycin N/A
pReceiver-Lv214 CMV Lentiviral N/A IRES2-mCherry N/A
pReceiver-Lv215 CMV Lentiviral N/A IRES2-eGFP N/A
pReceiver-Lv216 CMV Lentiviral Puromycin C-Flag-SV40-mCherry-IRES-puromycin N/A
pReceiver-Lv217 CMV Lentiviral Puromycin SV40-hLUC-IRES-puromycin N/A
pReceiver-Lv220 CMV Lentiviral Hygromycin IRES2-Hygromycin N/A
pReceiver-Lv224 EF1a Lentiviral Puromycin IRES2-mcherry-IRES-puromycin N/A
pReceiver-Lv225 EF1a Lentiviral Puromycin IRES2-eGFP-IRES-puromycin N/A
pReceiver-Lv228 PGK Lentiviral N/A IRES2-eGFP N/A
pReceiver-Lv229 PGK Lentiviral N/A IRES2-mcherry N/A
pReceiver-Lv230 PGK Lentiviral Puromycin IRES2-mcherry-IRES-puromycin N/A
pReceiver-Lv231 PGK Lentiviral Puromycin IRES2-eGFP-IRES-puromycin N/A
pReceiver-M45 CMV Mammalian Neomycin C-3xHA+IRES-eGFP N/A
pReceiver-M46 CMV Mammalian Neomycin C-Flag+IRES-eGFP N/A
pReceiver-M48 CMV Mammalian Neomycin N-Avi+IRES-Biotin ligase N/A
pReceiver-M51 CMV Mammalian Neomycin C-His+IRES-eGFP N/A
pReceiver-M61 CMV Mammalian Neomycin IRES2-eGFP N/A
pReceiver-M72 CMV Mammalian Neomycin C-Myc+IRES2-eGFP N/A

Learn more about expression-ready ORF cDNA clone collection available in bacterial, mammalian, insect, yeast and wheat germ cell free systems with more than 50 fusion tags.

 

References

  1. Moroz MA, Serganova I, Zanzonico P, Ageyeva L, Beresten T, Dyomina E, Burnazi E, Finn RD, Doubrovin M, Blasberg RG. Imaging hNET Reporter Gene Expression with 124I-MIBG. J Nucl Med. 2007 May;48(5):827-836.
  2. Allera-Moreau C, Chomarat P, Audinot V, Coge F, Gillard M, Martineau Y, Boutin JA, Prats AC. The use of IRES-based bicistronic vectors allows the stable expression of recombinant G-protein coupled receptors such as NPY5 and histamine 4.Biochimie. 2006 Jun;88(6):737-46. Mulky A, Sarafianos SG, Arnold E, Wu X, Kappes JC. Subunit-specific analysis of the human immunodeficiency virus type 1 reverse transcriptase in vivo. J Virol. 2004 Jul;78(13):7089-96.
  3. Harries M, Phillipps N, Anderson R, Prentice G, Collins M. Comparison of bicistronic retroviral vectors containing internal ribosome entry sites (IRES) using expression of human interleukin-12 (IL-12) as a readout.J Gene Med. 2000 Jul-Aug;2(4):243-9. Fujiwara T, Urata Y, Tanaka N. lomerase-specific oncolytic virotherapy for human cancer with the hTERT promoter. Curr Cancer Drug Targets. 2007 Mar;7(2):191-201.
  4. Martinez-Salas E. Internal ribosome entry site biology and its use in expression vectors. Curr Opin Biotechnol. 1999 Oct;10(5):458-64. D Trouet, B Nilius, T Voets, G Droogmans, J Eggermont. Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells. Pflugers Arch (1997) 434: 632-8.