pOnebyOne™ Bicistronic Mammalian Expression

Bicistronic expression vectors are used for the simultaneous translation of two genes of interest from the same mRNA transcript, involving IRES-containing vectors or 2A sequence-containing vectors.

IRES, a viral sequence of around 600-700 bp, has been widely used in bicistronic mammalian expression vectors, although its length could be a disadvantage in viral vectors with limited packaging capacity. IRES based expression vectors are characterized by the non-stoichiometric production of proteins and generally lower downstream gene expression.

2A sequence is a viral oligopeptide of approximately 20 amino acids that mediates cleavage of polypeptides during translation in eukaryotic cells. The designation “2A” refers to a specific region of the viral genome that contain the consensus motif Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro at the C-terminus. 2A sequence acts co-translationally. The mechanism of 2A sequence-mediated "self-cleavage" is that the formation of a normal peptide bond between glycine and proline residues at the C-terminal of 2A is prevented, resulting in ribosomal hopping that leads to cleavage of the nascent polypeptide. Compared with other strategies for multi-gene co-expression, 2A sequences lead to relatively high levels of downstream protein expression, and they are small in size thus bearing a lower risk of interfering with the function of co-expressed genes.

pOnebyOne™

pOnebyOne™ are efficient, accurate and flexible bicistronic mammalian expression kits that contain an expression cassette based in 2A sequence breakthrough technology. Its novel technology allows simultaneous expression of two proteins from the same mRNA. Cells transfected with bicistronic vectors ensure that if one of the proteins is present, the other one is also present.

Figure. 2A sequence-based vector.

Both genes must be in frame and the nascent peptide is cleaving between the glycine and proline. After the cleavage, the short peptide IGSGEGRGSLLTCGDAEENPG (21 amino acids) remains fused to the C-terminus of the protein of interest while the proline is added to the N-terminus of the reporter protein. 2A sequence used has high cleavage efficiency in some biological systems. Essential reverse primer sequence for directional cloning is underlined.

Advantages & Features

• Complete solution: a directional cloning vector to clone and produce a protein of interest in mammalian systems.
• Breakthrough technology: based in 2A sequence simultaneous expression of two proteins in mammalian cells (a protein of interest and reporter).
• Highly efficient: cloning system tested with up to 4 kb inserts.
• Time-saving cloning process: linearized vector ready-to-mix with PCR fragment.
• Easy-to-use: facilitates the selection of positive cells expressing the recombinant gene of interest. Only check that your gene is in frame with 2A-reporter.
• Cost avoidance: avoids the use of expensive restriction enzymes.
• Really low experimental background: lower than 1%.
• Convenient: available with different resistance marker cassettes.
• Directional cloning: of PCR with the gene of interest.
• Reporter checking: stoichiometric amount of your protein of interest and a reporter protein.
• Risk-free: product covered by our quality 100% guarantee.

Product Range

Amerigo Scientific offers a ready-to-clone solution of your gene of interest onto a wide collection of bicistronic vectors based on 2A sequence.

• Non-viral Mammalian Expression Kits

P_CMV, citomegalovirus promoter; P_EF1α, elongation factor 1 alpha promoter; △NGFR, truncated nerve growth factor receptor; eGFP, enhanced green fluorescent protein; LUC, firefly luciferase.

• Universal Non-Viral Mammalian Expression Kits

• Retroviral Expression Kits

• Lentiviral Expression Kits

• Multicistronic Expression Kits

Applications

pOnebyOne™ Non-viral Mammalian Expression vectors

Protein Expression of intracellular, extracellular, or transmembrane in higher cells in an equimolecular ratio with a surface marker that allows quantification.

pOnebyOne™ Retroviral Expression vectors

• Introduction of DNA in refractile transfection cell lines.
• Co-expression of your gene of interest and a reporter gene.

pOnebyOne™ Lentiviral Expression vectors

• Highest generation Lentiviral transfer vector to transform replicating and non-replicating cells, including stem and neuronal cells.
• Vector of choice for short-interfering RNA (siRNA) delivery and for gene therapy.