FastCONTROL™ Dual Reporters
In many fields of research, dual reporter systems are an essential and commonly used tool, although they are not cheaper and simpler than system with a single reporter gene. Among the approaches to achieve a dual reporter system, the most common one is the co-transformation of target coding plasmids and reference coding plasmids. But this approach does not guarantee that both plasmids enter each cell, or that they do so in consistent proportions. Differences in the size of each plasmid may also affect their relative transformation efficiencies. An improvement of the method is to encode the target protein and the reference protein on the same plasmid, which are regulated by respective promoter and transcriptional termination sequences. However, limitations of this modified approach are the increased plasmid size, differences activities of the two different promoters, and recombination between identical promoters or terminators. In addition, the order of gene expression in the vector may affects its expression level.
The third method is to encode the target and reference proteins on the same transcript, avoiding variation in the relative expression of target genes and reference genes, since transcription with both genes are equally affected. Multicistronic gene expression can be achieved by inserting an internal ribosomal entry site (IRES) or sequence encoding a self-cleaving 2A peptide between the target gene and the reference gene. The IRES sequence produces a secondary structure in the mRNA that allows translation to take place downstream. However, the translation efficiency for the proteins encoded upstream and downstream of the IRES varies widely depending on the selected IRES sequence. The protein expression levels of genes cloned downstream of the IRES are lower than those of gene cloned upstream. Unlike IRES based vectors, 2A peptide-based vectors allow both reporter proteins are produced in identical proportion. 2A sequences are much shorter than IRES. The 2A linked gene is expressed in a single open reading frame and "self-cleavage" co-translation occurs between glycine (G) and praline (P) at the C-terminus of the 2A polypeptide, producing equal amounts of co-expressed proteins. 2A-mediated cleavage is a universal phenomenon in all eukaryotic cells. The 2A sequences have been successfully used to express multiple proteins driven by a single promoter in some biological models such as plants, zebrafish, transgenic mice, and human cell lines.
FastCONTROL™ Dual Reporters
Amerigo Scientific offers FastCONTROL™ dual reporters developed by breakthrough technology based on 2A sequences, which express dual reporter proteins in stoichiometric proportion. FastCONTROL™ dual reporters are deal as fast, convenient and flexible target cell transfection controls with high co-expression of two reporter genes driven by ubiquitous, strong and constitutive promoters [cytomegalovirus promoter (Pcmv) or elongation factor 1 alpha Promoter (PEF1α)].
Advantages and Features
- Faster Solution: to test transfection/ transduction efficiency in unknown cell lines.
- Robust: available combination of two cell location markers in one vector.
- Convenient: available in lentiviral, retroviral, or non-viral vectors for immediate in vivo and in vitro expression.
- Compatible with transient or stable transfections.
- Flexible: available with different mammalian resistance markers and dual reporter genes combination for different cell location.
- Risk-free: 100% quality guarantee.
Product Range
Amerigo Scientific offers two versions of each FastCONTROL™ dual reporter product. The base version contains 15 µL FastCONTROL™ dual reporter plasmids, available for 100 assays. The plus version contains 15 µL FastCONTROL™ dual reporter plasmids and 0.2 mL CANFAST™ transfection reagent (1 mg/mL).
CANFAST™ transfection reagent is a new generation of cationic polymer with critical advantages for efficient transfection, such as optimal transfection efficiency, high delivery stability, reproducibility, minimal cytotoxicity, and ease of use. CANFAST™ can be widely used for both primary cells and established cell lines, including human cells (293-HEK, BOSC 23 and HepG2), mouse cells (NIH3T3, P815, B16F10), rat cells (RBL2H3, PC12), and hamster cells (CHO-K1), etc.
Applications
- Control for assessing the efficiency of transfection in mammalian cells
- Targeting of different cell locations
- Obtention of cell lines with reporter proteins
Quality Control
- The quantity and quality of purified DNA attend to:
Ratio 260/280 (1.8-2.0);
Agarose gel electrophoresis;
Digestion with restriction endonucleases
- Transient transfection CHO-K1 (625 cells / well with these vectors) provides a SEAP activity >1,000 fold higher than untransfected cells themselves.
- The surface expression of NGFR in transiently transfected CHO > 60%.
- Expression of eGFP in transient intracellular CHO > 60%.
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