Tags may be used for multicolor labeling in cell-based research.
Cellectis bioresearch entered a nonexclusive agreement with Evrogen of Moscow for the latter’s fluorescent proteins. The license allows Cellectis to incorporate these proteins into its own products and will expand the company’s offering of genome-engineering tools.
Evrogen fluorescent proteins range in color from blue to far-red. They can be used for multicolor labeling to observe different cellular events in a particular cell or a cell population.
“One of the key components of our kits is the integration matrix, which enables the user to express a gene of interest,” says Luc Selig, sales and marketing director of Cellectis bioresearch. “By extending the choice of promoters and tags in these matrices, we can offer our clients a full range of methods to achieve single-phase developments, thus saving valuable time during experiments.”
Targeted integration corresponds to the stable genomic integration of a DNA sequence, most of the time a transgene expression cassette, at a unique genomic position selected to guarantee an adequate transcriptional activity.
Any transgene expression cassette or shRNA can be integrated at the meganuclease recognition site. Overexpression is achieved following transgene integration, while RNA silencing studies can be conducted the same way with the same benefits of targeted integration.
Targeted integration involves a cut-and-paste process, performed by a meganuclease and homologous recombination, respectively. A meganuclease expression vector is co-transfected together with a DNA integration matrix. Following transfection, the meganuclease enters the nucleus and recognizes its recognition site, binds to it and induces a DNA double-strand break (cut).
The cell senses the DNA damage and uses homologous recombination to fix it. The co-transfected integration matrix, containing the 5´ and 3´ homology regions flanking the meganuclease recognition site, is used as a template by the cell for homologous recombination.
The integration matrix also contains a Multiple Cloning Site to easily clone a transgene expression cassette. Following homologous recombination, the transgene expression cassette gets stably integrated (paste) at the exact meganuclease recognition site.
Meganuclease-driven targeted integration is based on the presence of a meganuclease recognition site within the targeted cell genome. Although a natural meganuclease recognition site is not present in mammalian cells (since meganucleases are naturally occurring enzymes from single-celled organisms), meganuclease-driven targeted integration can be achieved two ways in higher eukaryotic cells.
Either the recognition site of a natural meganuclease is integrated in the targeted host cell as a single copy (this is the principle of Cellectis’ cGPS® line of targeted integration kits), or a natural meganuclease is engineered so as to change its recognition site and to target a native genomic locus (this is the principle of the firm’s cGPS Custom line of targeted integration kits).
Cellectis bioresearch was incorporated as a subsidiary of Cellectis in June 2008. It provides life-science researchers with ready- and easy-to-use tools for genome customization. These tools, based on meganucleases, enable the engineering of cells with optimized features for drug discovery, protein production, and gene functional studies.
Meganucleases act like molecular scissors, enabling targeted modifications to DNA. Applications span research, biomanufacturing, agrobiotechnology, and therapeutic sectors. The firm reportedly markets over one hundred products through an online store, an international distribution network, and a sales force.
Cellectis says that to date it has formed over 20 academic research partnerships and has established more than 50 agreements with pharmaceutical laboratories, seed producers, and biotech companies across the world.