Intres™ Expression Vectors
Selectable markers are widely used in lentiviral transgenesis and CRISPR/Cas-mediated genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Selectable markers work well for research involving single transgene, but adding more than one transgene to cells can be challenging to track. There are a limited number of fluorescence proteins, and subjecting cells to multiple antibiotic selections after each plasmid is delivered can harm them.
To address the issue, antibodies-online offers Intres Expression Vectors for split selectable markers (SSM). Intres stands for "Intein-split resistance", Intres Expression Vectors can indicate the successful integration of multiple transgenes. Intres Expression Vectors can be also used to facilitate genome engineering using the CRISPR/Cas system when it is used to “knock-in” (add) a genetic sequence at a specific location in the genome.
What are Split Selectable Markers?
SSM involve dividing the marker genes at specific sites and fusing them to protein splicing elements. The divided fragments are then added to separate plasmids, each of which also includes a transgene. When both plasmids are successfully delivered into a cell, a full-length marker protein is produced via protein trans-splicing. Cells that only receive one plasmid will not express the full marker protein and can therefore be selected out via antibiotic treatment or identified via lack of fluorescence. Cells receiving both plasmids could be selected at very high percentages, generally more than 90% and up to 100%, for subsequent transgenic research.
How are Selectable Markers Utilized?
Split selectable markers are created by dividing fluorescent proteins or antibiotic resistance proteins into several segments fused to protein splicing elements called “inteins” (fig.1). They can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors.
Fig.1: Split selectable marker can be used for co-selection of two separate transgenic vectors (table1). The coding sequence of a selectable marker is split into an N-terminal fragment (MarN) and a C-terminal fragment (kerC) and separately cloned upstream of an N-terminal fragment of a split intein (IntN) and downstream of a C-terminal fragment of the split intein (IntC), respectively, on two different vectors each carrying a different transgene. These vectors are delivered to cells yielding sub-populations of cells containing either one, or both of the vectors. Only cells with both vectors expressing the two intein-split selectable marker fragments (“markertrons”) undergo protein trans-splicing to reconstitute a full-length selectable marker, allowing specific selection and enrichment of the double transgenic cells.
What are the Advantages of Intres Expression Vectors
The system is not limited to simple two-plasmid applications, it is possible to select up to six transgenes using a single split selectable marker. In addition, split selectable markers can be used to facilitate genome engineering using the CRISPR/Cas system when it is used to “knock-in” (add) a genetic sequence at a specific location in the genome. Current precision editing and knock-in methods are inefficient, underscoring the need for effective marker and cell selection systems. Using split markers in concert with CRISPR/Cas editing again yielded very high percentages of cells with the desired genotypes following selection and growth.
Choose Intres Vectors Combinations
antibodies-online offers Intres Expression Vectors. Choose from vector combinations in the table to utilize your own inserts. Or discover all available split selectable marker vectors below.
| ABIN | Backbone | Selection Marker |
|---|---|---|
| ABIN3188233 | pLX-DEST-IRES-Hygro(1-89)-NpuDnaE(N) | Hygromycin |
| ABIN7539391 | pLX-DEST-IRES-NpuDnaE(C)-Hygro(90-341) | Hygromycin |
| ABIN7539406 | pLX-DEST-IRES-Neo(1-194)-NpuDnaE(N) | Neomycin |
| ABIN7539407 | pLX-DEST-IRES-NpuDnaE(C)-Neo(195-267) | Neomycin |
| ABIN7539412 | pLX-DEST-IRES-Puro(1-119)-NpuDnaE(N) | Puromycin |
| ABIN7539413 | pLX-DEST-IRES-NpuDnaE(C)-Puro(120-199) | Puromycin |
References
- : "Split selectable markers." in: Nature communications, Vol. 10, Issue 1, pp. 4968, (2020) (PubMed).
Intres™ is a registred trademark of the Jackson Laboratory
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