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Applications: 1. Transient knock-down of CBL-B in target cells
2. Generation of a stable CBL-B knock-out cell pool following puromycin selection and limited dilution
Description: CBL-B is an E3 ubiquitin-protein ligase which has been identified as a negative regulator of T-cell activation. Using CRISPR/Cas9 to inactivate CBL-B has been shown to be sufficient to inhibit T-cell expansion.The CBL-B CRISPR/Cas9 Lentiviruses are replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles that are ready to infect almost all types of mammalian cells, including primary and non-dividing cells. The particles contain a CRISPR/Cas9 gene driven by an EF1a promoter, along with 5 sgRNA (single guide RNAs) targeting human CBL-B driven by a U6 promoter.The integrating lentivirus integrates randomly into the cellular genome to express both Cas9 and the sgRNA. Puromycin selection forces high expression levels of both Cas9 and the sgRNA, and can be used with the integrating lentivirus to quickly and easily achieve high knockdown efficiencies in a cell pool. Efficiencies may vary, depending on the cell type and the gene of interest.
Formulation: The lentiviruses were produced from HEK293T cells in medium containing 90% DMEM + 10% FBS.
Storage Stability: Lentiviruses are shipped with dry ice. For long term storage, it is recommended to store the virus at -80°C. Avoid repeated freeze-thaw cycles. Titers can drop significantly with each freeze-thaw cycle.
Supplied As: Two vials (500 µl x 2) of lentivirus at a titer ≥1 x 106 TU/ml. The titer will vary with each lot; the exact value is provided with each shipment.
Warnings: Avoid repeated freeze/thaw cycles. Titers can drop significantly with each freeze/thaw cycle.Biosafety: None of the HIV genes (gag, pol, rev) will be expressed in the transduced cells. Although the pseudotyped lentiviruses are replication-incompetent, they require the use of a Biosafety Level 2 facility. BPS recommends following all local federal, state, and institutional regulations and using all appropriate safety precautions.
Biosafety Level: BSL-2
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