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Applications: 1. Transient knock-down of CBL-B in target cells
2. Generation of stable CBL-B knock-out cells using transient puromycin selection (48h maximum) followed by 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.Note: unlike CBL-B CRISPR/Cas9 Lentivirus (Integrating) (BPS Bioscience, #78343), the CBL-B CRISPR/Cas9 Lentivirus (Non-Integrating) is made with a mutated Integrase, resulting in only transient expression of the Cas9 and CBL-B-targeting sgRNA. It is expected that this will minimize potential off-target effects caused by either prolonged expression or random integration of Cas9 and the sgRNA. A short round of puromycin selection right after transduction may increase knockout efficiency, however puromycin should not be used for more than 48 hours post-transduction due to the transient nature of expression using the non-integrating lentivirus.
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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