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Applications: 1. Transient knock-down of CD47 in a target cell pool.
2. Generation of a stable CD47 knock-out cell line following limited dilution.
Description: CD47 (also known as Rh-associated protein, GP42, Integrin-Associated Protein (IAP), or Neurophilin) is an immunoglobulin-like protein that interacts with its receptor, Signal-regulatory protein alpha (SIRPα), on macrophages. This binding interaction regulates transmigration, oxidative burst cytokine production, and phagocytosis, generating a "don't eat me" signal. CD47 is ubiquitously expressed on the surface of normal cells, but is overexpressed in numerous cancer cells where it is thought to contribute to the resistance of tumors to phagocyte-dependent clearance.The CD47 CRISPR Lentiviruses are replication incompetent, HIV-based VSV-G pseudo-typed lentiviral particles that are ready to be transduced into 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 4 sgRNA (single guide RNA) targeting human CD47 (NM_198793.2) driven by a U6 promoter (Figures 1 and 2).The integrating lentivirus integrates randomly into the cell's genome to express both the Cas9 and sgRNA. Puromycin selection increases the knockout efficiency by forcing 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 also depend 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 10^6 TU/ml. The titer will vary with each lot; the exact value is provided with each shipment.
Warnings: Biosafety: None of the HIV genes (gag, pol, rev) will be expressed in the transduced cells. Although the pseudotyped lentiviruses are replication-incompetent, they do require the use of a Biosafety Level 2 facility. BPS recommends following all federal, state, local, and institutional regulations and using all appropriate safety precautions.
Biosafety Level: BSL-2
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