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Applications: 1. Transient knock-down of CTLA4 in a target cell pool.
2. Generation of a stable CTLA4 knock-out cell line following limited dilution.
Description: CTLA4 (Cytotoxic T-Lymphocyte Associated Protein), also known as CD152, is a protein receptor that functions as an immune checkpoint. It is expressed by activated T-cells and transmits an inhibitory signal to T-cells. CTLA4 is homologous to the T-cell co-stimulatory protein CD28, and both molecules bind to CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells. CTLA4 binds CD80 and CD86 with greater affinity and avidity than CD28, thus enabling it to out-compete CD28 for its ligands and act as an "off" switch when bound to CD80 or CD86. CTLA4 is an important immunotherapy target for the treatment of cancer and autoimmune diseases.
The CTLA4 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 CTLA4, GenBank Accession #NM_005214, driven by a U6 promoter.
Note: Unlike human CTLA4 CRISPR/Cas9 Lentivirus (Integrating) (BPS Bioscience, #78054), the Human CTLA4 CRISPR/Cas9 Lentivirus (Non-Integrating) is made with a mutated Integrase, resulting in only transient expression of the Cas9 and CTLA4 targeting sgRNA. While this may minimize potential off-targeting risks due to either prolonged expression or integration of the Cas9, puromycin selection should not be used for more than 48 hours post-transduction, which may lower knockout efficiency.
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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