To order a CRISPR/Cas9 lentivirus to knockout a kinase of interest, select the Gene Symbol of the kinase in the ordering window (for example, select AKT1 if ordering the lentivirus to knockout kinase AKT1). Download the table to view all available kinases.
The Kinase CRISPR/Cas9 lentivirus is designed to target a specific kinase of interest for knockout. The replication-incompetent, HIV-based, VSV-G pseudotyped lentiviral particles are ready to infect almost all types of mammalian cells, including primary and non-dividing cells. The SIN lentiviral backbone contains the Cas9 gene (Streptococcus pyogenes CRISPR associated protein 9) driven by an EF1A promoter, an sgRNA driven by a U6 promoter, and a puromycin selection marker. Each vial of lentivirus consists of a mixture of lentiviral particles targeting 5 different sgRNAs per gene.
The lentivirus integrates randomly into the cellular genome to express both Cas9 and the sgRNAs. Because it contains Cas9, the lentivirus can be used in any target cell regardless of whether the cells already express Cas9. Puromycin selection ensures high expression of both Cas9 and the sgRNAs. Knockout efficiencies will depend on the cell type and the gene of interest. Stable CRISPR-Cas9 knockout cell lines can also be generated following limiting dilution.
Figure 1: Schematic of the lentivector used to generate each kinase CRISPR/Cas9 Lentivirus.
Two vials (200 µl x 2) of lentivirus at a titer ≥1 x 107 TU/ml. The titer varies with each lot and is provided with each shipment.
Formulation
The lentivirus particles are produced from HEK293T cells. They are supplied in cell culture medium containing 90% DMEM + 10% FBS.
Storage/Stability
Lentiviruses are shipped with dry ice. For long-term storage, it is recommended to store the lentiviruses at -80°C.
Applications
Transient knockout of the kinase of interest in a cell pool
Generation of a stable knockout cell line following puromycin selection and limiting dilution
Shipping Temperature
-80°C
Notes
The CRISPR/CAS9 technology is covered under numerous patents, including U.S. Patent Nos. 8,697,359 and 8,771,945, as well as corresponding foreign patents applications, and patent rights.
Biosafety The lentiviruses are produced with the SIN (self-inactivation) lentivector which ensures self-inactivation of the lentiviral construct after transduction and after integration into the genomic DNA of the target cells. None of the HIV genes (gag, pol, rev) will be expressed in the transduced cells, as they are expressed from packaging plasmids lacking the packing signal and are not present in the lentivirus particle. Although the lentiviruses are replication-incompetent, they require the use of a Biosafety Level 2 facility. BPS Bioscience recommends following all local federal, state, and institutional regulations and using all appropriate safety precautions.
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