CTLA4 CRISPR/Cas9 Lentivirus (Non-Integrating)

Catalog #

78061

$995 *

Size: 500 µl x 2

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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 (Figures 1 and 2).

Figure 1. Schematic of the Lenti-vector used to generate the CTLA4 CRISPR/Cas9 Lentivirus.

Gene Target:	Primer ID:	sgRNA Sequence
CTLA4	CTLA4-1	TGCCCAGGTAGTATGGCGGT
CTLA4	CTLA4-2	CAAGTGAACCTCACTATCCA
CTLA4	CTLA4-3	TTCCATGCTAGCAATGCACG
CTLA4	CTLA4-4	GTGTGTGAGTATGCATCTCC

Figure 2. List of sgRNA Sequences in the CTLA4 CRISPR/Cas9 Lentivirus.

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.

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Synonyms

Cytotoxic T-Lymphocyte Associated Protein Lentivirus, CD152 lentivirus, CTLA4 CRISPR, CTLA-4

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Product Info

Storage and Usage

Citations

Species

Replication Incompetent HIV

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.

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 lentiviruses at -80°C for up to 12 months from date of receipt. Avoid repeated freeze/thaw cycles. Titers can drop significantly with each freeze/thaw cycle.

Instructions for Use

See assay protocol for detailed instructions.

Applications

1. Transient knock-down of CTLA4 in a target cell pool.
2. Generation of stable CTLA4 knock-out cells using transient puromycin selection (48h maximum) followed by limited 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.

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.

Scientific Category

Immunotherapy

Regulatory Status

Research Use Only

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CTLA4 CRISPR/Cas9 Lentivirus (Non-Integrating)

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