KC-4812

Jurkat-VAV1-HiBiT-KI Cell Line

61560

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Background of Jurkat-VAV1-HiBiT-KI Cell Line

VAV1 is a critical guanine nucleotide exchange factor in lymphocyte receptor signaling, regulating Rho GTPase activation and being essential for the development, activation, and function of T cells and B cells. Studying the dynamic expression, localization, and stability of VAV1 is important for understanding immune signaling networks and the mechanisms of immune-related diseases. However, traditional methods like Western blot or fluorescent protein tagging face limitations such as cumbersome procedures, low sensitivity, or potential interference with the native properties of the protein. The HiBiT tag is an 11-amino acid peptide tag that can complement an exogenously added LgBiT protein fragment to form a highly active NanoLuc luciferase, enabling highly sensitive and accurate quantification of target proteins within cells. Precisely inserting the HiBiT tag into the endogenous VAV1 gene locus via gene editing technology can endow VAV1 with a highly sensitive bioluminescent reporter function without significantly disrupting its normal structure and function. This strategy provides a revolutionary tool for real-time, dynamic, and quantitative study of endogenous VAV1 protein expression levels, degradation kinetics, subcellular localization, and interactions within signaling complexes in live cells under physiological conditions.

Specifications

Catalog Number	KC-4812
Cell Line Name	Jurkat-VAV1-HiBiT-KI Cell Line
Clone Number	2B2
Host Cell Line	Jurkat
Description	Stable Jurkat clone expressing VAV1 with an endogenous C-terminal HiBiT tag
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	NA
Morphology	Lymphoblast
Subculture	Split saturated culture 1:4-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 26 hours
Mycoplasma Status	Negative

Cell Line Generation

Jurkat-VAV1-HiBiT-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of Jurkat-VAV1-HiBiT-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of Jurkat-VAV1-HiBiT-KI cell line stable clone using RT-PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10% FBS) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640+20% FBS+10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Turner M, Billadeau DD. VAV proteins as signal integrators for multi-subunit immune-recognition receptors. Nat Rev Immunol. 2002 Jul;2(7):476-86. doi: 10.1038/nri840. PMID: 12094222.