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293.STAT1 BAX KO

CRL-1573-VHG

This STAT1 BAX double knockout HEK-293 cell line exhibits significantly increased adeno-associated virus (AAV) production capacity for gene therapy applications.
Product category
Human cells
Product type
Cell model
Organism
Homo sapiens, human
Morphology
epithelial
Tissue
kidney
Applications
Vaccine development
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

Biosafety Icon BSL 2

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Detailed product information

General

Specific applications
  • Adeno-associated virus (AAV) production for gene therapy applications
  • Viral vaccine production 
  • Virus propagation 
  • Production of high-titer viral stocks 
  • Viral vector transfection and viral particle production 
  • Transfection host

Characteristics

Cells per vial
Approximately 2.0 x 106
Volume
1.0 mL
Growth properties
Adherent
Age
embryo
Comments
This STAT1 BAX double knockout HEK-293 cell line was derived from the parental HEK-293 cell line (ATCC CRL-1573) at ATCC using CRISPR-Cas9 gene editing technology. This cell line carries short nucleotide insertions and deletions in both STAT1 gene and BAX gene. This cell line does not express STAT1 protein or BAX protein. STAT1 is a transcription factor required for the interferon-based cellular anti-viral response. BAX is a member of Bcl-2 family, which regulates cell apoptosis. The HEK-293.STAT1KO cell line has been validated at the genomic, transcript, and protein bio-functional levels. It exhibits significant increased viral titer and enhanced virus production capability when compared to its parental cell line.

Handling information

Complete medium
The base medium for this cell line is Eagle's Minimum Essential Medium (EMEM; ATCC 30-2003). To make the complete growth medium, add the following component to the base medium:  
  • Fetal bovine serum (FBS; ATCC 30-2020) to a final concentration of 10% 
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.  

  1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
  2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  3. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete culture medium. and spin at approximately 125 x g for 5 to 7 minutes.
  4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). pH (7.0 to 7.6).
  5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.

         

Subculturing procedure
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
    Cultures can be established between 1 x 104 and 4 x 104 viable cells/cm2.
  6. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 1 X 104 and 8 X 104 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:6 to 1:10 is recommended
Medium Renewal: 2 to 3 times per week
Reagents for cryopreservation
Complete growth medium supplemented with 5% (v/v) DMSO (ATCC 4-X)

Quality control specifications

Bacterial and fungal testing
Not detected
Mycoplasma contamination
Not detected
Virus testing
Cytomegalovirus (CMV): Not detected
Epstein-Barr virus (EBV): Not detected
Hepatitis B virus (HBV): Not detected
Human Immunodeficiency virus (HIV): Not detected
Human papillomavirus (HPV): Not detected
Functional tests
Genotype testing for STAT1 knockout and BAX knockout. Increased viral titer and enhanced viral production capability relative to parental HEK-293 cell line (ATCC CRL-1573).
Population doubling time
Approximately 20 hrs

History

Depositors
ATCC
Year of origin
2020

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

This material is subject to the following restrictions in addition to those outlined in the ATCC Material Transfer Agreement:

  1. CRISPR Label License 

For information on obtaining additional rights, please contact:

ATCC Licensing
Email: [email protected]

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

Horvath CM, Darnell JE Jr. The Antiviral State Induced by Alpha Interferon and Gamma Interferon Requires Transcriptionally Active Stat1 Protein. J Virol 70: 647-650, 1996. PubMed: 8523587

Meraz MA, et al. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84: 431-442, 1996. PubMed: 860859

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