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KC-5077

3A9-mouse-CD3E-mouse-CD3D-KO Cell Line

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Datasheet
54073
Home » 3A9-mouse-CD3E-mouse-CD3D-KO Cell Line

Background of 3A9-mouse-CD3E-mouse-CD3D-KO Cell Line

The mouse CD3D gene encodes the delta chain of the CD3 complex, an essential component of the T-cell receptor (TCR) signaling pathway. This protein plays a pivotal role in T-cell development and activation by facilitating signal transduction following antigen recognition. The CD3D molecule associates with other CD3 chains (epsilon, gamma, and zeta) to form a stable complex that interacts with the TCR alpha/beta or gamma/delta heterodimers. Mutations or deficiencies in CD3D can lead to impaired T-cell function and immune system disorders. Studies have shown that CD3D is crucial for maintaining peripheral T-cell homeostasis and ensuring effective immune responses against pathogens. Understanding the role of CD3D in T-cell biology provides insights into potential therapeutic targets for autoimmune diseases and cancer immunotherapy.

Specifications

Catalog NumberKC-5077
Cell Line Name3A9-mouse-CD3E-mouse-CD3D-KO Cell Line
Clone Number4A3
Host Cell Line3A9-mouse-CD3E-KO-4C2
DescriptionStable 3A9-mouse-CD3E-KO clone with mouse CD3D gene knockout, No.4A3
QuantityOne vial of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing MediumRPMI1640+20% FBS+10% DMSO
Propagation MediumRPMI1640+10% FBS
Selection MarkerNA
MorphologyLymphoblast
SubcultureSplit saturated culture 1:5-1:10 every 1-2 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 16 hours
Mycoplasma StatusNegative

Cell Line Generation

3A9-mouse-CD3E-mouse-CD3D-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 3A9-mouse-CD3E-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of 3A9-mouse-CD3E-KO cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of 3A9-mouse-CD3E-mouse-CD3D-KO cell line stable clone using PCR sequencing.

Figure 4: Characterization of 3A9-mouse-CD3E-mouse-CD3D-KO cell line stable clone using RT-PCR sequencing.

Figure 5: Characterization of 3A9-mouse-CD3E-mouse-CD3D-KO cell line stable clone using FACS.

Cell Resuscitation

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

Cell Freezing

  1. Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
  2. Keep the freezing medium on ice and label cryovials.
  3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
  4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
  5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
  6. Aliquot 1 mL of the cell suspension into each cryovial.
  7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
  8. Transfer vials to liquid nitrogen for long-term storage.

References

  1. Roifman CM. CD3 delta immunodeficiency. Curr Opin Allergy Clin Immunol. 2004 Dec;4(6):479-84. doi: 10.1097/00130832-200412000-00002. PMID: 15640687.
  2. Wucherpfennig KW, Gagnon E, Call MJ, Huseby ES, Call ME. Structural biology of the T-cell receptor: insights into receptor assembly, ligand recognition, and initiation of signaling. Cold Spring Harb Perspect Biol. 2010 Apr;2(4):a005140. doi: 10.1101/cshperspect.a005140. Epub 2010 Mar 17. PMID: 20452950; PMCID: PMC2845206.
  3. Dave VP, Cao Z, Browne C, Alarcon B, Fernandez-Miguel G, Lafaille J, de la Hera A, Tonegawa S, Kappes DJ. CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage. EMBO J. 1997 Mar 17;16(6):1360-70. doi: 10.1093/emboj/16.6.1360. PMID: 9135151; PMCID: PMC1169733.
  4. https://www.ncbi.nlm.nih.gov/gene/12500

Use License Agreement

Research Use Only.
Not for use in diagnostic procedures or therapeutic applications.
Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.

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