KC-3957

293T-cyno-CRLF2 Cell Line

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Background of 293T-cyno-CRLF2 Cell Line

CRLF2 (Cytokine Receptor Like Factor 2) is a Protein Coding gene,he encoded protein is a receptor for thymic stromal lymphopoietin (TSLP).T Together with the interleukin 7 receptor (IL7R), the encoded protein and TSLP activate STAT3, STAT5, and JAK2 pathways, which control processes such as cell proliferation and development of the hematopoietic system. Diseases associated with CRLF2 include Lymphoblastic Leukemia, Acute, With Lymphomatous Features and Down Syndrome.

Specifications

Catalog Number	KC-3957
Cell Line Name	293T-cyno-CRLF2 Cell Line
NCBI/UniProt Accession Number	XM_045383695.1
Clone Number	2#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous cyno CRLF2 gene
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	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-cyno-CRLF2 cell line was generated using a lentiviral vector expressing the cyno CRLF2 sequence.

Characterization

Figure 1: Characterization of cyno CRLF2 overexpression in the 293T-cyno-CRLF2 stable clone using QPCR.

Figure 2: Characterization of cyno CRLF2 in the 293T-cyno-CRLF2 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 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. Maciel ALT, Poubel CP, Noronha EP, Pombo-de-Oliveira MS, Mansur MB, Emerenciano M. CRLF2 expression associates with ICN1 stabilization in T-cell acute lymphoblastic leukemia. Genes Chromosomes Cancer. 2019 Jun;58(6):396-401.
2. Konoplev S, Lu X, Konopleva M, Jain N, Ouyang J, Goswami M, Roberts KG, Valentine M, Mullighan CG, Bueso-Ramos C, Zweidler-McKay PA, Jorgensen JL, Wang SA. CRLF2-Positive B-Cell Acute Lymphoblastic Leukemia in Adult Patients: A Single-Institution Experience. Am J Clin Pathol. 2017 Apr 1;147(4):357-363.
3. Nakajima S, Kabata H, Kabashima K, Asano K. Anti-TSLP antibodies: Targeting a master regulator of type 2 immune responses. Allergol Int. 2020 Apr;69(2):197-203.