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KC-0935

MIAPaCa2-KRAS-G12C-HiBiT-1B4 Cell Line

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Home » MIAPaCa2-KRAS-G12C-HiBiT-1B4 Cell Line

Background

Kirsten rat sarcoma viral oncogene homologue (KRAS) is the best-known oncogene with the highest mutation rate among all cancers and is associated with a series of highly fatal cancers, including pancreatic ductal adenocarcinoma (PDAC), nonsmall-cell lung cancer (NSCLC), and colorectal cancer (CRC). The identification of tumor driver genes and the development of specific inhibitors have revolutionized cancer treatment strategies and clinical outcomes.

Specifications

Catalog Number:
KC-0935
Cell Line Name:
MIAPaCa2-KRAS-G12C-HiBiT-1B4 Cell Line
Host Cell Line:
Human MIAPaCa2 cell line
Description:
MiaPaCa2 cell line stable clone with KRAS gene N-terminal knock-in HiBiT
Quantity:
One vial of frozen cells (5×106 per vial)
Stability:
Stable in culture over a minimum of 10 passages
Application:
Drug screening and biological assays
Freeze Medium:
70% DMEM+20% FBS+10% DMSO
Propagation Medium:
DMEM+10%FBS+2.5%HS
Selection Marker:
Morphology:
Epithelial
Subculture:
Split saturated culture 1:3-1:6 every 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 48 hours
Mycoplasma Status:
Negative

Cell Line Generation

MiaPaCa2-KRAS-HiBiT-KI cell line was generated using CRISPR method

Characterization

Figure 1: Characterization of MiaPaCa2-KRAS-HiBiT-KI Cell Line stable clone using PCR sequencing. Figure 2: Characterization of MiaPaCa2-KRAS-HiBiT-KI Cell Line stable clone using functional assay. V． Application In vitro and in vivo studies VI． Cell Resuscitation 1. Prewarm culture medium (DMEM supplemented with 10% FBS and 500ug/ml hygromycin) in a 37°C water bath. 0h 24h 48h 72h 0 1000 2000 3000 4000 HiBIT Degradation by K

Application

Figure 2: Characterization of MiaPaCa2-KRAS-HiBiT-KI Cell Line stable clone using functional assay.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 2.5% HS)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 1:3-1:6 every 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. Lamei Huang, Zhixing Guo, Fang Wang & Liwu Fu. "KRAS mutation: from undruggable to druggable in cancer". Signal Transduction and Targeted Therapy (2021) 6:386 ; https://doi.org/10.1038/s41392-021-00780-4.

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