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KC-6312

SP2/0-mRosa26-mCherry-M22-KI Cell Line

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Home » SP2/0-mRosa26-mCherry-M22-KI Cell Line

Background of SP2/0-mRosa26-mCherry-M22-KI Cell Line

Based on the previously constructed SP2/0-mRosa26-Lox71-GFP-Lox2272 tool cell line, we utilized the Cre-LoxP heterotypic recombination system to achieve precise site-specific substitution of the GFP expression cassette, thereby generating a stable mCherry-M22 labeled cell line. The successful cassette replacement verified that this tool cell platform enables accurate, unidirectional, and irreversible exogenous gene recombination without disrupting the endogenous genomic background. Benefiting from genetic editing at the mRosa26 genomic safe harbor locus, this system maintains stable genetic background and consistent transgene expression, effectively eliminating the defects of random integration, gene silencing and unstable expression commonly observed in traditional cell engineering. The established mCherry-M22 cell line possesses reliable fluorescent labeling characteristics, allowing real-time visual tracking and quantitative monitoring of myeloma cell growth, proliferation and migration both in vitro and in vivo. This standardized editing platform provides a valuable tool for investigating the molecular mechanisms of myeloma progression, facilitating cell tracing analysis, and supporting functional verification of target genes. Moreover, this flexible gene replacement system can be widely applied to subsequent cell engineering modification and functional research, providing a stable and universal experimental platform for myeloma basic research and related translational studies.

Specifications

Catalog NumberKC-6312
Cell Line NameSP2/0-mRosa26-mCherry-M22-KI Cell Line
Clone Number1A1
Host Cell LineSP2/0-mRosa26-Lox71-GFP-High-Lox2272-KI
DescriptionBased on the established SP2/0-mRosa26-Lox71-GFP-Lox2272 tool cells, the GFP expression cassette was site-specifically replaced via the Cre-LoxP heterotypic recombination system, and mCherry-M22 dual-labeled cell line was successfully obtained.
QuantityOne vial of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% RPMI1640+20% FBS+10% DMSO
Propagation MediumRPMI1640+10% FBS
Selection MarkerNA
Morphologylymphoblast
SubcultureSplit saturated culture 1:2-1:4 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 30 hours
Mycoplasma StatusNegative

Cell Line Generation

SP2/0-mRosa26-mCherry-M22-KI cell line was generated via the CRISPR method and Cre-LoxP heterotypic recombination system.

Characterization

Figure 1: Characterization of SP2/0-mRosa26-mCherry-M22-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of the stable SP2/0-mRosa26-mCherry-M22-KI cell clone by fluorescence microscopy.

Figure 3: Characterization of SP2/0-mRosa26-mCherry-M22-KI Cell Line stable clone using ELISA.

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 1:2-1:4 every 2-3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

  1. Prepare the freezing medium (70% RPMI1640 + 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. Daly, A. Z., Mortensen, A. H., Bando, H., & Camper, S. A. (2021). Development of Rosa26LSL-SV40-GFP mice. Endocrinology, 162(7), bqab073.DOI: 10.1210/endocr/bqab073. PMID: 33834207 PMCID: PMC8183496
  2. Tian, X. Y., & Zhou, B. (2021). Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution. Journal of Biological Chemistry, 296, 100509. DOI: 10.1016/j.jbc.2021.100509

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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