Construction Principle
The MDA-MB-231-Luc cell line is developed using genetic engineering techniques by inserting the luciferase (luc) gene into an expression vector, followed by stable transfection into parental MDA-MB-231 cells. The luciferase enzyme catalyzes the oxidation of luciferin, emitting bioluminescence with a wavelength of approximately 550-570 nm. The light intensity is linearly correlated with the enzyme expression level.
 
Common systems include the single Luc reporter system and the dual Luc system, which incorporates Renilla luciferase as an internal control to normalize experimental variability.

Common Construction Method
Lentiviral transfection is the most widely used and efficient approach for stable integration and long-term gene expression. The following steps outline a representative workflow (as applied in EDITGENE’s construction of the MDA-MB-231-Luc cell line):

• 1. Vector Construction
  a. The luciferase gene (luc) is cloned into a lentiviral vector (e.g., pLenti).
• b. The vector contains a CMV promoter driving luc expression, a puromycin resistance gene for selection, and a GFP marker for visualization.
• c. For dual-luciferase systems, Renilla luciferase can be co-expressed or integrated into the same plasmid (e.g., pGL3 or pmirGLO) as an internal control.
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• 2.Lentiviral Packaging
  • a. The recombinant vector is co-transfected with helper plasmids (packaging plasmid psPAX2 and envelope plasmid pMD2.G) into HEK293T cells to produce lentiviral particles.
  • b. Viral supernatants are collected, filtered, and titrated (typically 10⁸-10⁹ TU/mL).
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  • 3.Transfection of Parental Cells
  • a. Parental MDA-MB-231 human breast cancer cells are cultured to 70-80% confluence.
  • b. Lentiviral particles are added at an MOI (multiplicity of infection) of 10-50, and cells are incubated for 48-72 hours.
  • c. Transfection efficiency is evaluated by GFP fluorescence or preliminary luciferase activity assays.
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  • 4.Screening of Stable Clones
  • a. 24-48 hours post-transfection, puromycin (2-5 μg/mL) is applied for positive selection.
  • b. The selection process continues for 7-14 days until resistant clones form.
  • c.  EDITGENE employs 3D single-cell printing technology for precise monoclonal isolation, ensuring high accuracy and cell viability while avoiding mechanical stress and dilution errors associated with traditional limiting dilution methods.
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  • 5.Validation and Applications
  • a. Validation methods: qPCR is used to confirm gene integration, Western blotting to verify protein expression, and luciferase assays to assess enzymatic activity.
  • b. Quality criteria: Luciferase activity >1×10⁶ RLU/10⁵ cells, with expression stability maintained for >20 passages.
  • c. Applications: Suitable for in vivo bioluminescence imaging. Cells can be injected into nude mouse models, and after intraperitoneal administration of luciferin, tumor growth and metastasis can be monitored in real time using an IVIS imaging system.

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  IV

  Application Examples: Advancing Research Projects

  4.1 Breast Cancer Metastasis Mechanisms

  • ● Objective:Investigate the role of the Wnt signaling pathway in bone metastasis.
  • ● Method: β-catenin gene knockout was performed, followed by injection of MDA-MB-231-Luc cells. Bone metastasis was monitored in real time using the IVIS imaging system.
  • ● Result: Bioluminescence intensity was quantified to evaluate metastatic burden, providing mechanistic insights.

  4.2 Drug Screening

  • ● Objective: Screen novel PARP inhibitors for efficacy against TNBC.
  • ● Method: High-throughput in vitro drug screening, with luminescence signals used to assess inhibitory effects.
  • ● Result: Rapid generation of IC50 curves, providing robust data to support publication.

  4.3 Immunotherapy

  • ● Objective: Evaluate the therapeutic efficacy of PD-L1 inhibitors combined with nanocarriers.
  • ● Method: Combination therapy was applied in nude mouse models, and tumor burden was dynamically monitored using IVIS imaging.
  • ● Result: Real-time bioluminescence data supports translational research applications.
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  V

  Summary

  The MDA-MB-231-Luc stable cell line is a powerful tool for constructing TNBC metastasis models, enabling real-time monitoring of tumor progression, drug screening, and immunotherapy studies. Its high-sensitivity bioluminescence provides reliable experimental data, accelerating publication and project progress. Unlike time-consuming in-house cell line construction, MDA-MB-231-Luc product optimized by EDITGENE features >90% positive expression, high metastatic efficiency, and comes with a COA and SOP, allowing researchers to rapidly initiate breakthrough studies.

     

  VI

  References

  • 1. Li, X., et al. (2012). Establishment of a bioluminescent MDA-MB-231 cell line for human triple-negative breast cancer research. Zhonghua Bing Li Xue Za Zhi, 41(4), 267–271. DOI: 10.3760/cma.j.issn.0529-1356.2012.04.011.
  • 2. Jenkins, D. E., et al. (2005). Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice. Breast Cancer Research, 7(4), R444–R454. DOI: 10.1186/bcr1026.
  • 3. Holliday, D. L., & Speirs, V. (2011). Choosing the right cell line for breast cancer research. Breast Cancer Research, 13(4), 215. DOI: 10.1186/bcr2889.
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