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Stable Cell Line Generation

Custom Gene Expression Cell Lines
Custom Gene Knockdown Cell Lines
CircRNA Overexpression Stable Cell Line Development
Stable Knock-in Cell Line Generation
Stable Knockout Cell Line Generation
OverviewHighlightsServiceCase StudySupportFAQ

Overview

Creative Biogene is dedicated to delivering high-quality, rapid, and cost-effective stable cell line generation services to support protein expression, gene knockdown, and genome editing for specialized research needs. With a wealth of experience, our cell culture scientists focus on fine-tuning every parameter to meet specific requirements, using proprietary expression vectors, advanced high-throughput screening technologies, and animal-component-free processes to generate reliable and adaptable cell lines in reduced timelines.

Service Highlights

Comprehensive Solutions for Stable Cell Line Generation

Creative Biogene offers a robust range of services for stable cell line generation, covering applications such as protein expression, gene knockdown, genome editing, and circRNA overexpression. Our extensive experience spans over 150+ cell lines, including H1299, MCF-7, HEK293, 3D4/2, HT-29, and more, ensuring reliable and scalable solutions for diverse cell lines.

Platform Expertise and Process Optimization

Our service approach is based on a three-phase model:

1. Vector Design and Optimization

  • Codon optimization for enhanced expression.
  • Promoter selection tailored to specific expression needs.
  • Stability elements and advanced selection markers integration.

2. Cell Line Development and Screening

  • Strategic host cell line selection.
  • Transfection/transduction optimization.
  • High-throughput clone screening and expansion.

3. Advanced Gene Editing

  • CRISPR/Cas9 gene editing for precise knockout/knock-in.
  • Custom RNA interference for targeted gene knockdown.

Core Services

Gene Expression Cell Lines

Flexible cassette options, comprehensive clone selection, and high-level protein production for research applications.

Gene Knockdown Cell Lines

Precise shRNA design and validation for sustained gene silencing and extensive screening potential.

CircRNA Overexpression

Specialized service for disease research using structurally stable circular RNAs—comprehensive design, construction, and validation.

Stable Knock-in Cell Line Generation

Supports diverse mutations, comprehensive screening, and quality control across multiple cell types.

Stable Knockout Cell Line Generation

Precise gene knockout capabilities for functional genomics and disease modeling, adaptable to complex cell types.

Client Case Studies & Research Outcomes

Creative Biogene has collaborated with various biotechnology companies and academic researchers to create reliable, functional cell lines for different applications. Our case studies demonstrate how our customized cell line development has contributed to breakthroughs in areas like cancer research, drug discovery, and gene therapy.

Case Study 1

Chimeric Antigen Receptor (CAR) T cell therapy has shown promise in cancer treatment, but time-consuming manufacturing processes often hamper the development of autologous CAR T cells. Allogeneic CAR T cells, while offering a potential solution, are vulnerable to immune rejection and may not persist effectively in the host. In the study, Human hypoimmune (HIP) T cells were generated by CRISPR-Cas9 editing, targeting genes B2M, CIITA, and TRAC to overcome immune rejection. The HIP CAR T cells demonstrated comparable cancer-killing activity in vitro and exhibited similar tumor clearance in immunodeficient mice. In humanized, immunocompetent mice, HIP CAR T cells showed superior persistence, expansion, and durable anti-tumor responses compared to standard allogeneic CAR T cells.

The researchers used Nalm6 fluc+ cells(cat. no. CSC-RR0361, Creative Biogene) to study the effectiveness of CAR T-cell therapies, aiming to enhance tumor cell clearance through immune cell modifications. Nalm6 cells were transduced with firefly luciferase to enable bioluminescence imaging (BLI), providing a method for tracking tumor progression in vivo.

This figure describes the experimental setup, including the injection of Luc+ Nalm6 cells into NSG mice, followed by the administration of CAR T cells and tumor monitoring through bioluminescence imaging BLI.Figure 1. The researchers injected 1 × 10⁶ Luc+ Nalm6 cells into immunodeficient NSG mice via the tail vein, followed by BLI. On day 3, the mice received either allo CAR T cells or HIP CAR T cells. Spleen and bone marrow samples were collected after 27 or 63 days for analysis. (Hu X, et al., 2023)

Case Study 2

Polycystic ovarian syndrome (PCOS) is a prevalent endocrine condition that frequently causes estrogen insufficiency and insulin resistance. The researchers aimed to investigate the role of WNT signaling in regulating estrogen synthesis in PCOS. They found that FZD3 expression was significantly increased in cumulus cells (CCs) from PCOS patients. This upregulation, along with activation of the WNT2/β-Catenin pathway, was closely associated with estrogen deficiency. The researchers used FZD3-overexpressing cells from Creative Biogene to study its impact on estrogen production and found that it impaired FSH-induced estrogen synthesis. This study highlights the potential of targeting FZD3 in CCs as a therapeutic strategy for restoring estrogen production in PCOS. Our stable cell line services, like the FZD3-overexpressing system, offer researchers robust tools to study gene function and signaling pathways in various disease models.

Figure 2: Assessing FZD3 expression, β-Catenin activity, estradiol synthesis, and luciferase activity, Figure 2 shows how FZD3 overexpression affects long-term FSH-stimulated steroidogenesis in COV434 and HEK293 cells. (doi:10.1016/j.bbrc.2017.07.057)Figure 2. The researchers used FSHR-overexpressing HEK293 cells, transfected with FZD3 and related plasmids, to study FZD3's effect on FSH-stimulated steroidogenesis. (Qiao GY, et al., 2017)

Case Study 3

The SARS-CoV-2 epidemic has motivated researchers to look into how the virus infects lung cells, including its interaction with the human ACE2 receptor. The researchers utilized computational approaches to analyze the differences between SARS-CoV and SARS-CoV-2 spike proteins, revealing key alterations that enhance the binding affinity of SARS-CoV-2 to ACE2. To further explore the virus's impact, the researchers employed A549 cells expressing ACE2, which were procured and cultured using Creative Biogene's services.

Figure 3: The experimental setup used to examine the effect of interfacial water in viral infectivity under various temperature settings is shown in Figure 3, where SARS spike protein and SARS-CoV-2 spike protein conjugated to gold nanourchins were compared. (doi:10.1021/acs.langmuir.2c00671)Figure 3. The researchers conjugated SARS-CoV-2 spike protein on gold nanourchins and compared its effects with SARS spike protein using ACE2/TMPRSS-expressing A549 cells to analyze infectivity through FACS and ICP-MS. (Singh AV, et al., 2022)

Service Support

Customized Construction Methods

Stable cell line construction enables effective gene interference, especially for proteins with long half-lives, and prevents artificial results from excessive expression by selecting cells with optimal copy numbers. Stable cell lines also provide inducible expression for lethal genes and are crucial for in vivo applications like tumorigenesis studies. Creative Biogene offers customized construction methods to meet various research needs.

Table 1. Service Specifications Overview

ServiceDetailsMethods
Gene Overexpression Stable Cell LinesConstitutive Expression: Single/Multiple gene expressionLipofectamine™ Transfection
Electroporation
Lentivirus transduction
Inducible Expression Stable Cell LinesTet-On/Tet-Off system
Gene Knockdown Stable Cell LinesshRNA
miRNA Overexpression Stable Cell LinesmiRNA precursors
Gene Knock-out Stable Cell LineCRISPR/Cas9 system
Gene Knock-in Stable Cell LineHomology-directed repair (HDR)/Non-homologous end joining (NHEJ)

Figure 4. Workflow of stable cell line construction

Quality Control

We maintain stringent quality control throughout the development process:

  • Genetic Validation: Sequence verification, copy number analysis, and genomic integration confirmation.
  • Expression Validation: Quantification of RNA and protein expression, stability assessments, and functional verification.
  • Microbial Testing: All cell lines are mycoplasma-free, and sterility tests are conducted as part of our rigorous validation process.

Technical Process

Our services are designed to ensure smooth, efficient, and reliable results. We support every step of the cell line development process:

1Precise vector and gene target selection

2Custom strategy development

3Construct generation for expression/knockdown

4High-throughput vector production

5Stable clone establishment

6Comprehensive molecular validation (Western blot, PCR, sequencing)

7Continuous technical guidance

8Detailed protocol and data documentation

Contact Us

Creative Biogene is a national high-tech enterprise aiming to lead the global biopharmaceutical services industry. Having worked in the biopharmaceutical business for more than ten years, we provide specialized research platforms, state-of-the-art technology, and skilled project management to assist global industrial and academic development. We prioritize client demands and offer customized services to researchers and biopharmaceutical firms, promoting R&D in the sector.

Use Creative Biogene's skilled cell line creation services to expedite your study. Get in touch with us right now to find out how our tailored solutions can help you with your work.

FAQ

Q1: How is the optimal antibiotic concentration determined?

A1: We test various antibiotic concentrations, observe cell death over 7-14 days, and set the selection concentration slightly above the lowest lethal dose.

Q2: How do you assess stable cell line selection efficiency?

A2: We verify selection efficiency through Real-Time qPCR, Western Blot, or flow cytometry to examine target protein expression, and perform limiting dilution to isolate stable clones.

Q3: What selection systems are used for production cell lines?

A3: We utilize systems like QGS (for glutamine-deficient media) and GS/DHFR (for selective pressure with methotrexate) to ensure high stability and productivity in production cell lines.

Q4: What are the Differences Between Transient and Stable Cell Lines?

A4: Transient cell lines feature target genes that are temporarily expressed, as these genes are not integrated into the host cell genome. This results in short-term expression, making transient systems ideal for studies requiring quick, temporary results. Stable cell lines, on the other hand, incorporate the target gene into the host genome, allowing for sustained, long-term expression. For applications demanding reliability and consistent gene expression over time, stable cell lines are the preferred choice. Use transient systems for rapid, short-duration experiments and stable cell lines for long-term reliability.

* For research use only. Not intended for any clinical use.
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