Precise Point Mutation Cell Lines

Q: What's your approach for introducing silent mutations in PAM sites when engineering temperature-sensitive variants? How do you validate the temperature-dependent phenotype?

We design silent mutations using advanced computational tools to disrupt PAM sites without altering amino acid sequences. Temperature sensitivity validation involves phenotype assays measuring growth rate or protein activity at permissive versus restrictive temperatures, complemented by functional sequencing. For example, temperature-sensitive BRAF mutants would be assessed for MAPK pathway activity via p-ERK Western blot under varying temperature conditions.

Q: For BRAF V600E mutation in melanoma cell lines, how do you verify the activation status of the downstream MAPK pathway post-editing? Do you include Western blot validation?

We confirm BRAF V600E functionality and MAPK pathway activation through Western blot analysis of phosphorylated ERK (p-ERK), a key pathway marker. Additionally, we use VE1 antibody staining to validate the BRAF V600E mutation specifically. These validations ensure the edited cells exhibit expected pathway activation, which is critical for modeling therapeutic responses accurately.

Q: What's your protocol for introducing multiple point mutations within the same exon of TP53? Can you achieve this without affecting the surrounding regulatory elements?

Our approach combines multiplex gRNA design with our proprietary α-donor system to introduce multiple mutations within a single exon. We utilize homology-directed repair (HDR) to ensure precision and avoid disruption of adjacent regulatory elements. Post-editing confirmation includes Sanger sequencing and functional assays, such as CypD interaction checks for TP53 exon-6 truncations, to maintain genomic integrity.

Q: For iPSC lines, what's your strategy to maintain pluripotency markers (OCT4, NANOG) during the gene editing process? Do you provide flow cytometry data for stemness verification?

We prioritize pluripotency maintenance by optimizing the delivery of gene modification components to minimize cellular stress, and employing footprint-free editing through our proprietary α-donor system. Post-editing validation includes immunofluorescence staining for OCT4, NANOG, and SOX2. While our standard reports include immunofluorescence images, we can provide quantitative flow cytometry data for stemness verification upon request.

Q: Can Cyagen achieve homozygous KRAS G12D mutations in HCT116 cells while maintaining their microsatellite instability (MSI) status? What's your validation method for MSI verification post-editing?

Yes, our Cell Gene Editing System with targeted gene editing technology enables precise homozygous KRAS G12D mutations in HCT116 cells while preserving MSI status. We validate MSI stability using PCR-capillary electrophoresis to analyze five microsatellite loci (including BAT25 and BAT26), following standardized colorectal cancer research protocols. This ensures no unintended genomic disruptions occur during the editing process.

Point Mutations Cell Lines

Engineer disease-specific cell lines with Cyagen's targeted gene editing technology, delivering validated point mutations in just 6 weeks. Achieve up to 87% HDR efficiency across challenging cell types, backed by comprehensive QC documentation and guaranteed homozygous clones.

Comprehensive Deliverables

Receive homozygous clones with detailed QC reports and biweekly updates.

Flash Turnaround

Obtain homozygous clones in as fast as 6 weeks.

Optimized α-Donor System

Achieves HDR efficiency up to 87% for precise point mutations.

Overview

Workflow

FAQs

Overview

Point Mutation Cell Line: Precision Meets Efficiency

Cyagen's targeted gene editing technology delivers precise genomic modifications with unprecedented efficiency. Our validated protocol achieves 87% HDR rates and 70% homozygous outcomes across diverse cell lines, enabling rapid disease modeling and drug discovery with comprehensive quality validation.

Explore Ready-to-Use Mouse Models

Discover over 18,000 validated mouse strains—including knockout, conditional knockout, and humanized models—covering 20+ research areas such as oncology, neurology, and metabolism. All models are supported by detailed genotype data and guaranteed quality, helping you fast-track discovery with confidence.

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Workflow

Workflow and Delivery

Our cell line development services provide a comprehensive and streamlined workflow for generating genetically modified cell lines, ensuring quality, reproducibility, and fast delivery. Below, you will find detailed descriptions of the workflows, estimated timelines, and deliverables for each type of cell line, tailored to your research needs.

Point Mutation Cell Line Development & Delivery

Cyagen’s Point Mutation Cell Line development workflow ensures precise, efficient gene editing. From initial design to final QC, we offer a transparent, quality-controlled process to deliver reliable point mutation cell lines within the expected timeframe.

Deliverables & QC

Cell Type	Example Cell Lines	Deliverables	Quality Control (QC)
Tumor & Immune Cells	Jurkat, HepG2, SK-MES-1	Point mutation clones + matched WT controls (2 vials each, 10⁶ cells/vial) + validation report	PCR, Sanger sequencing
Non-Cancerous Cells	HK-2, AC16		PCR, Sanger sequencing

Workflow & Timeline

Stage	Description	Turnaround Time
Cell Expansion & QC	1. Mycoplasma and sterility testing. 2. Cell proliferation assessment. 3. Primer design for sequencing the target region.	1-2 weeks

Guide Molecule & Donor Vector Design	1. Design and synthesize guide molecules for gene targeting. 2. Construct donor template.	2-5 weeks
Electroporation & Clone Selection	1. Electroporate guide molecules into target cells. 2. Screen single clones, extract genomic DNA, and validate point mutation through PCR and sequencing.	4-6 weeks
Cryopreservation & Final QC	1. Sequence analysis of the mutation site. 2. Mycoplasma and sterility testing. 3. Viability testing.	1-2 weeks

Note:

Total Estimated Time: 8-15 weeks
Time may vary depending on cell type and project complexity.

iPSC Point Mutation Cell Line Development & Delivery

Our iPSC Point Mutation Cell Line development process is optimized for high-quality gene editing while maintaining the pluripotency of the cells. With a dedicated workflow and expert oversight, we ensure robust, validated iPSC lines that meet the rigorous standards of your research.

Deliverables & QC

Cell Type	Example Cell Lines	Deliverables	Quality Control (QC)
iPSC Point Mutation Cell Lines	H1, H9, iPSC	Differentiated or undifferentiated cell lines	PCR, Sanger sequencing, Immunofluorescence (IF)

Workflow & Timeline

Stage	Description	Turnaround Time
Cell Expansion & QC	1. Mycoplasma and sterility testing. 2. Cell proliferation assessment. 3. Primer design for sequencing the target region.	1-2 weeks
Guide Molecule & Donor Vector Design	1. Design and synthesize guide molecules for gene targeting. 2. Construct donor template.	2-5 weeks
Electroporation & Clone Selection	1. Electroporate guide molecules into target iPSCs. 2. Screen single clones, extract genomic DNA, and validate point mutation through PCR and sequencing.	4-6 weeks
Cryopreservation & Final QC	1. Sequence analysis of the mutation site. 2. Mycoplasma and sterility testing. 3. Viability testing. 4. Immunofluorescence staining to verify pluripotency markers.	2 weeks

Note:

Total Estimated Time: 8-12 weeks
Turnaround time may vary for specialized iPSC lines.

FAQs

Frequently Asked Questions (FAQs)

What's your approach for introducing silent mutations in PAM sites when engineering temperature-sensitive variants? How do you validate the temperature-dependent phenotype?

For BRAF V600E mutation in melanoma cell lines, how do you verify the activation status of the downstream MAPK pathway post-editing? Do you include Western blot validation?

What's your protocol for introducing multiple point mutations within the same exon of TP53? Can you achieve this without affecting the surrounding regulatory elements?

For iPSC lines, what's your strategy to maintain pluripotency markers (OCT4, NANOG) during the gene editing process? Do you provide flow cytometry data for stemness verification?

Can Cyagen achieve homozygous KRAS G12D mutations in HCT116 cells while maintaining their microsatellite instability (MSI) status? What's your validation method for MSI verification post-editing?

Citation Database

Molecular Therapy: Methods & Clinical Development, March, 2025

Intracranial AAV administration dose-dependently recruits B cells to inhibit the AAV redosing

【Other】

Gut, February, 2025

E-twenty-six-specific sequence variant 5 (ETV5) facilitates hepatocellular carcinoma progression and metastasis through enhancing polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC)-mediated immunosuppression

【Other】

Cell Death & Disease, February, 2025

Mcm5 mutation leads to silencing of Stat1-bcl2 which accelerating apoptosis of immature T lymphocytes with DNA damage

【Other】

Molecular Therapy, February, 2025

Single-cell data-driven design of armed oncolytic virus to boost cooperative innate-adaptive immunity against cancer

【Other】

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