Custom Knockin Cell Lines for Accurate Gene Studies

Q: For APOE4 point mutation knockins for Alzheimer's research, can you provide functional validation that the mutation affects protein folding?

We deliver fully sequenced and clonally validated APOE4-mutated cell lines, including protein expression level checks. While functional validation (e.g., folding assays) is not standard, we collaborate with clients to design tailored experiments or recommend downstream partners for phenotypic analysis. Our HepG2 case study highlights our ability to support functional research pipelines.

Q: What specific validation methods do you use to confirm the absence of off-target effects beyond the primary insertion site?

We employ: ●Sanger sequencing to verify target locus integrity. ●PCR-based screening to confirm insert size and orientation. ●Cell viability assays to rule out unintended functional impacts. While whole-genome off-target analysis is not explicitly mentioned, our stringent QC (100% contamination-free cells and dual mycoplasma/bacterial testing) ensures reliable outcomes.

Q: Do you offer humanized protein expression in mouse cell lines for antibody development projects?

Yes. Our KI services include humanized protein expression in mouse cell lines, enabling applications like antibody development. For example, we generated SR-BI mutant HepG2 cells to study humanized protein interactions in mice. Custom requests for species-specific protein swaps or epitope tagging are available.

Q: Can you perform multiple gene edits simultaneously in the same cell line, and how does this affect validation timelines?

Yes, we support simultaneous multi-gene edits, as demonstrated in our case study delivering a double-point mutation HepG2 cell line. Validation timelines may extend slightly due to additional sequencing and quality checks, but our standardized workflow (including single-cell cloning and multi-step sequencing) ensures thorough characterization. Timelines are project-specific and discussed during quoting.

Q: What is your typical efficiency rate for knockin of large inserts (>2kb) in hard-to-transfect cell lines like primary neurons?

Our Cell iGeneEditor™ system achieves up to 90% editing efficiency for gene knockins (KIs). While insert size and cell type influence outcomes, our optimized homologous recombination protocols and extensive experience with diverse cell types (including neuronal lines like SH-SY5Y and U251) ensure robust results. For hard-to-transfect cells, we conduct pre-experiments to assess feasibility and refine delivery methods. Primary neurons are not explicitly listed in our cell bank, but contact us for custom project evaluations.

Knockin Cell Lines

Drive your experiments using site-specific modifications that support gene regulation studies, disease modeling, and drug screening. Our knockin cell lines offer consistent insertion of reporters, point mutations, or fusion tags, delivering validated solutions for actionable insights in your next project.

Rapid Turnaround

Obtain your customized knockout cell line in as little as 2 weeks.

Expert-Guided

Free tech consultation on model selection and experimental alignment in minute

Guaranteed Outcomes

We deliver as promised—or your money back.

Overview

Workflow

FAQs

Overview

Knockin Cell Lines: Defined Genomic Modifications

Cyagen's targeted gene editing cell lines feature documented site-specific DNA insertions at predetermined loci. Each model undergoes rigorous validation with full QC documentation, enabling researchers to study gene function, track proteins, and model diseases with high experimental reproducibility.

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

Cyagen offers a streamlined process for generating Knockin (KI) cell lines, ensuring precise genetic modifications for various research applications. Our workflow covers all stages, from gene design to final cell line validation, with strict quality control at each step. We deliver well-characterized, stable cell lines along with comprehensive reports and sequencing data, providing reliable models for your research.

Knockin Cell Line Development & Delivery

Cyagen provides a streamlined process for Knockin (KI) cell line development, ensuring accurate gene insertion and high-quality models for various research applications. The following workflow outlines the typical steps involved in generating Knockin cell lines, with estimated turnaround time and deliverables.

Deliverables & QC

Cell Type	Example Cell Lines	Deliverables	Quality Control (QC)
Tumor & Immune Cells	Jurkat, HepG2, SK-MES-1	Knockin 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. Donor vector design and construction	5-7 weeks
Electroporation & Clone Selection	1. Electroporate guide molecules into target cells. 2. Screen single clones, extract genomic DNA, and validate the knockin via PCR and sequencing.	4-8 weeks
Cell Cryopreservation & QC	1. Sequence analysis of the knockin site. 2. Mycoplasma and sterility testing. 3. Viability testing.	1-2 weeks

Note:Total Estimated Time: 11-19 weeks

iPSC Knockin Cell Line Development & Delivery

For iPSC Knockin Cell Line development, Cyagen offers an optimized workflow to ensure precise gene insertion while preserving the pluripotency and differentiation potential of the stem cells. Below is the workflow with estimated timelines and deliverables.

Deliverables & QC

Cell Type	Example Cell Lines	Deliverables	Quality Control (QC)
iPSC Knockin 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. Donor vector design and construction	5-6 weeks
Electroporation & Clone Selection	1. Electroporate guide molecules into target iPSCs. 2. Screen single clones, extract genomic DNA, and validate the knockin via PCR and sequencing.	4-8 weeks
Cell Cryopreservation & QC	1. Sequence analysis of the knockin site. 2. Mycoplasma and sterility testing. 3. Viability testing. 4. Immunofluorescence staining to verify pluripotency markers.	1-2 weeks

Note:Total Estimated Time: 11-18 weeks

FAQs

Frequently Asked Questions (FAQs)

For APOE4 point mutation knockins for Alzheimer's research, can you provide functional validation that the mutation affects protein folding?

What specific validation methods do you use to confirm the absence of off-target effects beyond the primary insertion site?

Do you offer humanized protein expression in mouse cell lines for antibody development projects?

Can you perform multiple gene edits simultaneously in the same cell line, and how does this affect validation timelines?

What is your typical efficiency rate for knockin of large inserts (>2kb) in hard-to-transfect cell lines like primary neurons?

Citation Database

Molecular Therapy: Methods & Clinical Development, March, 2025

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

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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

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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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