Overexpression Stable Cell Line
VectorBuilder can engineer cell lines to stably over-express your gene of interest (GOI) to help you overcome variations associated with transient transfection. Gene overexpression cell lines are generated by lentiviral transduction followed by antibiotic selection. The GOI expression level is validated by RT-qPCR. Additionally, a series of standard QC assays such as sterility tests and mycoplasma detection are performed before releasing the final cell line products. Other validation assays such as Western blot and immunocytochemistry can be performed upon request.
Highlights
- Permanent integration: lentiviral transduction allows for stable integration into the genome of host cells and all progeny.
- Broad tropism: VSV-G pseudotyped lentivirus is able to bind to surface receptors on most mammalian cells, facilitating efficient transduction of your target cells.
- Fast turnaround: engineered cells can be delivered in as fast as 9 weeks starting from vector design.
Service Details
Figure 1. Typical workflow of gene overexpression stable cell line production.
Price and turnaround
| Service Type | Deliverable | Price (USD)* | Turnaround |
|---|---|---|---|
| Gene overexpression | Mixed pool (>106 cells/vial, 2 vials) | From $2,999 | 8-11 weeks |
| Two single clones (>106 cells/vial, 2 vials per clone) | From $3,999 | 13-16 weeks |
* Additional charge will apply for extra single clones or vials.
QC assays
| Assays | Methods |
|---|---|
| Overexpression validation (default） | RT-qPCR |
| Expression test (add-on) | WB, IF, FACS |
| Sterility (default) | PCR for mycoplasma detection, bioburden test for sterility detection |
Downstream services
We can perform various phenotypic assessments and functional validation of your engineered cell lines, including assays for proliferation, apoptosis, migration, viability, cytotoxicity, etc.
Case Studies
Figure 2. Validation of gene overexpression (OE) cell lines. (A) Transgene expression of ID8, CHO-K1, and HEK293 cells transduced with lentiviruses carrying MSLN, B7-H3, and CD19, respectively, analyzed by flow cytometry. (B) Protein expression level of GOI in Caco-2 cells by WB, and (C) luciferase overexpression in MCF7 cells by luciferase assay. (D) Luciferase-expressing Renca cell line-derived xenograft in mice were imaged to trace tumor migration in vivo.
How to Order
Resources
FAQ
What are the advantages and disadvantages of overexpression vs. gene knockin?
| Approach | Advantages | Disadvantages | |
|---|---|---|---|
| Overexpression | Lentivirus | Technical simplicity | Random insertion |
| Knockin | CRISPR (RNP electroporation) | Targeted insertion | Multiple components required |
What are the advantages and disadvantages in using lentiviral transduction over plasmid transfection?
| Advantages | Disadvantages | |
|---|---|---|
| Lentiviral transduction | Genomic integration, permanent expression; Ease of target cell transduction |
Lower carrying capacity (6.4 kb) |
| Plasmid transfection | High carrying capacity (27 kb) | Transient expression, plasmid will dilute after serial passages; Requires electroporation or other transfection process |
What cell types do you support?
We support the engineering of a wide range of immortalized cell lines and maintain an extensive collection of in-house cell lines. We also accept client-provided and commercially sourced cell lines. In addition, we have proven experience in isolating and engineering primary cells from a variety of human, rodent, porcine, and canine tissues.
How do you isolate single clones?
Following transduction or electroporation and subsequent antibiotic selection (if applicable), a pool of positive cells is established and characterized. Cells are then counted and seeded into 96-well plates by limiting dilution at an average density of <1 cell per well to isolate single-cell-derived clones. Individual clones are subsequently expanded and characterized to confirm the desired genetic modification or gene expression.
