| Field | Specification |
|---|---|
| Organism | |
| Applications | |
| Tissue | |
| Disease | Embryo |
| Growth properties | |
| Biosafety level | |
| Product format | Frozen |
| Storage | |
| Shipping | |
| Catalog no. (Mfr.) | |
| Main SKU |
Overview
This product is a stable CRISPR cell line in which a GFP reporter has been integrated at the AAVS1 safe harbor locus of HEK293T (Human Embryonic Kidney 293T) cells, with expression driven by the endogenous ACTB (β-Actin) promoter. This design enables real-time, quantitative reporting of endogenous ACTB expression levels without disrupting the native gene locus. Cells are supplied frozen (BSL-II), grow as adherent, epithelial-like monolayers, and are suitable for live-cell imaging, cytoskeletal dynamics studies, and gene expression analyses.
CRISPR Knockout Design
- Target gene / locus: ACTB (Beta-Actin); GFP integrated at the AAVS1 safe harbor locus under the endogenous ACTB promoter
- Engineering strategy: CRISPR/Cas9-mediated knock-in at AAVS1 to stably integrate a GFP reporter construct driven by the endogenous ACTB promoter
- Parental cell line: HEK293T — human embryonic kidney cell line; adherent, epithelial-like; derived from embryonic donor
Gene Background
ACTB (Beta-Actin) is one of the most abundantly expressed proteins in eukaryotic cells and a principal component of the cytoskeleton. As a member of the actin family, ACTB participates in cell motility, structural integrity, intracellular transport, and signal transduction pathways. β-Actin is present in both monomeric (G-actin) and filamentous (F-actin) forms; the dynamic equilibrium between these states is tightly regulated by actin-binding proteins. Due to its constitutive and high-level expression across cell types, ACTB is widely used as a housekeeping gene reference in gene expression studies (Western blot, qPCR, RNA-seq normalization). Research models exploring cytoskeletal dynamics, mechanobiology, and cell migration commonly employ ACTB-tagged reporters.
Cell Culture Specifications
- Recommended vessels
- PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422)
- Culture medium
- DMEM, High Glucose + 10% FBS (do not heat-inactivate) + 1% Penicillin/Streptomycin Solution
- Culture conditions
- 37.0°C, 5% CO₂
- Growth properties
- Adherent, epithelial-like
- Seeding density
- 20,000–30,000 cells/cm²
- Organism
- Human (H. sapiens)
- Tissue origin
- Kidney (embryonic)
- Biosafety level
- BSL-II
- Format
- Frozen; 1×10⁶ cells / 1.0 ml
Stable integration is confirmed by the presence of frameshift-inducing INDELs, verified by Sanger Sequencing.
Research Applications
- Cytoskeletal dynamics: Monitor real-time actin polymerization, depolymerization, and stress fiber formation using live-cell fluorescence imaging of ACTB-GFP.
- Gene expression reporting: Quantify changes in ACTB promoter activity under pharmacological treatments, genetic perturbations, or environmental stimuli by measuring GFP fluorescence.
- Protein co-localization: Use ACTB-GFP as a reference marker for co-localization studies with actin-binding proteins, motor proteins, or structural components.
- Drug screening: Screen compounds that disrupt actin dynamics (e.g., cytochalasin D, latrunculin, jasplakinolide) using GFP intensity and morphology readouts.
Important Notes
- Reporter interpretation: GFP signal reflects endogenous ACTB promoter activity and is not a direct measure of actin filament assembly; correlate with F-actin stains (e.g., phalloidin) for structural analysis.
- Donor/source: HEK293T parental cells originate from human embryonic kidney tissue.
- Viability warranty: Cell viability upon thaw is warranted for 30 days following shipment when handled according to abm's cell handling guidelines.
- Intended use: For laboratory research use only. Not intended for diagnostic, therapeutic, or clinical applications.
- Thaw cells quickly in a 37°C water bath while agitating gently (maximum 2 minutes). The vial cap should be kept above the water level to minimize the risk of contamination.
- Decontaminate the vial by spraying and wiping the exterior of the vial with 70% ethanol. From this point onwards, all operations should be strictly carried out inside a biological safety cabinet using aseptic conditions.
- Transfer the cell suspension into a 15ml sterile conical tube containing 5ml of pre-warmed, complete growth media. Centrifuge cells at 125xg for 5-7 minutes.
- Aspirate the supernatant without disturbing the cell pellet. Re-suspend the cell pellet in the recommended pre-warmed, complete growth media and dispense into a T25 culture flask.
- Incubate the cells at the recommended conditions.
- Aspirate the culture media, and add 2-3ml of pre-warmed 0.25% Trypsin-EDTA to the culture vessel.
- Observe the cells under a microscope to confirm detachment (typically within 2-10 minutes). Cells that are difficult to detach can be put in 37°C, for several minutes to facilitate detachment.
- Neutralize Trypsin-EDTA by adding an equal volume of the complete growth media into the culture vessel.
- Transfer the culture suspension into a sterile centrifuge tube, and centrifuge at 125xg for 5 minutes. The actual centrifuge duration and speed may vary depending on the cell type.
- Aspirate the supernatant, and re-suspend the pellet with pre-warmed fresh complete growth media. Add appropriate aliquots of the cell suspension to new culture vessels, as desired.
- Incubate the cells at the recommended conditions.
How should I handle live cells once I receive them?
https://www.abmgood.com/immortalized-cells-documents.html
Following these guidelines will help ensure optimal cell viability and performance.
What is your warranty or return policy?
Please refer to the following link for full information:
https://www.abmgood.com/terms
For additional questions, our Order team is happy to assist and can be reached at order@abmgood.com.
How many times can cells divide?
Primary cells have a limited lifespan and will undergo a finite number of population doublings before entering senescence. The exact number varies by cell type and culture conditions.
Immortalized cell lines are capable of extended or indefinite proliferation under proper culture conditions, although growth characteristics may vary between lines.
Do I need Applied Cell Extracellular Matrix (G422) if I am using PriCoat™ flasks?
Cell line sourcing and selection (species, tissue, and disease model matching) · Stable cell line engineering (overexpression, knockdown, knockout via CRISPR/Cas9, shRNA, sgRNA) · Reporter gene integration (GFP, RFP, luciferase, fluorescent/bioluminescent constructs) · Genome editing and knockin (point mutations, tagged endogenous proteins, conditional alleles) · Inducible expression systems (Tet-On/Off and regulatable constructs) · Drug resistance marker selection (puromycin, G418, hygromycin, and others) · Custom growth and media optimisation for specific assay requirements · Scale-up production for high-throughput screening campaigns · Authentication and QC services (STR profiling, mycoplasma testing, viability assessment). Talk to a Scientist or contact support@biohippo.com.
