HEK293 Cell Line: A Guide to Transfection Conditions, Applications, and Culture Setup
HEK293 Cell Line: A Guide to Transfection Conditions, Applications, and Culture Setup
The HEK293 cell line (Human Embryonic Kidney 293) is one of the most widely used mammalian cell lines in life science research and biotechnology. Its high transfectability, fast growth, and adaptability make it a top choice for both academic and industry labs.
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1. HEK293 Cell Line: Type, Growth Characteristics, and Confluency
Common HEK293 Types:
- HEK293 – The original adherent line.
- HEK293T – Expresses SV40 large T antigen for high plasmid replication.
- HEK293FT – A fast-growing version of 293T.
- HEK293F / HEK293E – Adapted for suspension culture, used in protein expression.
Growth Type:
- Most HEK293 variants grow as adherent monolayers under standard conditions.
- Suspension-adapted types (HEK293F) are used in shaker flasks or bioreactors.
Confluency at Transfection:
- For optimal transfection, seed cells to reach 70–90% confluency at the time of transfection.
- Avoid overconfluence (>100%) as it reduces efficiency and cell health.
2. Nucleic Acid Types Used in HEK293 Transfection
HEK293 cells are versatile and can be transfected with various types of nucleic acids:
- Plasmid DNA – For gene expression, protein production, or reporter assays.
- siRNA/shRNA – For gene knockdown studies.
- mRNA – For transient expression without genomic integration.
- CRISPR/Cas9 plasmids or RNPs – For gene editing.
- miRNA mimics/inhibitors – For studying post-transcriptional regulation.
3. Common Applications of HEK293 Transfection
Due to their ease of manipulation, HEK293 cells are widely used in:
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Protein expression: Produce recombinant proteins quickly and efficiently.
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Gene knockdown: Use siRNA or shRNA to silence target genes.
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Genome editing: Deliver CRISPR-Cas9 systems for gene modification.
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Reporter assays: Test promoter activity using luciferase or fluorescent proteins.
4. Culture Format for Transfection
| Culture Format | Use Case | Notes |
| 6-well / 12-well plates | Optimization and small-scale studies | Easy to control conditions and test multiple ratios |
| T25 / T75 flasks | Medium-scale expression | Good for producing protein or viral particles |
| 96-well plates | High-throughput screening | Requires miniaturized transfection protocols |