PEIone Transfection Reagent
PEIone transfection reagent
PEIone is a linear polyethyleneimine (Polyethylenimine Linear, PEI)-based transfection reagent specifically optimized for high-efficiency AAV production in HEK 293T or HEK 293 cells. As a chemically synthesized, animal-origin-free reagent, PEIone ensures minimal cytotoxicity while maintaining robust transfection performance. Supplied as a sterile, ready-to-use liquid, PEIone is compatible with our pAAVone plasmids, offering broad applicability in AAV vector manufacturing with AAVone system. Its key advantages include:
High DNA-binding capacity with our pAAVone plasmids (it also works with traditional triple-plasmid transfection method)
Low transfection complex ratio, reducing reagent waste
Exceptional complex stability, ensuring consistent transfection efficiency
These properties make PEIone an ideal choice for scalable AAV production, supporting both research and industrial-scale applications.
| Name | SKU | Price | Buy |
|---|
Shipping and Storage: PEIone Transfection Reagent products should be stored at 2-8℃ for 2 year.
PEIone has superior transfection efficiency at lower reagent-to-DNA ratios
PEIone demonstrates superior transfection efficiency at lower reagent-to-DNA ratios compared to conventional PEI reagents, reducing costs and minimizing cytotoxicity.
| Cell Culture Format | Transfection Reagent | Recommended PEI/pDNA Ratio |
| Suspension HEK293 cells | PEIone | ~0.6: 1 |
| Standard PEI | ~2 : 1 | |
| Adherent cells (e.g., HEK293 / HEK293T) | PEIone | ~1.5 : 1 |
| Standard PEI | ≥3 : 1 |
PEIone used as the transfection reagent for suspension-cultured cells. The pAAVone-AAV9-CMV-EGFP (2.8 kb) plasmid was transfected into suspension-cultured HEK293one cells at a DNA input of 0.35 µg per 1 × 10⁶ cells using PEIone, and EGFP expression was observed 72 hours post-transfection.
PEIone used as the transfection reagent for adherent-cultured cells. The pAAVone-AAV9-CMV-EGFP (2.8 kb) plasmid was transfected into adherent-cultured HEK293one cells at a DNA input of 0.5 µg/well (24-well plate) using PEIone or other PEI-Based competitor, and EGFP expression was observed 72 hours post-transfection.
PEIone produces high AAV titers in suspension HEK293one cells
The pAAVone-AAV9-CMV-EGFP (2.8 kb) plasmid was transfected into suspension-cultured HEK293one cells using varying DNA inputs and different PEIone-to-pDNA ratios. AAV titers were measured 72 hours post-transfection by qPCR using EGFP-specific primers.
PEIone produces high AAV titers in adherent HEK293T cells
PEIone achieves high AAV titers at an approximate PEIone:pAAVone ratio of 1.5:1 in adherent HEK 293T cells,
PEIone is capable of maintaining transfection efficiency even at high cell confluency in adherent HEK 293T cells
PEIone's performance at high cell confluency in adherent HEK 293T cells
Protocol of PEIone
1. Required Materials
- HEK 293 or HEK 293T cells (suspension or adherent).
• Plasmid DNA (pAAVone® plasmids or other plasmids for AAV production; 0.5–5 µg/µL stock).
• Appropriate cell culture medium.
2. Preparing Cells
- Suspension cells: Grow cells to a density of 4.0–6.0 × 10⁶ viable cells/mL with ≥95% viability. Dilute to 2.5–3.5 × 10⁶ viable cells/mL using pre-warmed medium at the time of transfection.
- Adherent cells: Seed cells to reach 70–90% confluence at the time of transfection.
3. Transfection Protocol for 240 ml culture
- Bring PEIone™ and culture medium to room temperature.
- Dilute plasmid DNA in culture medium to ~5% of the culture volume (e.g., 360 µg DNA in 12 mL medium for 240 mL culture). Mix gently.
- In a separate tube, dilute PEIone™ reagent in culture medium to ~5% of the culture volume (e.g., 126 µL PEIone™ reagent in 12 mL medium for 240 mL culture). Mix gently.
- Add diluted PEIone™ solution to the diluted DNA solution. Mix gently.
- Incubate for 15–30 minutes at room temperature to allow complex formation.
- Add complexes directly to the culture vessel.
- Incubate cells for about 68-72 hours, then harvest according to in-house AAV production protocols.
Recommended DNA and Reagent Amounts
- The amount of PEIone™ and DNA, and their ratios should be optimized for your AAV genomes and serotypes/capsids to obtain maximum productivity.
Typical PEIone™/ pAAVone® Ratios:
- Suspension HEK 293 or HEK 293T cells: PEIone™/DNA = 0.5:1–1:1
- Adherent HEK 293 or HEK 293T cells: PEIone™/DNA = 1:1–2:1
Typical pAAVone® amount:
- Suspension HEK 293 or HEK 293T cells: 0.3-0.6 µg per 1 × 10⁶ cells
- Adherent HEK 293 or HEK 293T cells: 0.3-0.6 µg per well (24-well plate)
Typical PEIone™ amount:
- Suspension HEK 293 or HEK 293T cells: 0.2-0.5 µl per 1 × 10⁶ cells
- Adherent HEK 293 or HEK 293T cells: 0.5-1.0 µl per well (24-well plate)
Table 1. Recommended DNA amounts and reagent volumes for transfection of pAAVone® plasmid at various scales in suspension HEK 293/293T cells
| Vessel type | ||||
6-well | 125 mL flask | 250 mL flask | 500 mL flask | 1 L flask | |
Cell number | 6 × 10⁶ | 90 × 10⁶ | 180 × 10⁶ | 360 × 10⁶ | 720 × 10⁶ |
Culture volume | 2 mL | 30 mL | 60 mL | 120 mL | 240 mL |
DNA amount | 3 µg | 45 µg | 90 µg | 180 µg | 360 µg |
PEIone™ | 2.1 µL | 31.5 µL | 63 µL | 126 µL | 252 µL |
Dilution medium | 2 × 100 µL | 2 × 1.5 mL | 2 × 3 mL | 2 × 6 mL | 2 × 12 mL |
Table 2. Recommended DNA amounts and reagent volumes for transfection of pAAVone® plasmid at various scales in adherent HEK 293/293T cells.
| Vessel type | |||||
24-well | 12-well | 6-well | T-25 flask | T-75 flask | 100 mm dish | |
Culture medium | 0.5 mL | 1 mL | 2 mL | 4 mL | 10 mL | 10 mL |
DNA amount | 0.5 µg | 1 µg | 2 µg | 4 µg | 8 µg | 8 µg |
PEIone™ | 0.75 µL | 1.5 µL | 3 µL | 6 µL | 12 µL | 12 µL |
Dilution medium | 2 × 25 µL | 2 × 50 µL | 2 × 100 µL | 2 × 200 µL | 2 × 0.5 mL | 2 × 0.5 mL |
Notes
For triple-plasmid AAV systems (AAVtri), the total plasmid amount required is typically higher than that for pAAVone®. The ratio of the three plasmids should be optimized for maximum AAV productivity. A starting molar ratio of 1:1:1 for each plasmid is recommended for initial testing.