What does PEI do in transfection?
What does PEI do in transfection?
DNA can be introduced into a host cell by transfection with polyethylenimine (PEI), a stable cationic polymer (Boussif et al., 1995). PEI condenses DNA into positively charged particles that bind to anionic cell surfaces.
How do you transfect a virus?
A typical transduction protocol involves engineering of the recombinant virus carrying the transgene, amplification of recombinant viral particles in a packaging cell line, purification and titration of amplified viral particles, and subsequent infection of the cells of interest.
How do you know if the transfection was successful?
Generally, reporter gene assays are performed 1–3 days after transfection; the optimal time should be determined empirically. A functional test for the protein of interest, such as an enzymatic assay, may be another method to determine transfection success.
How do you get the supernatant virus?
Collect viral supernatant by gentle pipetting to remove tissue culture media. All procedures should be carried out in a laminar flow hood to maintain sterility of the viral supernatant of subsequent infection of tissue culture or ex vivo cells.
Can PEI Max be frozen?
PEI-HCl-40K at 1mg/ml working solution: stable at 4 degree for up to 6 months. For long- term storage, put 3-5ml aliquots in -80 or -20 degree. Once thawed completely, filter through 0.22um filter, aliquot and keep at 4 degree. Do not freeze again.
What does PEI Pro do?
Summary. PEIpro® product range (PEIpro®, PEIpro®-HQ and PEIpro®-GMP) is the transfection method of choice to achieve reliable viral vector production and high infectious titer yields, with direct scalability and seamless transition from process development up to large-scale clinical-grade manufacturing.
What is the purpose of transfection?
The main purpose of transfection is to study the function of genes or gene products, by enhancing or inhibiting specific gene expression in cells, and to produce recombinant proteins in mammalian cells [3].
Can you freeze transfected cells?
Cryopreservation of transfected cells decreases the time required to perform assays by reducing the time spent transiently transfecting cells. Therefore, reducing the assay to data cycle time enables chemists to make synthetic decisions more rapidly and thus facilitates the lead opti- mization process.
When can I collect lentivirus after transfection?
After transfection with Lenti plasmids we harvest the supernatant after 4 days.
What is virus loading?
Viral load refers to the amount of virus that can be detected in an infected person. High viral loads are concerning because they can mean the person is more infectious.
Why is Pei used for DNA transfection?
Consequently, the DNA:PEI complex is endocytosed by the cells and the DNA released into the cytoplasm (Sonawane et al., 2003). Our laboratory uses PEI over other cell transfection reagents because of its low cost. This protocol is appropriate for two suspension cell lines, CHO-S and HEK 293 GnTi-.
What cell lines can be transfected with Pei?
This protocol is appropriate for two suspension cell lines, CHO-S and HEK 293 GnTi-. Many cell lines can be transfected successfully with PEI but in our experience these two cell lines express the highest level of protein compared to other cells.
How can I prepare cells for transfection from dissolved Pei?
Once fully dissolved PEI can be sterile filtered using a 0.22 µM syringe filter, aliquoted, and frozen at -20°C until needed. For best transfection efficiency, cells should have a viability of >95% at the time of transfection. incubator with shaking at 37°C. Cell density should be ~2 x 106 at the time of transfection.
What is the concentration of Pei in freestyle™ 293 medium for transfection?
A dilution of the 1 mg/ml PEI stock is prepared to a final concentration of 0.5 µg/µl in Freestyle™ 293 Medium. Example: PEI stock concentration = 1 mg/ml For 50 ml transfection (final volume = 100 ml) you need 450 µg PEI at a concentration of 0.5 µg/µl.
