How do you transfect CHO cells?
The optimized protocol for a 24-well plate to transfect CHO cells is here:
- Plate 10,000-15,000 CHO cells per well in 0.5 ml of complete growth medium 12-24 hours prior to transfection.
- Wash with 1xPBS and add 0.5 ml of fresh growth medium.
Are CHO cells easy to transfect?
From there, they are ideal for transfection technology: CHO cells have a high transfection efficiency rate compared with other mammalian expression systems.
What is the purpose of transient transfection?
Transient transfection often is used for studying the effects of short-term expression of genes or gene products, such as gene knockdown or silencing with inhibitory RNAs, or protein production on a small scale.
What is transient transfection assay?
Abstract. In conclusion, transient transfection is an efficient and powerful method to determine quickly whether a gene has a detrimental effect on cell survival. We have described a variety of assay systems from which to choose. Each system has its own advantages and disadvantages.
How do CHO cells grow?
CHO Subculture Protocol (adherent)
CHO cells grow quickly and easily and cell doubling time is 14-17 hours. Add 3 mL of Trypsin-EDTA to flask and watch for cell layer detachment under an inverted microscope. This should occur within 5-15 minutes. Do not agitate cells during this type as agitation encourages clustering.
What is lipofectamine transfection?
Lipofectamine™ Transfection Reagent is a value alternative for transfection of plasmid DNA into eukaryotic cells with a simplified protocol. For optimal results in a wider range of plasmid DNA transfection conditions, we recommend Lipofectamine™ LTX Reagent, due to its optimal balance of potency & low-cytotoxicity.
How are CHO cells obtained?
The CHO and CHO-K1 cell lines can be obtained from a number of biological resource centres such as the European Collection of Cell Cultures, which is part of the Health Protection Agency Culture Collections.
Why are CHO cells so commonly used?
The Advantages of CHO Cells
Grow well in suspension and as adherent culture, rendering the cells ideal for GMP procedures. Their tolerance to variations in pH, oxygen levels, temperature or pressure make them the ideal cell for large-scale culture. High recombinant protein yields and specific productivity.
What is the difference between stable and transient transfection?
In stable transfection, the plasmid DNA successfully integrates into the cellular genome and will be passed on to future generations of the cell. However, in transient transfection, the transfected material enters the cell but does not get integrated into the cellular genome.
What are the two types of transfection?
Generally, transfection can be classified into two types, namely stable and transient transfection (Kim & Eberwine, 2010; Stepanenko & Heng, 2017).
What is the doubling time of CHO cells?
The doubling time is typically 24 hours for contact-inhibited adherent CHO-K1 cultures but can decrease to nearly half that for suspension CHO cells.
How do CHO cells grow in suspension?
CHO Cell Culture. CHO cells can be maintained as a suspension or as adherent to a substrate. For suspension, maintain cells in culture by revolving cultures continuously at approximately 50 RPM. Cell lines should be kept in such 10 mL “roller cultures”.
Is Lipofectamine a LNP?
Lipofectamine transfection reagent is currently the most popular LNP forming reagent used to successfully deliver CRISPR/Cas9.
What is the difference between lipofectamine 2000 and 3000?
Lipofectamine 3000 reagent yields higher transfection efficiencies than Lipofectamine 2000 reagent when tested in a variety of cell lines.
What media is used for CHO cells?
CHO cells should be cultured in Ham’s F12K (ATCC suggestion) or DMEM modified with 10% FBS. If cells are not doubling every 14-17 hours, supplement the medium with 1-2% FCS. Subculture Protocol for CHO: CHO cells grow quickly and easily and cell count should have a doubled within 14-17 hours.
Why CHO cells are preferred?
Their tolerance to variations in pH, oxygen levels, temperature or pressure make them the ideal cell for large-scale culture. High recombinant protein yields and specific productivity. Thanks to genetic optimization, protein yields of 3-10 grams per liter of cell culture.
Why are BHK 21 cells used?
The BHK-21 cells are useful for transformations and for stable and temporary transfections. BHK cells are also used to study viral infections.
How do you get transient transfection?
Transient 293 and CHO cell transfection
- Rapidly go from cloned gene to large quantities of protein in 3-7 days.
- Obtain fully post-translationally modified and active mammalian proteins.
- Use your available mammalian cell culture facilities with shake flasks and a platform shaker.
What are advantages of stable transfection over transient transfection?
What is the difference between transient and stable transfection?
In transient systems, foreign DNA, unable to replicate independently from the host’s DNA, persists only for a few days. In contrast, with stable transfection, foreign DNA is integrated into the genome, replicated alongside it, and, more importantly, passed down to the progeny.
How do you freeze down CHO cells?
CHO Cell Storage
Cells should be frozen at a slow rate using a freezing vial (i.e. -1°C per minute in a -80°C freezer). Store the frozen cell vials in the liquid nitrogen vapor phase.
Are CHO cells adherent?
Fact #7 – CHO cells are adherent … and so much else
Such cell types can be subcultured by simply taking a small volume of the parent culture and diluting it in a fresh growth medium. But to get back to the heart of the matter: CHO cells are adherent and can also be grown in suspension.
Are CHO cells adherent or suspension?
Why is lipofectamine used?
Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection.
How do you increase lipofectamine transfection efficiency?
Improving the Success of Your Transfection
- Transfect healthy, actively dividing cells at a consistent cell density.
- Transfect using high-quality DNA.
- Optimize the amount of DNA used to transfect cells.
- Optimize the transfection reagent:DNA ratio.
- Optimize cell number per well when transfected.