Table of Contents
How are CHO cells transfected?
CHO cells have a high transfection efficiency rate compared with other mammalian expression systems. Can be transfected through viral means to introduce genomic DNA for target proteins.
How do you do a stable transfection?
Ensure that only one cell is present per well after the transfer.
- Step 1 : Transfect cells. Transfect the cells using the desired transfection method.
- Step 2 : Passage cells with antibiotic.
- Step 3 : Monitor for cell “islands”
- Step 4 : Isolate colonies.
- Step 5 : Transfer single cells.
How do you create a stable transfection?
Stable cell line generation protocol
- Generate a kill curve to determine the optimal selection antibiotic concentration.
- Transfect cells with desired plasmid construct(s)
- Select and expand stable polyclonal colonies.
- Identify single clones by limited dilution and expansion.
- Transfer clones and assess expression.
How do CHO cells work?
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.
How are CHO cell lines made?
Genetic manipulation This genetic selection scheme remains one of the standard methods to establish transfected CHO cell lines for the production of recombinant therapeutic proteins. The process begins with the molecular cloning of the gene of interest and the DHFR gene into a single mammalian expression system.
How are Cho immortalized?
Already used as a laboratory animal in previous decades, these hamsters had been a good animal model in, for example, radiation studies. Upon culture in vitro, the cells spontaneously immortalized3, paving the way for unlimited expansion and use at the largest scales.
Are CHO cells adherent or suspension?
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.
What is a stable cell line?
Generation of a stable cell line refers to the process of developing homogenous populations of cells that demonstrate expression of a transfected gene insert. The transfected gene integrates into the genome of the host cell, and as a result, are able to express the transfected genetic material.
How do you make a cell stable?
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.
Are CHO cells transformed?
The expressed transgene products enabled the transformed CHO cell lines to grow in up to 1000 ng mL(-1) oligomycin, while untransformed sensitive CHO cells were eliminated in 1 ng mL(-1) oligomycin.
How do you make CHO cells adherent?
CHO Cell Storage Freeze CHO cells in complete growth medium with 5-10% DMSO at a concentration of 0.5-1 x 107 cells/mL if growing in suspension. For adherent cells, freeze cells in modified medium containing 10% DMSO with a cell concentration of approximately 1 x 106 cells/vial.
Why are CHO cell lines the most suitable host system for recombinant protein production?
What is stable transfection?
Stable Transfection. Unlike transient transfection, in which introduced DNA persists in cells for several days, stable transfection introduces DNA into cells long-term. Stably transfected cells pass the introduced DNA to their progeny, typically because the transfected DNA has been incorporated into the genome,
How is CHO cell line used in transfection?
Transfection experiments using the CHO cell line are used to produce recombinant proteins by clinical researchers trying to develop novel drugs and therapies.
What is the Optimized Protocol for a 24-well plate to transfect CHO cells?
The kit is optimized to transfect miRNA, siRNA or DNA plasmid following either a standard or reverse protocol. 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 Optional: Add 2 µl of Complex Condenser.
Why are preliminary experiments with transient expression performed in CHO cells?
Because making suitable CHO cell lines is time-consuming and costly, -preliminary experiments with transient expression are usually performed to optimize as many protein -production parameters as possible.