A majority of cell biology experiments employ transient transfection techniques that allow maximum gene expression from 24-96 hours after transfection. If sustained genes are required over longer time periods and require the creation of Stable Cell Line can be a viable alternative. In addition, AAV Cell Line chosen through minimal Dilution can result in a genetically identical and a clonal population.
Host Cell Line generation is possible due to the use of selection markers that are positive, like hygromycin and G418/Geneticin, zeocin and blasticidin resistance to antibiotics. You can release selection markers via the same plasmid that includes the gene of importance (in cis) or on a separate the plasmid (in trans) then you should transfect with the plasmid that contains the gene of the interest.
The Cis method is usually more straightforward and is more likely of generating stable transfect ants that are drug-resistant which have the gene that is of the interest. The trans method of co-transfection is a good alternative in instances where the target construct does not have the antibiotic-resistance gene in the vector backbone. In these cases the plasmid mixture comprising 5 to 10 parts of gene expression plasmids and one of the antibiotic-selection marker could introduce into the cells. This ratio of plasmids will ensure that the chosen cells will be expressing both the gene of interest as well as the marker for selection.
Stable Cell Line Generation Protocol
The process of generating AAV cell line requires a series of steps, as follows:
- Make a kill curve to determine the most effective dosage of antibiotics.
- Transfect cells using desired plasmid construct(s)
- Expand polyclonal colonies that are stable
- Find single clones using a limited diluting and expansion
- Transfer clones and test the expression
- Expand and then freeze down high expression copies
Create a kill curve in order to determine the best dosage of antibiotics
The first step crucial to Cell Line Development is determining the ideal dosage of antibiotics for choosing viable cells. You can expose Kill curves as experiment to increasing doses of antibiotics in order to determine the minimum concentration to kill all cells within one week. Biotech Company suggest a kill curve test for each new cell type, or whenever an antibiotic that is a selection or other amount of selection antibiotics.
Transfect cells using desired plasmid construct(s)
When performing killing curves (1 week) make sure you are optimizing the conditions for transfection within the T75 flask, by transfecting an plasmid that is a reporter (such as an GFP encoded expression plasmid) into cells with high confluence. Find the right dosage of the plasmid (5-15 mg) and the transfection the reagent (15-45 ul) within the T75 flask. Monitor reporter gene expression (GFP) and toxicities at regular intervals for at least a 48-hour period, and optimally over 72 hours.
Expand stable polyclonal colonies
After 48-72 hours after transfection, you can add the chosen antibiotic in the highest dosage, ideal and minimal dose in each one of your T75 flasks transfected. T75 flasks.
As a test to determine the reaction of the antibiotic both in transfected and non-transfected cells of the 6-well Tissue Culture Plate, mix in the antibiotic in the same doses as those used in the T75 flasks (as shown on the following table). Add a no-antibiotic control.
Find single clones with only a tiny amount of dilution, and then expansion
Place the polyclonal cells derived of the selection process with an average concentration of 10 cells/mL the 96-well plate for tissue culture with 100 ul for each well (i.e. 1-cell per well).
Check how many cells in each well after 18-24 hours. Then noting the wells with just one cell.
Transfer clones to test the expression
Expand the single-colony wells within the 96-well culture plate until selection they reach high confluence before transferring to a 12 well Tissue culture plate. Biotech company test GFP positive clones in the 96-well culture plate, while others evaluated for expression as of yet (depending upon the reporter test).
After the 12 well cell culture plate cells are expanded to a high confluence, they are able to be transferred to a six-well Tissue culture dish. A small percentage of cells will be tested to determine the level of expression of the targeted protein at this when the plate is.
Expand and then freeze down high express copies
When 12 well cell culture plate cells expanded to a high confluence, you can transfer it to a six-well Tissue culture dish. And then test a small percentage of cells to determine the level of expression of the targeted protein at this when the plate is.
After expansion, you can freeze cell stocks using the appropriate freezing medium, but not the antibiotic.
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