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4/26/22, 11:14 AMProtocol of Stable Cell Line Generation - Creative BioMartMenuKeywords SearchRGene SearchGoogle SearchPlease type your keywords. RESOURCEP R OTO C O L O F S TA B L E C E L L L I N E G E N E R AT I O NHome / Resource / Principle & Protocol / Protocol of Stable Cell Line GenerationProtocol of Stable Cell Line GenerationBackgroundThe generation of stably-transfected cell lines is critical for a wide range of applications. Itis applied to the production of recombinant proteins, gene function studies, as well asdrug discovery assays. Stable expression of target gene in cell lines overcomes the lowtransfection efficiency of transient expression and produces more target proteins.There are two types of stable cell lines. One is achieved by eukaryotic vectors that harbor elements for episomal maintenance in the nucleus of a transfected cell. Another isachieved via direct integration of the transfected plasmid into the target cells ration-373.htm1/8
4/26/22, 11:14 AMProtocol of Stable Cell Line Generation - Creative BioMartEpisomal stability is often limited and episomalplasmid elements is often restricted to certainspecies, therefore integration into the host cellchromosome is in common use. Althoughintegration into the host cell chromosome is a rareevent and, for most purposes, clonal events have tobe isolated, stability of the intended geneticmodification usually is much higher.Major challenges for generation of stable cell linesare low transfection efficiency and/or integrationfrequency. Stable expression can be influenced bythe transfection method used. The transfection method determines the cell type forstable integration. It is known that liposome reagents can be used to transfer DNA intoadherent cell lines. While viral methods or electrotransfection are used to deliver DNAinto primary cells or notoriously difficult-to-transfect suspension cell lines. Unfortunately,viral methods suffer from several limitations, such as time consuming production ofvectors and safety concerns, while electrotransfection suffer from the low cell survivalrate.Selection MarkerIn order to select stably-transfected cells, a selection marker must be co-expressed withthe target protein. The marker gene could be on either the same plasmid vector or asecond, co-transfected vector. There are a variety of systems for selecting transfectedcells, including resistance to antibiotics puromycin, neomycin, DHFR, and glutaminesynthetase. After gene transfer, cells are developed in medium containing the selectiveagent. Only those cells which have contained the drug resistant gene survive.Methods to Generate Stable Cell LinesDepending on the scope of the experiment, several options are used for the generationof a stable cell line. A mixed population of drug resistant cells can be used directly forexperimental analysis with the advantage of generating fast results, but also thedisadvantage of dealing with an undefined and genetically mixed cell population.Another option is to generate a monoclonal cell line. In this method, it is necessary to dilute the resistant cells by plating in 96-well plates in such a way that culture as singleand isolated cells. Subsequently, the cloning of single cell may be repeated several tion-373.htm2/8
4/26/22, 11:14 AMProtocol of Stable Cell Line Generation - Creative BioMartto obtain 100% clonal purity. This culture method can be used for screening experimentsor conduction studies by using a homogenous and defined cell system.In conclusion, depending on the type of expression you’re interested in and the constructthat you are incorporating, there are many different approaches for generation of stablecell lines. This protocol is specific for the generation of a monoclonal cell line thatresistance to antibiotics G418 (neomycin). The end result that you are looking for is apopulation of cells in which 100% of cells are expressing your fusion protein.Culture ConditionsCulture conditions (passage, split rhythm, number, etc.) of your selected cell type arecritical for generation of stable cell lines. For optimal results, we recommend followingthe cell culture recommendations of the supplier (e.g. ATCC) for the respective cell type.In general, for promoting good proliferation and cell physiology, the cell line should bepassaged two days before the experiment. Besides, cell passage should not be higherthan 30 due to the possibility of interference with integration efficiency.Experimental OutlineDesign experimentDesign experiment and choose expression vector, cell type and transfection method.Make sure that your expression vector and transfection method are suitable for your celltype.Choose the G418 concentration and cell number Determine appropriate G418 concentration and cell number per well by matrix titration.Pay attention to the active concentration of stock G418 is vary from batch to ation-373.htm3/8
4/26/22, 11:14 AMProtocol of Stable Cell Line Generation - Creative BioMartTransfectionTransfect expression plasmid into cells. Pease follow the manufacturer’s instruction ofyour transfection system. Do not add G418 to culture medium immediately aftertransfection.Cell selectionPlate transfected cells and cultivate cells in medium with G418 in appropriateconcentration. A mixed population of drug resistant cells is obtained.Monoclonal cell line screeningDilute cells into 96 well culture plates in appropriate cell density per well. Feed every 10days with selection medium. Refreshed selection medium is important to avoid falsepositive cells.Analyze Make sure to choose a suitable method for your application to analysis your stabletransfected ation-373.htm4/8
4/26/22, 11:14 AMProtocol of Stable Cell Line Generation - Creative BioMartProtocol1. Choose the G418 ConcentrationSusceptibility to G418 is different among cell lines, which many even vary with cellpassage numbers. The selection condition (e.g. G418 concentration, plating density) foryour specific cell type needs to be determined experimentally. Determine the minimumlevel G418 concentration to guarantee the minimum impact to cell growth. Note that theactive concentration of stock G418 can vary considerably from batch to batch. Therefore,we recommend testing G418 sensitivity for every new batch. The final plating densitydepends on the specific cell type and the G418 concentration. We therefore recommendmatrix titration of G418 and titration of cell number for determination of plating density inone 96-well plate.Pre-plate 100 μl medium in each well of the plate.Add 100 μl of cell suspension containing 4000 cells per well to the first column (#1).After gentle up and down pipetting, carry over 100 μl to the next column, thereby dilutingin a ratio of 1:2. Repeat this procedure for each consecutive column.Discard 100 μl from the last column (#12) after completing. The first column should thencontain about 2000 cells per well, the last column contain around one cell per well onaverage.Add 100 μl of G418 containing medium (2.8 mg/ml) to the first row (A) for a final G418concentration of 1.4 mg/ml. Add G418 to the following rows in decreasing concentrations of G418 in steps to 0.2mg/ml. For the last row (H) add medium without G418.Incubate cells at standard generation-373.htm5/8
4/26/22, 11:14 AMProtocol of Stable Cell Line Generation - Creative BioMartAnalyze cell growth by microscope. In some cases, cell growth can also be observed bychange of medium color.If you observe cell growth (after 10 days) in the wells without G418, it is reasonable toassume that those cells can grow out starting as single cells.Choose the G418 concentration which is just above the one which shows complete celldeath as the appropriate G418 concentration for selection.2. TransfectionFor transfection, please follow the manufacturer’s instruction of your transfection system.The important thing is to transfect the expression plasmid containing the target gene andthe sequence for a drug resistance gene into your cells. We suggest setting a negativecontrol of untransfected cells for selection. Besides, it is much better to check thetransfection efficiency and integration frequency of your experiment with a GFP-controlplasmid.3. Cell Selection Post-TransfectionAfter transfection, allow cells to grow and to express the protein for G418 resistanceunder non-selective conditions for about 24-48 hours (reach the peak of proteinexpression according to your transfection system).Use suspension cells directly or trypsinize adherent cells for analysis. Analyze fortransfection efficiency 24-48 hours post-transfection by microscopy or western blot ofyour target protein and positive control.Count living cells via trypan blue staining or other appropriate methods.Using standard medium and the appropriate amount of G418 pretested for your cell type,plate cells in a 96-well plate with different cell numbers per well (e.g., 0.5, 1, 2, 5, 10) in avolume of at least 100 μl per well. Depending on cell concentration determined before,conduct several serial dilution steps as applicable. It is important to thoroughly suspendcells before seeding, but avoid harsh treatment by frequent pipetting.Incubate cells under selective conditions and feed cells regularly with fresh selectionmedium.Cell clones can be analyzed or further expanded as soon as cells in the non-transfectedcontrol wells have completely died. In order to help assuring that selected cell populations are clones derived from a singlecell, another round of limiting dilution under selection is recommended.4. Analysis of Stable Cell tion-373.htm6/8
4/26/22, 11:14 AMProtocol of Stable Cell Line Generation - Creative BioMartOnce you have obtained resistant cell lines, you should expand the cells and assay yourtarget gene compared with positive and negative control. You can detect the expressionof fusion protein by an appropriate analysis method such as western blot, microscopy,ELISA, as well as flow cytometry.Creative BioMart provides stable cell line services to meet your specific needs. Not onlyhave we developed cell lines stable at expression level, but also established stable celllines ready for assay development.Tags: NoneApply For A Coupon 5 0 O F F YO U R F I R S T P U R C H A S EAPPLY FOR A COUPONENTER YOUR EMAIL HERE TO SUBSCRIBE.you@email.comSUBMITCREATIVE BIOMART INC. 45-1 Ramsey Road, Shirley, NY 11967, USA Global neration-373.htm7/8
Home / Resource / Principle & Protocol / Protocol of Stable Cell Line Generation Protocol of Stable Cell Line Generation Background The generation of stably-transfected cell lines is critical for a wide range of applications. It is applied to the production of recombinant proteins, gene function studies, as well as .
