1. Protocol for Generation of Stable Cell LinesStable Cell Lines

2. 1 Applications, types, major challenges, transfection methods, selection marker, methods and culture conditions of stably- transfected cell lines. Background

3. Applications of Stably-Transfected Cell Lines Recombinant Proteins Drug DiscoveryGene Function Studies

4. TYPE 1 Episomal Maintenance TYPE 2 Direct Integration Episomal Maintenance Episomal stability is often limited and episomal plasmid elements is often restricted to certain species Direct Integration Although integration into the host cell chromosome is a rare event and, for most purposes, clonal events have to be isolated, stability of the intended genetic modification usually is much higher. Two Types of Stable Cell Lines

5. Major Challenges Major challenges for generation of stable cell lines are low transfection efficiency and/or integration frequency. Major Challenges And Transfection Methods Transfection reagents Viral methods Electro- transfection Transfection Methods Stable expression can be influenced by the transfection method used. The transfection method determines the cell type for stable integration.

6. Antibiotics (From ancient Greek αντιβιοτικά, antiviotika) also called antibacterials, are a type of antimicrobial[1] drug used in the treatment and prevention of bacterial infections. Antibiotics Neomycin is an aminoglycoside antibiotic found in many topical medications such as creams, ointments, and eyedrops. Neomycin Dihydrofolate reductase, or DHFR, is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid. DHFR Glutamine synthetase (GS) (EC 6.3.1.2)[3] is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. Glutamine Synthetase Selection Marker

7. Methods to Generate Stable Cell Lines 01 02 A Mixed Population of Drug Resistant Cells Generate A Monoclonal Cell Line A mixed population of drug resistant cells can be used directly for experimental analysis with the advantage of generating fast results, but also the disadvantage of dealing with an undefined and genetically mixed cell population. This culture method can be used for screening experiments or conduction studies by using a homogenous and defined cell system.

8. For optimal results, we recommend following the cell culture recommendations of the supplier (e.g. ATCC) for the respective cell type. Culture Conditions Passage Split Rhythm Number Enviroment

9. 2 This protocol is specific for the generation of a monoclonal cell line that resistance to antibiotics G418 (neomycin). The end result that you are looking for is a population of cells in which 100% of cells are expressing your fusion protein. Protocol

10. Minimum Level Plating Density Susceptibility Note Susceptibility to G418 is different among cell lines, which many even vary with cell passage numbers. Note that the active concentration of stock G418 can vary considerably from batch to batch. Determine the minimum level G418 concentration to guarantee the minimum impact to cell growth. The final plating density depends on the specific cell type and the G418 concentration.. 1. Choose the G418 concentration

11. ① Pre-plate 100 μl medium in each well of the plate. ③ After gentle up and down pipetting, carry over 100 μl to the next column, thereby diluting in a ratio of 1:2. Repeat this procedure for each consecutive column. ⑤ Add 100 μl of G418 containing medium (2.8 mg/ml) to the first row (A) for a final G418 concentration of 1.4 mg/ml. ② Add 100 μl of cell suspension containing 4000 cells per well to the first column (#1). ④ Discard 100 μl from the last column (#12) after completing. The first column should then contain about 2000 cells per well, the last column contain around one cell per well on average. 1. Choose the G418 concentration ⑦ Incubate cells at standard conditions. ⑥ Add G418 to the following rows in decreasing concentrations of G418 in steps to 0.2 mg/ml. For the last row (H) add medium without G418. ⑨ If you observe cell growth (after 10 days) in the wells without G418, it is reasonable to assume that those cells can grow out starting as single cells. ⑧ Analyze cell growth by microscope. In some cases, cell growth can also be observed by change of medium color. ⑩ Choose the G418 concentration which is just above the one which shows complete cell death as the appropriate G418 concentration for selection.

12. Transfection For transfection, please follow the manufacturer’s instruction of your transfection system. We suggest setting a negative control of untransfected cells for selection.. The important thing is to transfect the expression plasmid containing the target gene and the sequence for a drug resistance gene into your cells. Besides, it is much better to check the transfection efficiency and integration frequency of your experiment with a GFP-control plasmid. 2. Transfection

13. ① After transfection, allow cells to grow and to express the protein for G418 resistance under non-selective conditions for about 24-48 hours. ③ Count living cells via trypan blue staining or other appropriate methods. ⑤ Incubate cells under selective conditions and feed cells regularly with fresh selection medium. ② Analyze for transfection efficiency 24-48 hours post- transfection by microscopy or western blot of your target protein and positive control. ④ 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 a volume of at least 100 μl per well. ⑥ Cell clones can be analyzed or further expanded as soon as cells in the non-transfected control wells have completely died. 3. Cell Selection Post-Transfection ⑦ In order to help assuring that selected cell populations are clones derived from a single cell, another round of limiting dilution under selection is recommended.

14. Western Blot ELISA Microscopy Flow Cytometry 4. Analysis of Stable Cell Lines

15. 3 We establish a experimental outline of the protocol for generation of stable cell lines. Experimental Outline

16. 01 02 03 04 05 06 Design experiment Design experiment and choose expression vector, cell type and transfection method.. Analyze Make sure to choose a suitable method for your application to analysis your stable- transfected cells.. Monoclonal cell line screening Dilute cells into 96 well culture plates in appropriate cell density per well. Feed every 10 days with selection medium.. Choose the G418 concentration and cell number Determine appropriate G418 concentration and cell number per well by matrix titration. Transfection Transfect expression plasmid into cells. Pease follow the manufacturer’s instruction of your transfection system. Cell selection Plate transfected cells and cultivate cells in medium with G418 in appropriate concentration. Experimental Outline

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