Protocol for Generation of Stable Cell Lines

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Protocol for Generation of Stable Cell Lines
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Background
Applications, types, major challenges,
transfection methods, selection marker, methods
and culture conditions of stably-transfected
cell lines.
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Applications of Stably-Transfected Cell Lines
Recombinant Proteins
Drug Discovery
Gene Function Studies
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Two Types of Stable Cell Lines
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.
Episomal stability is often limited and episomal
plasmid elements is often restricted to certain
species
Episomal Maintenance
Direct Integration
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Major Challenges And Transfection Methods
Major Challenges
Transfection reagents
Major challenges for generation of stable cell
lines are low transfection efficiency and/or
integration frequency.
Transfection Methods
Viral methods
Electro-transfection
Stable expression can be influenced by the
transfection method used. The transfection method
determines the cell type for stable integration.
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Selection Marker
Antibiotics
Neomycin
Antibiotics (From ancient Greek a?t?ß??t???,
antiviotika) also called antibacterials, are a
type of antimicrobial1 drug used in the
treatment and prevention of bacterial infections.
Neomycin is an aminoglycoside antibiotic found in
many topical medications such as creams,
ointments, and eyedrops.
DHFR
Glutamine Synthetase
Dihydrofolate reductase, or DHFR, is an enzyme
that reduces dihydrofolic acid to tetrahydrofolic
acid.
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.
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Methods to Generate Stable Cell Lines
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A Mixed Population of Drug Resistant Cells
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.
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Generate A Monoclonal Cell Line
This culture method can be used for screening
experiments or conduction studies by using a
homogenous and defined cell system.
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Culture Conditions
Passage
Number
For optimal results, we recommend following the
cell culture recommendations of the supplier
(e.g. ATCC) for the respective cell type.
Split Rhythm
Enviroment
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Protocol
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.
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1. Choose the G418 concentration
Susceptibility
Susceptibility to G418 is different among cell
lines, which many even vary with cell passage
numbers.
Minimum Level
Determine the minimum level G418 concentration to
guarantee the minimum impact to cell growth.
Note
Note that the active concentration of stock G418
can vary considerably from batch to batch.
Plating Density
The final plating density depends on the specific
cell type and the G418 concentration..
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1. Choose the G418 concentration
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After gentle up and down pipetting, carry over
100 µl to the next column, thereby diluting in a
ratio of 12. Repeat this procedure for each
consecutive column.
Pre-plate 100 µl medium in each well of the plate.
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.
Incubate cells at standard conditions.
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.
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Add 100 µl of cell suspension containing 4000
cells per well to the first column (1).
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.
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.
Analyze cell growth by microscope. In some cases,
cell growth can also be observed by change of
medium color.
Choose the G418 concentration which is just above
the one which shows complete cell death as the
appropriate G418 concentration for selection.
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2. Transfection
For transfection, please follow the
manufacturers instruction of your transfection
system.
The important thing is to transfect the
expression plasmid containing the target gene and
the sequence for a drug resistance gene into your
cells.
Transfection
Besides, it is much better to check the
transfection efficiency and integration frequency
of your experiment with a GFP-control plasmid.
We suggest setting a negative control of
untransfected cells for selection..
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3. Cell Selection Post-Transfection
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After transfection, allow cells to grow and to
express the protein for G418 resistance under
non-selective conditions for about 24-48 hours.
Count living cells via trypan blue staining or
other appropriate methods.
Incubate cells under selective conditions and
feed cells regularly with fresh selection medium.
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.
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Analyze for transfection efficiency 24-48 hours
post-transfection by microscopy or western blot
of your target protein and positive control.
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.
Cell clones can be analyzed or further expanded
as soon as cells in the non-transfected control
wells have completely died.
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4. Analysis of Stable Cell Lines
Western Blot
Flow Cytometry
Microscopy
ELISA
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Experimental Outline
We establish a experimental outline of the
protocol for generation of stable cell lines.
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Experimental Outline
Design experiment
Design experiment and choose expression vector,
cell type and transfection method. .
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Choose the G418 concentration and cell number
Determine appropriate G418 concentration and cell
number per well by matrix titration.
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Analyze
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Make sure to choose a suitable method for your
application to analysis your stable-transfected
cells..
Transfection
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Monoclonal cell line screening
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Transfect expression plasmid into cells. Pease
follow the manufacturers instruction of your
transfection system.
Dilute cells into 96 well culture plates in
appropriate cell density per well. Feed every 10
days with selection medium..
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Cell selection
Plate transfected cells and cultivate cells in
medium with G418 in appropriate concentration.
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Contact Information
Email contact_at_creative-biomart.com Website
www.creativebiomart.net Address 45-1 Ramsey
Road, Shirley, NY 11967, USA
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Any questions? You can find me at
contact_at_creative-biomart.com
Thanks!