Stable cell lines have a wide range of applications both for research and manufacturing purposes. The protocol of stable cell line generation is critical to collect and clone target stable cell lines. This presentation begins with the background that why the generation of stable cell lines is so important and shows a clear and comprehensive protocol to generate a specific cell line that resist against antibiotics G418 (neomycin). https://www.creativebiomart.net/resource/principle-protocol-protocol-of-stable-cell-line-generation-373.htm

1. Protocol
for
Generation of Stable 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
MicroscopyFlow 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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