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Establishing Stable Cell Lines

This document provides a procedure for establishing stable cell lines that constitutively express GFP-tagged proteins. Key steps include: 1. Cloning the protein of interest into a GFP vector and confirming expression and localization in transiently transfected cells. 2. Transfecting cells with the GFP construct and selecting for stable integrants using G418 antibiotic selection over 1-2 weeks. 3. Isolating individual colonies, expanding them, and screening for clones with uniform GFP expression in >95% of cells. 4. Freezing samples from early passages to maintain stable cell lines over many passages.

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Yamuna Joseph
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25 views2 pages

Establishing Stable Cell Lines

This document provides a procedure for establishing stable cell lines that constitutively express GFP-tagged proteins. Key steps include: 1. Cloning the protein of interest into a GFP vector and confirming expression and localization in transiently transfected cells. 2. Transfecting cells with the GFP construct and selecting for stable integrants using G418 antibiotic selection over 1-2 weeks. 3. Isolating individual colonies, expanding them, and screening for clones with uniform GFP expression in >95% of cells. 4. Freezing samples from early passages to maintain stable cell lines over many passages.

Uploaded by

Yamuna Joseph
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Establishing Stable Cell Lines

There are many different approaches for establishing stable cell lines, depending on the type of
expression you’re interested in (inducible vs. constitutive) and the construct that you are
incorporating. This protocol is specific for the establishment of cell lines that constitutively express
GFP-tagged proteins. The end result that you are looking for is a population of cells in which >95% of
cells are expressing your fusion protein at approximately the same level. This allows large-scale
biochemical analyses of the fusion protein and monitoring of localisation throughout the cell cycle,
and greatly simplifies many other microscopy techniques as well.

Procedure
1. Clone your protein of interest into one of Clonetech’s Living Colors vectors (EGFP, EFYP,
ECFP, etc.). These vectors encode kanamycin resistance for selection in bacteria and
neomycin (G418) for selected mammalian cells.
2. Characterize expression of the fusion protein in your cell type of interest using transient
transfections (we use HeLa cells for most of our stable cell lines, and QIAGEN’s Effectene
transfection reagent to give >80% transfection efficiency). You want to be sure that your
fusion protein is full length and not degraded (can probe with GFP antibody on a western
blot; Roche’s monoclonal anti-GFP works well at 1:1000). If you have an antibody to the
endogenous protein you can also compare the level of expression of the fusion protein to
the endogenous protein. Many proteins mislocalise when overexpressed. Include coverslips
in the dish that can be fixed to check localisation by fluorescence microscopy.
3. To set up for the stable cell line selection, split cells and transfect with your construct.
4. The following day, replace the standard media with media containing G418 (G418
(Geneticin) is an aminoglycoside antibiotic similar in structure to gentamicin B1.) Over time
this will select for cells that have stably incorporated the GFP plasmid into their genomic
DNA. The amount of G418 required to kill cells not expressing the construct, will vary from
cell line to cell line. We have titrated the amount required for our HeLa cell lines, and this
can be a starting point for the other cell lines. Initially we use 400µg/ml G418 in media when
selecting for stable clones. This selection can take anywhere from 1-2 weeks.
5. Carefully change the media in the dish every day, taking care not to pipette directly onto the
cells. At some point there will be a massive cell death and most of the cells will wash off the
bottom of the dish, leaving colonies of stable cells behind.
6. To pick colonies, prepare 24-well dishes with 1ml media containing G418 in each well.
7. Rinse the dish with PBS and then add warm PBS containing 5% trypsin (1ml standard
trypsin-EDTA plus 19ml PBS). Colonies can be picked on an inverted light microscope,
although an inverted fluorescence microscope allows you to monitor the fluorescence
signals in the colonies that you’re picking.
8. Using a Gilson pipette with a sterile yellow tip, lower the tip to the surface of the colony of
interest and scrape and suck gently until you’ve pulled it up into the tip.
9. Transfer to a well in a 24-well plate. Repeat with other colonies.
10. In a day or two, when the wells are confluent, rinse with PBS and trypsinize with 100µl of
trypsin-EDTA.
11. Split into one well of a 6-well plate (for passaging) and one well of a 24-well plate that also
contains a 13mm coverslip. These coverslips can be fixed in a day or two to screen the
colonies to decide which should be put down and which are worth keeping (and whether
they require further sub-cloning to reach >95% homogeneity).
12. All colonies that you are interested in keeping should be passaged from the 6-well plate into
at least 3 dishes: one for freezing as Passage 0 (in case something goes horribly wrong in the
future you can always go back to this), one for further passaging (if the line is already clonal)
and one for sub-cloning (if required).
13. Seed at clonal density for sub-cloning (e.g. 10 µl from 1:5 split of the 6-well dish into a 10cm
dish).
14. When you have your clone or sub-clone of interest, you can then use a lower amount of
G418 for maintenance (200µg/ml for HeLa cells). Keep track of the passage numbers as
stability of clones will vary and some may need to be thrown out after a few passages.
15. Periodically freeze down samples from early passages so you can always go back to a young
population of cells.

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