Baltimore PKD Center-Core D
Cell Culture Protocols and Handling
Instructions for PKD1 and PKD2 Conditional Rosa 26R Cell Lines
Drs. Erik Schwiebert and Eric Seales
Revised Jan 2013
Background on the Development of These Clonal Cell Lines: Core D has established two
panels of clonal immortalized renal epithelial cell lines from Pkd1cond/cond; ROSA26R+ and
Pkd2cond/cond; ROSA26R+ mice. Cells were immortalized using a combination of telomerase and
SV40 large T antigen immortalization process.
In order to delete Pkd1 or Pkd2, these cell lines require treatment with an adenovirus bearing
Cre recombinase (CreR) or an adenovirus containing an empty CMV promoter as a control (see
protocol below). With the help of Dr. Feng Qian, Ph.D. of the Baltimore PKD Center’s Core B, we
have a beginning method (starting point) for use of these Vector Biolabs products to ‘knockout’
polycystin-1 or polycystin-2 in your hands.
Some specific information about these panels of clonal mouse renal epithelial cell lines is
provided below:
There are 20 clonal renal epithelial cell lines from the Pkd1cond/cond; ROSA26R+ mouse model:
clones 2a, 2b, 3a, 3b, 4, 5a, 5b, 6, 7, 11, and 13 derived from tissue culture plastic-based culture
and clones 16-25 from permeable filter support-derived culture.
There are 3 clonal renal epithelial cell lines from the Pkd2cond/cond; ROSA26R+ mouse model:
clones 2, 4, and 5. Two of these clones (clones 2 and 5) grow relatively slowly and show a
differentiated epithelial cell phenotype of ‘island-like’ growth. One clone (clone 4) is a more
rapid grower and shows a less differentiated phenotype. They were all permeable filter support
derived.
Methods ‘starting point’ for in vitro introduction of CreR and ‘knockout’ of the gene:
Briefly, we added Adv-CMV-CreR or Adv-CMV-Null at different titers from 106 to 108 PFU
(constructs purchased from Vector BioLabs-see PDFs available on the web site) over 24, 48 and
72 hours. We found that 72 hours was an optimal time period. We also observed that 106 PFU
was not sufficient to completely ‘knockout’ PKD2 (determined via Western Blot) and 108 PFU of
either the Cre containing or control adenovirus was partially cytotoxic.
We found that 107 PFU of CreR knocked out PKD2 and therefore we would recommend treating
cultures with 107 PFU of CreR or control adenovirus for 48-72 hours – this should result in null
cells but if there are a small number of cells with remaining expression of Pkd1 or Pkd2 it may be
necessary to introduce the CreR /Null vectors more than once to elicit permanent knockout over
time.
Protocols for Pkdcond/cond; ROSA26R+ Immortalized Cell Lines
Cell Culture Medium (when received from Core D or revived from cryopreservation)
For a 500 ml bottle of DMEM/F12 medium, add…
- FBS, 50 mls
- Pen/Strep, 5-10 mls (note we do 2X Pen/Strep when shipping or after revival for a time)
-L-Glutamine, 5 mls
-Amphotericin B, 0.5-1.0 ml (note we do 2X Fungizone when shipping or after revival for a time)
-Gentamicin Solution, 0.125-0.25 mls (note we do 2X Gent when shipping or after revival for a
time)
Filter sterilize with a 0.2 micron filter bottle system – This medium is provided in the package
with a filter bottle system – The living T25 flasks (if provided) are also filled to the neck with this
medium (it can be pipetted off and filtered for use as well).
We advise using plasmocin for a brief period (1-2 weeks) to insure a clean cell culture to begin
and then withdraw it from the standard medium above.
You may wish to introduce mitogens or other stimulants at the appropriate time in your
bioassays.
Passaging of Cell Cultures
(1) Aspirate the serum-containing cell culture medium from the flask, well or plate.
(2) Wash 1X with excess CaMg-free PBS or HBSS. Aspirate.
(3) Incubate in an appropriate volume of CaMg-free PBS or HBSS in the cell culture
incubator at 37 oC for 15-30 minutes. (Note: The ‘Mono’ clones require at least 30
minutes and then addition of trypsin-EDTA solution or the equivalent to promote full
detachment – the other clones do not in our hands and detach in CaMg-free saline)
(4) Pellet the cells using a centrifuge set at 3,000 RPM for 5 minutes. Aspirate supernatant
while leaving a bit of volume to break up the pellet by finger flicking/vortexing or by
gentle vortexing with a vortexer.
(5) Resuspend in cell culture medium desired and plate as desired for your experiment.
Cryopreservation of Cell Cultures
(1) Aspirate the serum-containing cell culture medium from the flask, well or plate.
(2) Wash 1 X with excess CaMg-free PBS or HBSS. Aspirate.
(3) Incubate in an appropriate volume of CaMg-free PBS or HBSS in the cell culture
incubator at 37 oC for 15-30 minutes. (Note: The ‘Mono’ clones require at least 30
minutes and then addition of trypsin-EDTA solution or the equivalent to promote full
detachment – the other clones do not in our hands and detach in CaMg-free saline)
(4) Pellet the cells using a centrifuge set at 3,000 RPM for 5 minutes. Aspirate supernatant
while leaving a bit of volume to break up the pellet by finger flicking/vortexing or by
gentle vortexing with a vortexer.
(5) Resuspend in Crypreservation Medium (see recipe below). Note: DBM routinely freezes
6 vials from a confluent T75 flask of cells or a vial per 24 mm filter support (the large
filter supports held in wells of a 6-well plate or 6 vials for 6 such filter supports).
Cryopreservation Medium (-80 oC freezer or liquid nitrogen dewar)
We recommend that you ‘cryopreserve’ multiple vials of each clone for safekeeping at your
facility – The Cores have only finite resources for sending packages to investigators in the
Research Base. This also mitigates risk of loss of the clonal cell lines due to unforeseen events.
For a 500 ml bottle of Advanced MEM medium, add 20% DMSO cryoprotectant by volume and
add:
- FBS, 100 mls
- Pen/Strep, 10 mls
-L-Glutamine, 5 mls
-Amphotericin B, 1.0 ml
-Gentamicin Solution, 0.25 mls
Feel free to e-mail Dr. Erik Schwiebert, Core D Director, at erik@discoverybiomed.com or Dr.
Eric Seales, Core D co-Director, at eric.seales@discoverybiomed.com or call (205) 918-8138 with
questions and/or feedback.