ES and MEF feeder cells
The facility has stocks of ES cells and mouse embryonic fibroblast (MEF) feeder cells for gene targeting experiments. Our ES cells include (1) R1 cell line originally derived from 129/SvJ x 129/Sv strain background, passage #18 and #19; (2) CGR8.8 line derived from 129ola strain background, passage #19.
We have two kinds of feeder cells, both of which are primary cells derived from mouse embryos. They are Neomycin-resistant and DR-4 (neomycin, hygromycin, puromycin and 6-thioguanine resistant) feeder cells.
To obtain these cells and reagent, investigators need to fill out an on-line requisition form and arrange to pick up the cells with Dr. Yanru Chen-Tsai or Dr.Hong Zeng (724-9556).Please see our Guidelines for culturing R1 ES and MEF feeder cells before you start your cell cultures.
Guidelines for culturing R1 ES and MEF feeder cells
For 500 ml of ES complete media:
100 ml fetal bovine serum (germline tested from Applied StemCell,
Inc. Sunnyvale, CA)
5 ml 100 x non-essential amino acids
5 ml 100 x Pen-Strep
5 ml 100 x sodium pyruvate
5 ml diluted beta-mercaptoethanol (3.5 ul to 5 mls of water and
made fresh)
380 ml DMEM (ES cell tested from Applied StemCell, Inc., Sunnyvale,
CA)
1 ml 106 Units/ml LIF
For positive and negative selections, add:
5 ml 100x G418 (20mg/ml active concentration in PBS)
0.1 ml 5000x Ganciclovir (Cytovene, Syntex, 10 mM in PBS)
Store media at 4 degrees C and warm to 37 degrees C before use.
2x Freezing media
20% fetal bovine serum
20% DMSO
60% R1 ES cell media
Store freezing media at -20 degrees C and thaw to 37 degrees C or room
temperature before use.
Complete media for mouse embryonic fibroblast (MEF) feeder cells
For 500 ml media:
445 ml DMEM
50 ml fetal bovine serum (10% final concentration)
5 ml 100x Pen-Strep
Gelatinize plastic ware
All dishes need to be treated with 0.2% gelatin for 5 minutes at room
temperature. 0.2% gelatin is made from diluting 2% gelatin (Sigma# G1393)
with PBS and autoclave to sterilize. The volume added to dishes is not
critical as long as there is enough to cover the whole surface area.
Ordering information
FBS (germline tested): Applied StemCell #ASM-5007
trypsin (STV): UCSF # GP110
PBS: UCSF # BG200
Murine LIF: Gibco # 13275-011
10mM (100x) Non-essential amino acids: Gibco # 11140-050
100x Penicillin-streptomycin: Gibco # 15140-122
100x Sodium pyruvate: Gibco # 11360-070
DMEM: Applied StemCell #ASM-5001
beta-Mercaptoethanol: Sigma # M-7522
2% Gelatin: Sigma# G1393
Ganciclovir: 500 mg, Stanford Pharmacy
G418: Cellgro# MT61-234-RF
Expansion and irradiation of MEF feeder cells
• Plate one vial primary MEF cells (obtained from the transgenic
facility) in one T75 flask with 20 ml MEF media.
• Expand 1x T75 flask of cells into 1x T175 flasks.
• Split 1x T175 flask of cells into 3 T175 flasks.
• Split 3x T175 to 6xT175 flasks if more feeders are needed.
• Trypsinize and pool all cells, spin down and resuspend cells
in 50 ml MEF media.
• Irradiate cells (gamma irradiation in Radiology Department,
3000 rads).
• Spin down and resuspend cells in MEF media.
• Add an equal volume of 2x Freeze media.
• Mix well and freeze for overnight in 0.5 ml aliquots at -80
degrees C.
• Store in liquid nitrogen.
Each T175 flasks of cells can be aliquoted into 6 vials. Each vial can then be plated onto 1x T25 flask. You may need to adjust these numbers according to plating efficiency.
Routine maintenance of ES cells
To maintain a healthy and undifferentiated culture, cells need to grow
at high density (above 106
to 107/ 8cm2
or one well of a 6-well plate) and they should be passed often. As a general
rule, the media should be changed daily and cells should be passed every
other day with a 5 fold split. If the cells have just been thawed you
may need to wait until the third or fourth day for passage. Just observe
the dish and see if nests of ES cells are starting to touch each other.
Undifferentiated ES cells should be compact with prominent nucleoli and
grow in a colony or a nest. Individual cell borders should not be distinguishable
but the colony should have a well defined boundary and may appear a little
refractile under a phase microscope.
If desired, feeder cells can be removed from ES cultures by differential
adhesion. Plate ES and feeder cell suspension on a non-gelatinized plate
for 30 minutes at 37 degrees C. This is enough time for feeder cells to
attach and the supernatant should be relatively pure ES cells.
To pass ES cells:
• Aspirate media off the plate and rinse once with PBS.
• Trypsinize cells with STV (0.05% trypsin) at 37 degrees C for
3 to 5 minutes.
• Add 5 ml of ES media and pipet vigorously to get single cell
suspension.
• Spin at 1000 rpm for 5 minutes.
• Plate cells out onto irradiated feeder cells.
Thawing and culturing MEF and ES cells
• Thaw 1 vial of MEF feeders cells into 1x T25 flask the day before
or on the same day.
• Thaw 1 vial of R1 ES cells and plate in the T25 flask that contain
feeder cells.
• Split ES cells into 3-5x T25 flasks.
• Freeze down an aliquot and use the rest of ES cells for electroporation.
Preparation of DNA for electroporation
• Linearize 20-25 ug of targeting vector DNA.
• Phenol/chloroform extraction.
• Precipitate DNA with 7.5 M ammonium acetate and ethanol.
• Wash pellet with 70% ethanol and leave at this step until ready
to use.
• Resuspend DNA in 100 ul of DME-H21 before mixing with the ES
cells.
Electroporation of ES cells
• Trypsinize and pool ES cells from 3x T25 flasks.
• Spin down and resuspend the cells in 600 ul of ES media.
• Combine ES cells with DNA in an electroporation cuvette.
• Electroporate cells with 500 uF, 250 Volts (Time constant should
be around 7-8).
• Let cuvette sit in the hood for 10 min.
• Resuspend cells in ES media and plate cells onto 4-5x 6-well
plates with feeder cells.
• After 24 hours put cells under selection with G418 and/or gancyclovir.
• Change media with selection daily until colonies form. It usually
takes 7 to 10 days for visible colonies.
Picking ES cell clones
• Prepare 3 to 4 x 96-well plates with feeder cells.
• Prepare another set of fresh 96-well plates, add 10 ul trypsin
STV into each well.
• Remove media from electraporation plates and rinse once with
PBS. Do one well at a time from this step on.
• Pick colony in 5 ul PBS by scraping forcefully and pipetting
at the same. Trypsin may be added if mechanical picking becomes too difficult.
• Add colony to the trypsin well.
• Pick all colonies in one well of a 6-well plate at once.
• Let colonies sit in trypsin for 5 to 10 minutes.
• Disaggregate clones with 150 ul ES media.
• Add each clone to a well with feeder cells.
Splitting cells for freezing and DNA prep
• After 2 to 3 days clones need to be passed.
• For each master plate prepare 5 x new 96-well plates: 2 with
feeder cells (100 ul/well) and 3 gelatinized only with 100 ul media/well.
• Wash master plate once with PBS.
• Add 25 ul STV and incubate at 37 degrees C for 5 minutes.
• Disaggregate cells in 140 ul ES media.
• Add 30 ul cells to each of the 5 new plates.
• Allow the cells to grow to confluence in the well.
Freezing ES cells on 96-well plates
Aspirate media and wash once with PBS.
Freeze down the 3 x gelatinized only plate at -20 degrees C for DNA prep.
For the 2 x plates with feeder cells, add 25 ul trypsin STV and incubate
at 37 degrees C for 5 minutes.
Disaggregate cells with 100 ul ES media and spin cells at 1K for 5 minutes.
Take off supernatant. Be careful not to suck cells from bottom.
Resuspend cells in 75 ul ES media.
Add 75 ul 2x freezing media.
Cover plates with parafilm, place plates in plastic bags and then in Styrofoam
box and put in -80 degrees C freezer.
Thawing clones
• Prepare feeder cells (50 ul /well) on 96-well plates.
• Remove plates from -80 degrees C and let plates sit in warm
bath.
• After clones are thawed spin plates at 1K for 5 minutes.
• Remove supernatant from well.
• Resuspend clone in 100 ul ES media and add clone to fresh well
with feeder cells.
• After cells grow to confluence pass cells to 1 well of 24-well
plate.
DNA preparation from ES cells grown on 96-well plates
• Remove 96-well plates from -20 degrees C freezer.
• Add 50 ul of lysis buffer: 10mM Tris pH7.5
• 10mM EDTA
• 10mM NaCl
• 0.5% Sarkosyl
• 100 ug/ml Proteinase K (added fresh)
• Place plates on top of a platform inside a tupperware container
that has some water in the bottom. Incubate at 55 to 60 degrees C overnight.
• To precipitate DNA, add to each well 100 ul of a mixture comprised
of: 100 ul cold EtOH and 1.5 ul 5M NaCl.
• Sit 15 to 30 minutes at room temperature. DNA should become
visible.
• Spin the plates 10 minutes at 2000 rpm.
• Gently dump out ethanol and wash three times with 100 ul of
70% EtOH.
• After last wash, leave tilted upside down for 15 to 20 minutes
to air dry.
• Add 30 ul of TE to each well. Incubate in CO2 incubator at least
24 hours to resuspend.
Preparation of ES cells for microinjection
• Thaw ES cells 4 to 5 days before the injection day.
• Pass cells once.
• On the day of injection, feed cells with fresh ES media in the
morning.
• After 1 to 2 hours, trypsinize ES cells into single cell form.
• Wash trypsinized ES cells once with ES cell complete media.
• Resuspend cells in 1 to 2 ml M2 media (provided by the transgenic
facility).
• Keep cells on ice and bring the cells to Room AF025 in the animal
facility before 11 AM.
