The main reference for growing these cells is chapter 2 by Wurst and Joyner from Gene targeting: a practical approach, ed. A.L. Joyner. The R1 line we use was originally established on STO feeders, and the Rossant lab has since switched to either MEFs and media supplemented with LIF or media supplemented with LIF alone.
Our principal modification is growing the cells exclusively on gelatinized plastic in a slightly different media supplemented with LIF.
The cells came from the Rossant lab on feeders and were adapted to growth off of feeders. Following the instructions that came with the line for establishing a pool of R1's on feeders, 4x100 mm plates were generated. Three of these were frozen and the fourth was passaged onto gelatinized plastic in medium supplemented with LIF. Most of these adapted cells were frozen to create a large pool of consumable vials at passage 11, but some were kept going for 4-5 more passages to establish a karyotype. Ultimate proof was provided by a number of targeting experiments. The targeting rate in our hands has been roughly 50% of constructs.
ES Cell Media:
Iscove's modified Dulbecco's medium, Gibco #12440-020 for 500 ml; 12440-046 for 1 L; aliquotted 80 ml in small media bottles (125 ml); store at 4 degrees and protect from light. This comes in liter bottles which are aliquotted into smaller bottles: make sure it's a little orange in color. Discard any aliquots that have turned 'Koolaid Red'.
Hyclone FBS tested for ES cells, heat inactivated, 40 ml aliquots, stored at -20.
To heat inactivate, thaw serum overnight in refrigerator. Put into a full (i.e. water up to the level of the serum in the bottle) 37 degree water bath and allow to thaw until only a large ice cube remains (approximately 1 hour). Mix the bottle gently and return it to the fridge. Heat the water bath up to 56 degrees (set the safety thermostat to maximum). This also takes about an hour. Make sure you can get to the serum promptly after heat inactivating. Incubate the serum at 56 degrees for 30 minutes, mixing once or twice during this half hour. Mix and immediately aliquot the serum into 40 ml aliquots and freeze. Some white particulate junk floating in the serum is normal.
100 x 2-mercaptoethanol (Sigma M7522, 14.3 M stock): dilute 70 microliters into 100 ml of sterile ddH2O and filter sterilize; 10 ml aliquots; store 4 degrees.
100 x MEM nonessential amino acids: Gibco #11140-019 for 100 ml; 10 ml aliquots; store 4 degrees.
100 x Pen/strep: Gibco #15070-014 for 100 ml; 10 ml aliquots; store -20.
recombinant LIF protein, produced as per the V. Prideaux protocol; filter sterilized; put into 30 microliter aliquots; flash frozen in liquid nitrogen and stored -80; each batch tested for efficacy against an older batch.
Alternatively LIF can be purchased from Gibco (ESGro) following the Wurst and Joyner chapter (see above).
Gelatinized tissue culture dishes:
.1% gelatin (type B from bovine skin, Sigma G9382 ); .3g in 300 ml ddH2O autoclaved; cover dishes with solution and aspirate off; allow to air dry in hood--approximately 1/2 hour (alternatively can allow to air dry overnight): use within 24 hours.
ES cell culture medium/100 ml:
80 ml Iscove's MDM (contains 4 mM L-glutamine and 1 mM sodium pyruvate)
To an aliquot of IMDM add in exactly the order listed:
20 ml FBS (20% final)
1ml 100 x (10 mM) 2-mercaptoethanol (.1mM final)
1ml 100 x MEM nonessential amino acids (.1 mM final)
1ml 100 x PEN/STREP (50 U or µg/ml final)
30 µl of LIF (one -80 aliquot)
I prefer not to feed cells with cold medium. The un-supplemented IMDM is therefore warmed up by sitting in the hood for 10-20 minutes with the light off (L-glutamine is light sensitive). Likewise fully supplemented media can be warmed from the refrigerator this way. I prefer not to warm media in the water bath.
R1's are relatively pokey in growth. Standard split is 1:6. Unless otherwise noted one vial should equal one confluent 35 mm dish (i.e. 1/2 of a 60 mm or 1/6 of a 100 mm). To thaw, remove vial from liquid nitrogen and vent, then thaw ASAP in a 37 degree water bath. Gently mix thawed vial and transfer to 8 ml fresh medium in a 15 ml conical tube. Spin down on setting 4 in the clinical centrifuge for five minutes and aspirate supernatant. Plate out one vial onto one fresh 60 mm dish (1:2). The next day feed. The following day they should be ready to split 1:6 as usual.
G418 (Geneticin)
|
Geneticin, G418-SO4 powder |
Gibco |
11811-049 or 11811-015 or 11811-023 or 10131-019 |
100 mg or 500 mg or 1 g or 20 ml x 50 mg/ml soln. |
Batches are purchased in bulk from the same lot #. We normally purchase the liquid stock and put into 1 mL aliquots--keeps in the fridge forever. Mock electroporated cells are plated out at electroporation density and subjected to daily G418 selection of varying concentrations for a week to ten days. Minimum concentration that guarantees 100% killing is chosen. Some differences between cell lines have been observed, e.g. 100 µg/mL for R1 cells versus 150 µg/mL for Cast #1-12, but not always.
Puromycin
|
Puromycin |
Sigma |
P-7255 |
10 mg or 25 mg |
12.05 or 25.65 |
This is made up in 1 mg/mL stock in PBS and put into 100 µL aliquots which are stored at -20oC. Requires gentle warming at 37oC to get completely into solution. Each new batch is tested for efficacy in kill curve experiments. So far no variation has been seen in several batches. Some variation between cell lines. R1 cells are markedly less sensitive to puromycin: while other lines typically die with .75 micrograms per ml, R1 cells require twice that amount for equivalent killing (i.e. 1.5 micrograms per ml). Very fast drug--death is complete in three days.
Gancyclovir (Cytovene)
Because this is a prescription drug, it can purchased with a prescription from an obliging M.D.
Stock is 22 mM in PBS, distributed into 1 mL aliquots and frozen at -20oC. Large aliquots are then re-aliquotted at 10 µL and re-frozen without any problems. Tested for efficacy on mock electroporated Tk+ targeted cell lines. The stock is reported to be 10,000 x. However, kill curves have shown repeatedly that 1/2 x or even 1/4 x is sufficient for complete killing in as little as three days in Tk+ cell lines. Lot of concern with bystander killing with this drug: best proof of this is in Jim Thomas' paper (1998) Proc Natl Acad Sci U S A 95, 1114-9) where he got decidedly fewer colonies with GANC than with FIAU.
FIAU (1-2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl-5-iodouracil)
|
FIAU |
Moravek Biochemicals |
M-251 |
1 mg or 5 mg |
65.00 or 205.00 |
Stock is 200 µM and requires an elaborate protocol to get into solution. Add 388 mg of FIAU to 9 mL PBS and add NaOH until the FIAU has dissolved. Make total volume to 10 mL. This solution is 100 mM and must be diluted 1:500 in PBS to give the 1000 x stock solution. Store at -20oC, usually in 100 µL aliquots. (this recipe comes from the Bradley chapter in Methods in Enzymology Vol. 225). Scale down this recipe as appropriate for smaller amounts of FIAU. The published 1 x concentration, 0.2 µM FIAU, has always proven correct in kill curve experiments (probably the only drug for which this is true). The drug of choice when selecting rare Tk- variants from a predominantly Tk+ population. Some concern about bystander killing when cells are on feeders (see You, Y., Browning, V. L. & Schimenti, J. C. (1997) Methods 13, 409-21). Most published accounts of selecting rare Tk- variants from a predominantly Tk+ population have in fact been done feeder free, but see Li, Z.W., et al. Proc Natl Acad Sci U S A 93, 6158-62 (1996) for an exception.
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