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Day 0:
Prepare one T-150 flask of Neo-Resistant ( R ) Mefs and two T-25 flasks of irradiated Neo-R Mefs. The second T-25 is a backup.
Day 1:
Thaw one vial of the targeted, expanded ES clone to one of the T-25 flasks of irradiated Neo-R Mefs freshly fed with ES media. Expand the ongoing T-150 flask of Neo-R Mefs to 2 x T-150s.
Day 2:
In the morning, feed your ES cells with ES media. Harvest the Neo-R Mefs in the two T-150s with trypsin/EDTA, plate back half of the cells into
the same two T-150s, and irradiate the rest at 3000 rads. Use the irradiated cells to prepare 5 x 10 cm dishes at a concentration of 1x106 cells per dish. In the
afternoon, if the ES cells look ready, you can electroporate them.
Harvest all the cells from the T-25 containing the targeted ES cells using trypsin /EDTA. Be CERTAIN that all cells are thoroughly disaggregated , because it is critical that individual transfected ES cells (not pairs or clusters of cells) are plated after electroporation. Otherwise, "clones" will consist of groups of cells in which some will have cre-mediated removal of the PGK-Neo cassette, and some will not. This heterogeneity will be picked up later, during Southern analysis of the transfected "clones".
Spin the totally disaggregated cells at 1000 rpm for 5 minutes, and resuspend in 1 ml ice cold 1x Hebs buffer. Add 1 ml of the targeted cell suspension to 20ug of pTurbo-cre (supercoiled) in 20ul 1xTE and pipette up and down several times. Add the entire cell/plasmid solution to a Flatpack cuvette and electroporate at the standard setting (185V,500 uF). Set the electroporator (BTX 600 or equivalent) as follows:
500V/Capacitance and resistance
500uF Capacitance timing
360 ohms R8 Resistance timing
Add the electroporated cells to 3 ml of ES medium in a 15 ml tube (final volume 4 ml).
Plate the cells as follows:
Using the five 10 cm dishes of irradiated Mefs (previously fed with ES medium)Dish 1: 2.0 ml cells plus 10 ml ES medium
Dish 2: 1.0 ml of cells plus 11 ml of ES medium
Dish 3: 400 ul of ES cells plus 11.6 ES medium
Dish 4: 200 ul cells plus 11.8 ml ES medium
Dish 5: 100 ul cells plus 11.9 ml ES medium
Day 3:
Remove medium and add 12ml fresh ES medium to all plates.
Day 4:
Refeed cells with 12 ml ES medium per plate and prepare 3 x 24 well plates with irradiated Mefs. The third plate is a back up plate.
Day 5 or 6:
Feed ES cells with ES medium and, if the cells are ready (as determined by size and appearance), pick 48 individual clones and replate into the 2 x 24 well plates containing feeder Mefs. The cells selected should be of small to medium size, with the circumference entirely shiny and with no signs of differentiation (i.e. no cells protruding from inside the clone). REMEMBER, THERE IS NO G418 SELECTION USED AND THAT IS WHY SIZE/APPEARANCE IS SO IMPORTANT. If clones are not picked on day 5, this procedure is performed on day 6.
Day 7:
Disaggregate all clones with trypsin/EDTA.
Day 8:
Feed disaggregated cells with ES medium, 1 ml per well.
Day 9:
Feed cells again with the same medium.
Day 10:
Some wells may be ready to freeze by the standard method (see the earlier section on this web page for this procedure). The remaining wells must be disaggregated again (trypsin/EDTA).
Day 11:
Feed cells which were disaggregated on day 10 with ES medium.
Day 12:
Feed or freeze individual wells, depending on the number of colonies.
Day 13 or 14:
Freeze remaining wells.
Special note: When freezing individual clones there will be 200 ul remaining in each well of the 24 well plates, after removing 500 ul of cell suspension to each freezing vial. Coat 2 x 96 well plates with 0.1% gelatin. Transfer 20 ul of the remaining 200 ul into each well of the 96 well plate. Do this for all wells, numbering each 96 well plate appropriately so as to be able to identify each clone. Add ES medium to both the 24 well (1ml) and 96 well (100 ul) plates. The next day (day 11) feed the 96 well plates with 100 ul of G-418 selection medium, leaving the 24 well plate to grow to confluency without further feeding. Monitor the 96 well plates for cell death. Define the wells that fail to grow in G418 (which indicates that the neo marker has been removed ). Extract DNA for Southern analysis from the confluent 24 well plates. Using the information gained from the 96 well plates, you will have a clue as to which clones have lost the neo cassette. Analyze these clones first. However, ALWAYS be sure to check by Southern analysis that neo has been removed and that the targeted mutation has been retained in the targeted locus. It is useful to design a genetic 'mark' (i.e. a new restriction site) in the mutant locus to be sure that the doubly-manipulated ES cells still contain the desired mutation in the mutant locus.
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