Start this protocol six days after electroporating the ES cells (see Protocol on Electroporation of ES Cells).
View the 10 cm dishes containing the transfected ES cells through the inverted microscope. The clones are visible as small nests of rapidly growing cells. They have tight borders and are closely packed. Larger cells within the colony, with well-defined membranes are the cells that are beginning to differentiate. Do not pick these clones! This is Day 6 of the selection process. If the clones are big enough you may pick on this day, or wait until Day 7.
To pick the clones, set up an inverted microscope in a laminar flow hood.
Prepare the 24-well plates (made in Section D of the Protocol on Electroporation of ES Cells) by taking out the old media and replacing with 1.0 ml ES Selection Media per well.
Place the five 24-well dishes back into the incubator.
Replace the ES Selection Media in the first 10 cm dish with fresh ES Selection Media.
Place the dish under the microscope and view the cells at 4X and 10X magnification. Try to be sure to only identify clones that have not yet started to differentiate (see Hint #1). Isolate the clone to be picked in the viewing field.
Using a 0 to 160 μl barrier pipette tip, gently push the ES clone forward from the surrounding MEFs. With the pipettor set between 30 and 50 μl, and the plunger button already depressed, pluck the clone using a forward scooping suction motion. If the pipettor is set on 30 μl, there should be enough suction to dislodge the clone from the plate. However, if the inactivated MEF layer is too dense, it may be difficult to dislodge the ES clones. In this case, carefully tease the surrounding MEFs away from the clone without disrupting the clone with the pipettor tip. Then harvest the clones.
Place each clone into one of the 24 wells containing ES Selection Media.
Continue to pick clones and place in the wells of the prepared 24-well plate until 12 clones are picked or the 10 cm dish has been out of the incubator for 15 min (see Hint #2).
Place the plate containing the picked clones in the incubator.
Continue picking clones until all 24 wells have been filled in all of the prepared dishes (see Hint #3).
Place the 24-well dishes in the incubator overnight.
See the Protocol on Dissaggregation, Expansion, and Freezing of Transfected ES Clones.
Solutions and Buffers
G418 Stock
Prepare in 3 ml PBS
Filter sterilize with 0.2um nitrocellulose filter
ES Selection Media
2.5 ml of 2 M HEPES pH 7.2
5 μl of 2-Mercaptoethanol
Make fresh every week.
5 ml of 10 mM Non-Essential Amino Acids
85 ml Heat-Inactivated Fetal Calf Serum
5 ml of 200 mM L-Glutamine
Filter sterilize with 0.2um Nitrocellulose Filter
50 μl of ESGRO (Murine Leukemia Inhibitory Factor,LIF) (1000 units/ml final)
405 ml of Dulbecco's Modified Eagle's Medium (MEM)
300 μg/ml active concentration G418
BioReagents and Chemicals:
Non-Essential Amino Acids
Dulbecco's Modified Eagle's Medium (DMEM)
HEPES
G-418 (Geneticin)
2-Mercaptoethanol
ESGRO (Murine Leukemia Inhibitory Factor, LIF)
L-Glutamine
Fetal Calf Serum (FCS), Heat Inactivated
Protocol Tips:
1. After electroporation and subsequent selection, it is important to carefully pick stable clones for later expansion and DNA analysis. Clones (which contain many small nests of cells) should increase in size during selection but not undergo differentiation. The cells will pile up and maintain a discrete colony on top of the feeder cells. The cells within a clone will adhere tightly to one another, making it difficult to see individual cells; a tight border should surround the clone. The ES clones will grow well on the MEF feeder layer, forming various shapes, such as oblong or triangular. Clones that exhibit cellular heterogeneity, such as endodermal cell formation on the outside free surface of the colony, or a spreading of the cells at the periphery and general flattening, should not be selected.
2. The clones are sensitive to pH and temperature changes.
3. If you were not able to pick all your clones on Day 6 of selection, you may continue to pick on Selection Day 7. The contributors of this protocol do not recommend picking clones on Day 8.
