Biology Reference
In-Depth Information
3.3. Identification of Correctly Targeted ES Clones
1.
Wash cells twice with sterile PBS and prepare the following items: 96-well
V-bottom plates (“dissociation plate”), 24-well plates (“DNA plate”); 96-well
flat-bottom plates (“stock plate”); trypsin, selection medium, gelatin, EF cells,
multichannel pipetor, and sterile pipet tips. Coat the “DNA plate” and the “stock
plate” with gelatin and add 2 mL/well of selection medium to the former and
150
µ
L/well of EF cells resuspended in the same medium to the latter. Add
20
L/well of trypsin to the “dissociation plate” prepared just before picking
the ES colonies.
µ
2.
L; transfer to the “dissocia-
tion plate” and resuspend the cells by multiple pipeting. Continue to pick clones
(in groups of 8, 16, or 24 depending on your speed) and add 175
Pull up colonies with a 10-
µ
L micropipet set at 3.5
µ
L of selection
medium after a few minutes, making sure all components are thoroughly mixed.
µ
3.
Transfer 30
L/well from the “dissociation plate” into the “stock plate;” transfer
remaining 150
µ
µ
L/well into the middle of the wells in the “DNA plate.”
4.
Change the medium of the “stock plate” daily until the first clones reach
≥
80%
confluency (2-3 d). At that time, wash all wells with PBS, add 30
µ
L/well of
trypsin and 150
L of freezing solution a few minutes later; seal plates around the
edges with parafilm and place them in a seal-a-meal plastic bag. Freeze and store
cells at -80
µ
°
C for up to six mo.
5.
Wash the “DNA plate” with PBS once the cells reach confluence (~7 d). Add
0.5 mL/well of DNA lysis buffer and incubate overnight at 37
C
(8)
. Place the
plate on a shaker for 30' at room temperature; add an equal volume of isopro-
panol and continue to shake for ~3 h until DNA is completely precipitated. Trans-
fer DNA from the wells to Eppendorf tubes. Wash DNA with 80% ethanol, let
air-dry, and resuspend the pellet in 5
°
µ
L of TE by overnight incubation at 55
°
C.
6.
Digest 5
L with 20 U/mg of
DNA of the appropriate restriction enzyme and then perform Southern blot
hybridization according to the standard protocol
(7)
.
µ
L of the DNA sample (~10
µ
g) overnight at 37
°
C in 50
µ
3.4. Expansion of Positively Targeted Clones
Thaw the 96-well “stock plate” in a 37
C waterbath after placing it in another
seal-a-meal bag. Transfer cells from the stock plate to a 24-well plate which
has been covered with EF cells in ~2.0 mL of ES medium. Split the culture
once the colonies reach a reasonable size. ES cells require a minimal density to
grow and it is best to expand them in a step-wise manner (using 24, 12, and
6-well plates, etc.). If the density is too low, “split back” the cells into a smaller
plating area; be careful because the dense layer of EF cells may cause some
problems. Clones are usually grown enough to obtain several aliquots at ~10
7
cells/mL which are stored frozen until microinjection.
°
3.5. Microinjection of ES Clones into Blastocysts
1.
Organize the set-up for ES microinjection into blastocysts; it consists of a fixed-
stage inverted microscope with a cooled injection chamber and two microman-
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