Protocols

1) Freezing medium

2) Thawing medium

0.25 M sucrose in M2 (860 mg sucrose/10 ml M2). Prepare fresh and sterilize by filtering.

THAWING:

• Cut the straw open and remove the id-stick from the straw.
• Thaw immediately in +37 oC water bath (appr. 3 seconds), the freezing medium turns from opaque to clear.
• Dry the straw with a Kimwipe, cut the end open.
• Transfer the morulae to 2ml thawing medium (with a mouth pipette). Incubate 10 min at RT without disturbing the embryos.
• Transfer the morulae to 2 ml of M2 medium. Incubate 10 min at RT.  
• Wash the morulae in four drops of M2 medium and one drop of KSOM medium.
• Incubate the morulae 30 min in KSOM medium under mineral oil in a +37 oC CO2 incubator.
• Transfer the morulae to pseudopregnant females

NOTE ! Depending on the strain 1-2 straws are thawed. Usually one straw is sufficient and contains 12-16 morulae for transfer to a pseudopregnant female.

ES cells should be split every other day 1:5-1:10. On the day of splitting the ES cell colonies may almost touch one another, but the plate should not be confluent.

ES cell medium is changed every other day and couple of hours before aggregation. For reagents of ES cell medium, contact the GM mouse unit.

For maintenance and freezing, ES cell culture on feeder cells is recommended

For aggregation, ES cells are cultured on gelatinized plates:

First aggregation in 24 hours: dilutions 1:3, 1:6, 1:12

Second aggregation in 72 hours: dilutions 1:6, 1:12, 1:24, 1:48

Note! Dilutions depend on the growth characteristics of your cell line. It is recommended to make several dilutions and pick the best for aggregation

SPLITTING OF ES-CELLS (6cm plate)

• Aspirate the old ES cell medium
• Wash with 5 ml PBS (without Ca and Mg)
• Add 1ml 0,05% trypsin-EDTA solution and incubate in +37oC a few minutes until the colonies start to slough off when you tilt the plate.
• Resuspend in 5 ml ES cell medium and transfer to 15 ml tube (avoid extensive pipetting up and down)
• Centrifuge 5 min 1000rpm, aspirate supernatant
• Add a couple of drops of ES medium. Resuspend the cells in a small volume by tapping the tube semi-vigoruosly. Add 6ml of ES medium. Plate different dilutions on freshly gelatinized plates/feeder cell plates.
• Pick plates containing small colonies for aggregations 24h and 72h after splitting

Take several ES cell plates and 50 ml of prepared ES cell medium to the GM mouse unit.

Note ! On the day of aggregation, the trypsinizing of ES cells to generate ES cell clumps is done in the GM mouse unit.

• Cut out all plasmid sequences from the construct. Use a restriction enzyme leaving cohesive ends (NO “blunt ends”). In the digest you should have at least 30 micrograms of the plasmid insert to be injected.
• Agarose gel electrophoresis: use low current to better separate the fragments. To avoid contamination, clean the electrophoresis equipment thoroughly and use clean reagents.
• Take a picture of the gel to be taken to the GM mouse unit with the DNA. Cut out the insert fragment from the gel and extract the DNA (for example Qiagen QIAEX or QIAquick Gel Extraction –kit).
• Dry the DNA pellet and resuspend in filtered buffer (10mM Tris, 0.1 mM EDTA, pH7.5). Check concentration and integrity by agarose gel electrophoresis with DNA-standards. Take a picture and take it to the GM mouse unit with the DNA. Adjust concentration to 5 ng/microliter (in 10mM Tris, 0.1 mM EDTA, pH7.5), the total volume should be min 200 microliters. This concentration is on the borderline of being toxic to the oocytes, but gives the best insertion frequency. Therefore, careful adjustment of the concentration is important.
• Centrifuge the DNA in a microcentrifuge full speed (13 000 rpm) for 10 min. Transfer 90% to a clean sterile tube. Avoid repeated freezing and thawing and long-term storage.
• Note ! You can get the filtered buffer from the GM mouse unit (10mM Tris, 0.1 mM EDTA, pH7.5)