ES cell targeting

ES cells were grown, thawed and expanded in complete E14 medium containing LIE and supplemented with BRL conditioned medium (see below). The cells were then

electroporated with the targeting construct and after a 24 hr recovery period, the

electroporated cells were grown under G418 selection. Each of 412 colonies obtained was picked on day 9 of selection and expanded. DNA from a sample of each colony was extracted and probed by southern blot hybridisation for correctly targeted clones. The remainder of each colony was split and expanded and samples were frozen down at each step. Correctly targeted clones were expanded further and frozen down for use in blastocyst injection and chimera production.

2.17.1 Production of conditioned media for El 4 ES ceii iines

BRL cell conditioned medium was produced as described below: BRL cells were cultured in E14 complete medium:

480 ml DMEM High glucose/no L-Glutamine added 120 ml ES cell tested foetal bovine serum (FBS)

6 ml L-Glutamine

6 ml Non-essential amino acids

4.4 p.1 2-mercaptoethanol

The surface of a 150 mm diameter tissue culture (TC) dish was covered with 0.1% gelatine solution and left for 5 min. The gelatine solution was then aspirated and 50 ml of BRL cell growth medium was added to each dish.

A frozen vial of BRL cells was thawed in a 37°C water bath and the contents were transferred to a 15 ml centrifuge tube. 9 ml of growth medium was slowly added whilst agitating the tube and then the tube was centrifuged at 250xg for 5 min. The medium was aspirated and fresh medium was added to resuspend the cell pellet. The cell suspension was added to the 150 mm TC dish

Cell were grown to 80-90% confluence and split 1:10 by the following steps.

The medium was collected from the original 150 mm TC dish, passed through a 0.2 pm filter and stored at -20°C. The plate was washed with PBS. 3 ml o f 37°C trypsin/EDTA solution was added and the plate incubated at 37°C for 3-4 min. At least 15 ml of complete media was added to neutralise the trypsin. 1/10 of the cell suspension was placed into 9 150 mm gelatinised TC dishes.

The remaining cell suspension was spun at 250 g for 5 min, then aspirated, resuspended in 1 ml of 90% FBS/10% DMSO and transferred to a labelled cryovial. The cryovial was placed in a Nalgene freezing pot containing isopropanol and the pot placed at -80°C overnight before transferring to liquid nitrogen.

The nine plates were grown for 2 days. Then the conditioned medium was collected and replaced by fresh unconditioned medium. This process was repeated two more times before discarding the plates. All collected medium was passed through a 0.2 pm filter and stored at -20°C.

Chapter 2: Methods and materials

2.17.2 Growth of E14.TG2A ES cells

Medium for growing ES cells LIE to 10^ units/ml

3 parts BRL conditioned media

2 parts of the following E14 complete media

A vial of E14TG2A cells was thawed into a gelatinised TC dish as describes for BRL cells. The cell population was feed every one-two days as necessary and was grown for 3-4 days until it was 70%-80% confluent. Cells were expanded and frozen as needed as described for BRL cells.

2.17.3 Electroporation of ES cells

A 75 cm^ contains flasks containing 3x10^ cells was used for each electroporation

The flask was washed with PBS (CMF). Cells were trypsinised with 1 ml of trypsin/EDTA solution then washed and centrifuged as described earlier. The cell pellet was resuspended in PBS (CMF) and centrifuged again before being resuspended in 0.8 ml DMEM. 40 pg of

targeting construct DNA, suspended in 0.1 ml H2O, was mixed with the cell suspension and

the entire mix added to a 0.4 cm electroporation cuvette, where it was left to stand for 10 min. The mix was electroporated twice;

1) 240 V at SOOpFD (time constant ~6 ms) 2) 230 V at 500pFD (time constant ~6 ms)

The cuvette was tapped and placed on ice for 20 min, after which the cell suspension was made up to 33 ml volume. 3 ml were added to a 60 mm TC dish (with a gridded bottom to help make counting colonies easier) and 10 ml were added to each o f three further 100 mm TC dishes.

Selection was started after 24 hr. Concentrations of 200 pg/ml o f G418 and 2 pM gangcyclovir were added to the ES cell growth media to achieve selection. Medium was changed everyday washing with PBS (CMF) if needed (for the removal of cell debris). Colonies were picked on day 9 of selection.

2.17.4 Picking, expanding, freezing and thawing ES ceii ciones

Picking clones;

A flat bottomed 96 well plate was gelatinised for 10 min, after which the gelatin was

removed and 50 p-1 of selection ES cell growth medium was added to each well. Plates were put into the 37°C incubator to allow pre-warming of the plate and medium ready for the addition o f ES cells.

25 pi of trypsin/EDTA were added to round-bottomed 96 well plates. Medium was removed from the 100 mm TC dish containing colonies and replaced with 10ml of PBS (CMF). Colonies were picked by scraping with a 10 pi pipette tip and transferred to the plate containing the trypsin/EDTA. After 48 colonies had been picked 75 pi of medium was added to the first row. The colonies in this row were triturated well and transferred to the plate from the incubator containing media. This was repeated for all four rows and the whole process was repeated until all 412 colonies had been picked.

The plates containing the colonies were fed as required until the majority of clones were ready to split.

Expanding ES cell clones;

The plates were fed 2-4 hours before splitting. Four 48-well plates per one 96 well plate were gelatinised for 10 mins. The gelatin was then replaced with 400 pi of media.

Cells were washed with PBS (CMF). 50 pi trypsin/EDTA was added and cells were left to incubate until they had lifted. 200 pi of medium was added to neutralise the trypsin and the cell suspension was triturated to achieve a single cell suspension. 100 pi of the single cell suspension was added to each well in each of the two 48 well plates. The whole process was repeated until all clones were transferred. A further 100 pi medium was added to each well of the 96 well plate. The 96 well plates were fed as required for 7 days and used for DNA southern blot analysis.

Freezing the ES cell clones;

The 48-well plates were feed daily or as required until ready for freezing (-3-4 days). Cells were then washed with PBS (CMF). 100 pi of trypsin/EDTA was added and cells were

Chapter 2: Methods and materials

incubated until they lifted. Trypsin was neutralised with 100 pi FBS and the cells were triturated to achieve a single cell suspension. 200 pi of 80% FBS/20%DMSO solution was added and the whole suspension was mixed well. The process was repeated until all clones were in freezing mixture. The edge of the plate was wraped with parafilm and the plate was placed in a polystyrene box after which it was transferred to a -80°C freezer.

Thawing the ES cell clones;

The fi'ozen 48 well plates were thawed at 37°C. 600 pi of media was added to each thawed well and triturated well to obtain a single cell suspension. The cell suspension in each well was split 1/2 and each half (500 pi) of the suspension was transferred to a separate

gelatinised well of a 24-well plate. 24 well plate and the plates returned to the incubator. Cells were fed daily or as required and then expanded and frozen as required.