Expansion of ES cells

Electroporation and expansion

Infection of ES cells with retroviral vectors

ß - gal - staining

Recombinase delivery into ES cells

FRT-PCR

FlipROSAbetageo Inversion

Electroporation of ES cells ("Gene-Trap Conditions"; kindly provided by Susanne Bourier)

Expansion of ES cells

• 1. Thaw one vial of early-passage ES cells, wash as usual and plate the cells on a 60 mm gelatin-coated petri dish with primary embryonic feeder cells (EMFI). EMFI cells should be confluent on the plate.
• 2. Change the ES-cell medium once each day.
• 3. After 2 days trypsinize and expand the cells on one to two 90mm gelatin-coated petri dishes (with EMFI cells) dilution 1:3 up to 1:8, depending on the cell density.
• 4. Wait another 2 days and transfer the cells to fresh feeder plates again dilution 1:3 or 1:4.
• 5. After 2 days expand the cells to at least 2x200 mm or 6x90 mm gelatinized petri dishes (only a dilution of 1:2) and start the transfection 36 hours later in one electroporation cuvette. The cell number on 2x200 mm subconfluent petri dishes should be approximately 1x10⁸ cells. Change medium in the plates 6 hours before the electroporation.

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Electroporation and selection

• 1. Trypsinize ES cells for 10 minutes in 3 ml trypsin per dish, and pipet them gently up and down to aqcuire a single cell suspension, add 3 ml medium per dish and transfer the cells to 2 Falcon tubes .
• 2. Centrifuge the cells for 3 minutes at 270 g.
• 3. Resuspend the cells in 10 ml PBS.
• 4. Dilute an aliquot 1:10 and count the cells (keep the cells on ice).
• 5. Centrifuge 1x10⁸ cells 5 minutes at 270 g for one cuvette.
• 6. Add to the pellet 500 ul cold PBS and 100 ul of the DNA (120 ug), no more than 700-800 ul in total (Vector linearization: digest with adaequate enzyme 1 U/ug at least for 4 hrs, phenol-extract and precipitate the DNA at 70C for 10-15 minutes. Wash in 70% ETOH and air-dry under sterile conditions).
• 7. Transfer the suspension to the electroporation cuvette.
• 8. Set up the electroporation conditions in advance (0.8 kV, 3 uF for the Bio Rad gene pulser).
• 9. Transfer the cuvette into the cuvette-holder with electrodes facing the output leads and deliver electric pulse.
• 10. Remove the cuvette from the cuvette-holder and leave it at room temperature or on ice for 10 to 20 minutes.
• 11. Transfer the cell suspension from one cuvette into 12 ml ES cell medium. Seed the electroporated cells at a density of 2.5-5x10⁶ cells / 90 mm dish on a gelatinized plate in medium containing LIF. The cell concentration per plate must be adjusted depending on the vector such that no more than 200-500 neoR colonies are obtained on each plate.
• 12. Change the ES medium the next day.
• 13. Two days after electroporation, add the drugs for selection to the ES medium (e.g. G418: 200 ug/ml (active); puromycin: 1 mg/ml).
• 14. Change the selection medium every day for the first 3 days, than every other two days.
• 15.  About 6-8 days of selection, drug resistant colonies should have appeared.
• 16.  After 8 - 9 days of selection colonies are picked and plated on 96-well-feeder plates, containing ES-medium. Stop the selection-pressure and exchange the ES-medium each day. After one day of growth tryplate the colonies, after another 2 days trypsinize and dilute the clones 1:3. After another two days, split each 96 well replica-plate 1:1 on 2 x48 well feeder plates.

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Infection of ES cells with retroviral vectors (Wurst and Gossler, 2000)

• 1. Plate ES cells on gelatinized tissue culture dishes at a density of 3 x 10⁶ cells per 90 mm dish in ES cell medium supplemented with LIF or BRL conditioned medium.
• 2. After 24 hours aspirate medium, add 5 ml fresh medium containing the retroviral particles at a m.o.i < 1 and 5 g/ml polybrene to obtain single integration per clone.
• 3. After over night culture (14 h) remove virus containing medium, add 10 ml fresh medium and culture for an additional 24 h.
• 4. Change the medium to selection medium and change medium every other day. Drug-resistant colonies should become visible between 7-10 days.

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ß - gal - staining

ß - galactosidase staining of cultured cells, whole embryos and tissues

(Wurst & Gossler (2000): Gene trap strategies in ES cells. In: A. Joyner (ed.): "Gene Targeting", A practical approach, 2^nd edition. Oxford University Press.

• 1. Wash in PBS: for cells: aspirate the medium and replace with PBS; repeat. For embryos: transfer into PBS, gently swirl around. For tissues: transfer into PBS, gently swirl around.
• 2. Fix in buffer B: for cells: add sufficient buffer B to the plate such that the cells are well covered and leave for 5 minutes at room temperature For embryos: up to day 9.5 add 1 ml buffer B for 10-20 embryos and leave for 5 minutes at room temperature. For day 10.5 to 12.5 embryos add 5-10 ml buffer B for 10 embryos and leave for 15 minutes at room temperature (see Note 1). For tissues: add about 10 times the volume of the tissue of buffer B and leave 15 to 60 minutes (depending on size) at room temperature.
• 3. For all fixation steps: aspirate well wash buffer before adding buffer B to prevent dilution.
• 4. Wash 3 times with 10 ml buffer C at room temperature: for cells: 5 minutes each. For embryos up to day 9.5, 5 minutes each. For embryos up to day 10.5 to 12.5, 15 minutes each. For tissues: 15 to 60 minutes (depending on size).
• 5. Replace buffer C with buffer D and incubate at 37C. Before adding buffer D, aspirate well buffer C for cells: add sufficient buffer to the plate that cells are well covered and that solution will not evaporate (see Notes 2, 3, 4 and 5): for embryos up to day 9.5 add 1ml buffer; for 10-20 embryos, for day 10.5 to 12.5 add 5-10 ml for 10 embryos. For tissues: add about 10 times the volume of the tissue.
• 6. After staining wash samples 3 times in 10 ml buffer C.
• 7. Samples can be stored for short term (a few days) in solution C at 4C, but for prolonged storage the specimens should be fixed again in 4% paraformaldehyde for 2 hours at room temperatureand kept in 70% ethanol at 4C.

Notes

• 1. Always prepare fresh fixing solution before use or store at -20C; other fixations can also be used [e.g. 2% glutaraldehyde or 4% paraformaldehyde (PFA) are possible]
• 2. When solution D is prepared freshly, chill on ice for 10 minutes and spin down precipitate, aliquot supernatant.
• 3. The staining solution D can be reused several times; filter after each use and keep in dark at -20 C.
• 4. A 50 mg/ml X-gal stock solution (=100x) can be made both in Dimethylsulfoxid or Dimethylformamide (DMF). Keep at -20C. DMF has the advantage to stay liquid at -20C.
• 5. K₃[Fe(CN)₆] and K₄[Fe(CN)₆] should be kept as 0.5 M stock solutions (=50x) in dark bottles at (-20C).

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Recombinase delivery into ES cells (DNA transfection)

This protocol is used for removing targeted DNA fragments flanked by frt or loxP sites using site-specific FLPe- or Cre-recombinases, respectively. For higher efficiency of recombination (or reducing work load) the recombinases are bicistronically expressed with a puromycin resistance gene (pac) under the control of the CAGGs promoter. Thereby, transient selection for 48 h can be used to reduce background level (Taniguchi et al., 1998). The expression vector, pCAGGs-Flpe-IRES-puro-pA has been described elsewhere (Schaft et al., 2001). Similar vectors for Cre and ΦC31 integrase (pCAGGs-Cre-IRES-puro-pA and pCAGGs-ΦC31-IRES-puro-pA) are available. Usually the flanked fragments include selection cassettes, so that the efficiency of recombination can be checked afterwards by sensitivity assays to the selection pressure used previously.

• 1. Split confluent ES cells (usually 1x107 cells per 10 cm TC dish) and start with 2x106 cells per 10 cm TC dish so that after 24 h they are in the log phase of grow and having a density of 4-5 x 107 cells per dish.
• 2. After 24 h, wash cells with PBS and trypsinize with 1 ml 0.5% (v/v) Trypsin solution for 5 min. at 37ºC.
• 3. Inactivate Trypsin with 9 ml ES medium and pipette the suspension using a 10 ml pipette (8 - 10 times) in order to get single cells.
• 4. Take an aliquot of 10 μl and count cells.
• 5. Centrifuge in 15 ml Falcon tube at 1000 rpm for 5 min at RT.
• 6. Resuspend the pellet in PBS in a concentration of 1x107 cells in 900 µl PBS.
• 7. Mix the 900 μl suspension with the DNA (40 μg, circular) and transfer to an electroporation cuvette (4 mm gap).
• 8. Leave on ice for 5-10 min.
• 9. Electroporate using the following conditions: 250 Volt, 500 μF (Gene pulser X-cell; BioRad)
• 10. After electroporation immediately flick the cuvette to stabilize the pH.
• 11. Place the cuvette on ice.
• 12. Transfer the electroporated suspension using 1 ml pipette in a 15 ml Falcon tube containing 9.5 ml ES medium. Total volume is around 10.4 ml.
• 13. Distribute the cells in 10 cm TC dishes (1 ml per dish - 10 dishes) that are already filled with 10 ml ES medium each.
• 14. After 24 h replace the ES medium with one that contains 1 μg/ml puromycin (Sigma P-8833)
• 15. Keep the selection for 48 h, without changing the medium.
• 16. Remove the selection medium and replace with normal ES medium and change every day.
• 17. 10-12 days after electroporation colonies become visible.

Low-density seeding of ES cells

In cases selection is not possible or colonies exhibit mosaic genotype, cells have to be diluted and seeded onto feeder layers for growing clonal cell colonies.

• 1. Proceed as described above until step 13
• 2. Wash cells with PBS and trypsinize with 1 ml Trypsin for 5 min. at 37°C.
• 3. Inactivate Trypsin with 9 ml ES medium and pipette the suspension using a 10 ml pipette (8 - 10 times) in order to get single cells.
• 4. Take an aliquot of 10 μl and count cells.
• 5. Seed 500 cells onto gelatinized 6 cm dishes containing feeder layers.
• 6. Grow cells for 10-12 days with daily change of medium until colonies reach an appropriate size for picking.

Transfection by FuGene

Seed 2 x 105 cells onto 6 well plates and trasfect with 2 μg Caggs Flip IRES Puro and 6 μl FuGene according to the manufacturers protocol. After 24 h start selection with puromycin for 48 h. Wait until colonies reach the optimal size for picking and procees as described.

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FRT-PCR

protocol as PDF

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FlipROSAbetageo Inversion

protocol as PDF

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