Use of the Sleeping Beauty Transposon System for Stable Gene Expression in Mouse Embryonic Stem Cells

Reagents

D3 mES cells (ATCC collection or University of Michigan Transgenic Core)

Expand High Fidelity PCR System (Roche)

Hanks’ Balanced Salt Solution (1X HBSS) (without CaCl₂ , MgCl₂ , or MgSO₄ ) (GIBCO/Invitrogen)

Hygromycin B (Invitrogen)

Lipofectamine (Invitrogen)

Lipofectamine Plus Reagent (Invitrogen)

mES cell growth medium

mES cell growth medium with Leukemia Inhibitory Factor (LIF)

OPTI-MEM (+HEPES, +sodium bicarbonate, +glutamine) (GIBCO/Invitrogen)

Sleeping Beauty transposon and transposase plasmids (available from the Center for Genome Engineering, University of Minnesota, http://www.cge.umn.edu )

It is important to include a selection marker for the isolation of clones that harbor the transposon. The vector described in this protocol contains a hygromycin cassette for selection of stable clones. Alternative selection markers can be chosen from various antibiotic resistance genes or fluorescent markers such as enhanced green fluorescent protein (EGFP). Prepare plasmid DNA (~ 1 µg/µL) using a QIAGEN miniprep kit. To obtain high concentration yields, use a 5-mL overnight bacterial culture, wash the prep with the optional wash provided in the kit, and elute the DNA twice (see also Hermanson et al. 2004 ). For a diagram of the expression construct used in this protocol, see Figure 2 .

Equipment

Aspirator

Centrifuge (benchtop)

Hemacytometer

Hood (sterile for tissue culture)

Incubator (humidified, 37ºC, 5% CO₂ )

Micropipettors (e.g., Gilson Pipetman P10, P20, P200, P1000) and disposable barrier tips (sterile, DNase/RNase-free)

Microscope (inverted, equipped with bright-field optics)

Pipettes (sterile serological; 2-, 5-, 10-, and 25-mL)

Plates, dishes, and flasks (sterile tissue culture, gelatin-coated; 48-well, 24-well, 12-well, six-well, 10-cm, T25)

Prepare gelatin-coated plasticware just before use by coating the bottom of each dish with 0.1% (w/v) gelatin solution (Sigma). Incubate the dishes for 15 min in a humidified, 5% CO₂ incubator at 37ºC, and wash twice with 1X HBSS. For a 10-cm dish, use 5 mL of gelatin solution and 2 x 5 mL of HBSS. Use the 10-cm dishes as soon as they are prepared; T25 flasks can be stored at 4ºC for up to a month.

Tubes (sterile conical, 15-mL; Falcon)

Tubes (sterile microcentrifuge, 1.5-mL)

METHOD

Use standard aseptic technique for all procedures.

Passaging mES Cells onto Gelatin-Coated Dishes for Transfection

1. Grow D3 mES cells in a gelatin-coated T25 tissue culture flask in a humidified, 5% CO₂ incubator at 37ºC until 90%-95% confluent (2 d).
D3 mES cells may also be grown in 10-cm, gelatin-coated tissue culture dishes or flasks. Other ES cell lines may require different culture methods. For example, mouse embryonic fibroblasts (MEFs) may be used as an alternative to gelatin, in combination with LIF (Nagy et al. 2003 ).
 

2. Wash the cells twice with 1X HBSS.
 

3. Add 2 mL of trypsin/EDTA to the flask and incubate for 2 min in a humidified, 5% CO₂ incubator at 37ºC.
 

4. Add 4 mL of mES cell growth medium to the flask to stop the trypsin. Triturate (pipette up and down) with a 5-mL pipette to mechanically dissociate the cells from the flask.
 

5. Transfer the dissociated mES cells to a 15-mL conical tube, and centrifuge at 1200 rpm for 5 min at room temperature.
 

6. Aspirate the supernatant. Resuspend the pelleted cells in 1 mL of mES cell growth medium with LIF; dissociate the cells mechanically into a single-cell suspension.
 

7. Count the cells using a hemacytometer (see Estimation of Cell Number by Hemocytometry Counting [Sambrook and Russell 2006a ]).
At least 6 x 10⁶ cells are needed to proceed with the transfection.
 

8. Plate 3 x 10⁶ mES cells in 10 mL of mES cell growth medium with LIF into a gelatin-coated, 10-cm tissue culture dish. Prepare two 10-cm dishes per transfection.
 

9. Incubate the cultures in a humidified, 5% CO₂ incubator overnight at 37ºC.

^{Transfection of mES cells}

^{10. Grow the mES cells from Step 9 to 50%-80% confluence.}

^{11. Prepare the Lipofectamine transfection mixture:}

^{i. In a microcentrifuge tube labeled "A," add 750 µL of OPTI-MEM, 20 µL of Lipofectamine Plus Reagent, 3 µg of transposon plasmid, and 1 µg of transposase expression plasmid.}

^{ii. In a microcentrifuge tube labeled "B," add 750 µL of OPTI-MEM and 30 µL of Lipofectamine.}

^{iii. Incubate both tubes in a sterile tissue culture hood for 15 min at room temperature.}

^{iv. Combine the contents of tubes A and B and incubate in a sterile tissue culture hood for 15 min at room temperature.}

^{12. Aspirate the growth medium from the plated cells and add the Lipofectamine mixture from Step 11.iv to the cells.}

^{13. Incubate the cells with the Lipofectamine mixture in a humidified, 5% CO₂ incubator for 6 h at 37ºC.}

^{14. Replace the Lipofectamine mixture with 10 mL of mES cell growth medium with LIF. Continue to incubate the cells in a humidified, 5% CO₂ incubator at 37ºC.}

^{15. 48 h after transfection, add hygromycin B at a final concentration of 200 µg/mL to the culture to select for transfected cells. Maintain the cells in a humidified, 5% CO₂ incubator at 37ºC, and split them at a 1:2 ratio into four gelatin-coated, 10-cm dishes when they are 90%-95% confluent.
See Troubleshooting.}

^{Isolation of Individual Clones}

^{16. When colonies are ready to be picked, aspirate the medium from each 10-cm dish and replace it with 2 mL of 1X HBSS.}

^{17. Manually pick the colonies using an inverted microscope equipped with bright-field optics:}

^{i. Draw up 20 µL of 1X HBSS with each colony and place it into one well of a gelatin-coated, 48-well plate with 50 µL of trypsin/EDTA.}

^{ii. Gently pipette up and down several times to dissociate the colony into single cells.}

^{iii. Use the microscope to verify that there are no clumps.}

^{18. Add mES cell growth medium with LIF to a final volume of 300-350 µL per well to inactivate the trypsin. Grow the cells in a humidified, 5% CO₂ incubator at 37ºC until they are 90%-95% confluent. Change the medium every 2 d.}

^{19. Passage the cells of each clone into one well of a gelatin-coated, 24-well sterile tissue culture plate. Use a final per-well volume of 500 µL of mES cell growth medium with LIF plus 200 µg/mL hygromycin B. Grow the cells in a humidified, 5% CO₂ incubator at 37ºC until they are 90%-95% confluent. Change the medium every 2 d.}

^{20. Passage the cells of each clone into one well of a gelatin-coated, 12-well sterile tissue culture plate. Use a final per-well volume of 1 mL of mES cell growth medium with LIF plus 200 µg/mL hygromycin B. Grow the cells in a humidified, 5% CO₂ incubator at 37ºC until they are 90%-95% confluent. Change the medium every 2 d.}

^{21. Passage the cells of each clone into one well of a gelatin-coated, six-well sterile tissue culture plate. Use a final per-well volume of 2 mL of mES cell growth medium with LIF plus 200 µg/mL hygromycin B. Grow the cells in a humidified, 5% CO₂ incubator at 37ºC until they are 90%-95% confluent. Change the medium every 2 d.}

^{22. Passage the cells of each clone at a 1:2 ratio into two wells of a gelatin-coated, six-well sterile tissue culture plate. Use a final per-well volume of 2 mL of mES cell growth medium with LIF plus 200 µg/mL hygromycin B. Grow the cells in a humidified, 5% CO₂ incubator at 37ºC until they are 90%-95% confluent. Change the medium every 2 d.}

^{23. Passage the cells of each clone (two wells of a six-well sterile tissue culture plate) into a gelatin-coated T25 flask. Use a final volume of 3 mL of mES cell growth medium with LIF plus 200 µg/mL hygromycin B. Continue to maintain the cells in a humidified, 5% CO₂ incubator at 37ºC.}

^{Verification of Gene Expression}

^{24. Check the gene expression of each clone by standard reverse transcriptase polymerase chain reaction (RT-PCR). See Amplification of cDNA Generated by Reverse Transcription of mRNA (Sambrook and Russell 2006b ).}

^{Determination of Transposon Integration Number by Southern Blot Analysis}

^{25. Isolate genomic DNA from the transgenic mES cells, perform a restriction digest of the DNA, and use Southern blot analysis to determine the number of transposons that have integrated into the genome. For details of these procedures, see Southern Blotting: Capillary Transfer of DNA to Membranes (Sambrook and Russell 2006c ), Southern Blotting: Simultaneous Transfer of DNA from a Single Agarose Gel to Two Membranes (Sambrook and Russell 2006d ), and Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes (Sambrook and Russell 2006e ).}

^{Determination of the Transposon Integration Site with Inverse PCR}

^{26. Verify that the integration is SB-mediated by performing inverse PCR with genomic DNA from the transgenic mES cells. For details of this procedure, see Inverse PCR (Sambrook and Russell 2006f ).
We recommend the Expand High Fidelity PCR System (see also Hermanson et al. 2004 ). Splinkerette PCR is an alternative method for identifying the transposon insertion site (Dupuy et al. 2002 ).}

^{TROUBLESHOOTING}

^{Problem: Cell death occurs during transfection.}

^{[Step 15]}

^{Solution: To reduce cell death, try the following:}

^{1. Transfect ES cells at higher confluence.}

^{2. Use less Lipofectamine.}

^{3. Use less DNA.}

^{4. Include serum at concentrations used to maintain the cells normally (10% serum for mES cells).}

^{Problem: Transfection efficiency is low.}

^{[Step 15]}

^{Solution: To increase transfection efficiency, consider the following:}

^{1. The plasmids might be degraded. Verify that the plasmids are intact by running each plasmid on an agarose gel before transfecting.}

^{i. If the plasmids are intact, check that the plasmid concentration is at least 1 µg/µL.}

^{ii. If the plasmids are degraded, regrow the plasmid and isolate a fresh plasmid preparation.}

^{2. The ES cells might have differentiated. To avoid transfecting differentiated cells, only grow the cells for 24 h. Increase the plating density if the cells are not confluent enough within this 24-h period.}

^{3. Mycoplasma could be contaminating the ES cells. To detect Mycoplasma contamination, use a commercially available PCR kit.}

^{4. Lengthen the time that the cells are exposed to Lipofectamine.}

^{5. Retransfect the cells.}

^DISCUSSION

^{The use of viral vectors for gene transfer, although highly efficient, is not always an ideal choice due to recombination, immune responses, and other safety concerns associated with these vectors. DNA transposons offer an effective, alternative method for nonviral gene transfer in mES cells, because they avoid the safety concerns associated with viral vectors and are more efficient than plasmid-based methods. The result of using a transposon system is stable integration and long-term expression of a transgene. SB as described in this protocol is an advantageous method for gene delivery and integration in mES cells. It is important to identify the insertion locus and confirm that the integration is SB-mediated. For additional applications, such as delivering expression cassettes that are >10 kb or mutagenic vectors to mES cells, other transposon systems such as Tol2 and piggyBac may also be considered.}

^{ACKNOWLEDGMENTS}

^{This work was supported by a postdoctoral fellowship to A.E.D. from Tissue Engineering at Michigan (TEAM), by startup funds to C.E.K. from the School of Dentistry/Biologic and Materials Sciences, a National Institutes of Health (NIH) grant to C.E.K., NS-050142, and NIH grants to K.S.O., GM-069985, NS-048187.}

^REFERENCES