Protocol


Nature Protocols 2, 792 - 796 (2007)
Published online: 5 April 2007 | doi:10.1038/nprot.2007.105

Subject Categories: Cell and tissue culture | Cell and developmental biology | Genetic modification

A method for genetic modification of human embryonic stem cells using electroporation

Magdaline Costa1,4, Mirella Dottori2,4, Koula Sourris1, Pegah Jamshidi2, Tanya Hatzistavrou1, Richard Davis1, Lisa Azzola1, Steven Jackson1, Sue Mei Lim1, Martin Pera2,3, Andrew G Elefanty1 & Edouard G Stanley1

The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell amplification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.

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Introduction

The value of genetically modified embryonic stem cells (ESCs) as a research tool is demonstrated throughout the literature1, 2, 3. In mouse ESCs, electroporation is the most widely used technique for the generation of site-specific genetic modification, usually by homologous recombination4, 5, 6. Although homologous recombination can be achieved using other transfection procedures7, a survey of studies involving mouse ESCs demonstrates that electroporation is the preferred technique for achieving homologous recombination outcomes. Studies in mouse ESCs also indicate that the success of gene targeting is influenced by factors including the locus of interest, whether the homology arms of the vector are isogenic with the recipient cell line and the length of homology. One common theme concerning gene targeting in both mouse and human ESCs is that homologous recombination occurs in only a small fraction of stably transfected cells (usually less than 10% and more often ~1%)8, 9, 10, 11. In this regard, it is important to use protocols that allow for the repeated generation and screening of large numbers of stably transfected clones.

To date, the number of reports in which electroporation has been applied to HESCs have been limited10, 11, 12, most likely reflecting difficulties in culturing large quantities of HESCs required for this procedure and the high cell mortality associated with electroporation. In this report, we describe a reliable electroporation protocol for HESCs that uses parameters similar to those used for gene targeting in mESCs13. A critical aspect determining the success and reproducibility of this protocol for HESCs is the tissue culture regime used to generate large quantities of HESCs that are able to survive the electroporation procedure. Although collagenase type IV is commonly used for the large-scale passaging of HESCs, this enzyme yields cell clumps, making it difficult to determine precise cell numbers. However, if HESCs are passaged with trypsin14 (Nissim Benvenisty, personal communication) or TrypLE select, it is possible to generate a single-cell suspension, the preferred substrate for electroporation. The protocol described here has proved to be robust using different vector constructs as well as different HESC lines and has been routinely used in our laboratory to generate genetically modified stable HESC lines, including the ENVY cell line12.


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Materials

Reagents

  • Vectors for transfection, pEFBOS-GFPneo (encoding a GFPNeo) fusion protein and human β-actin promoter-GFP-IRES-Neo12
  • hES2, hES3 or hES4 cells: these lines are supplied by ES Cell International and are on the National Institutes of Health Stem Cell Registry
  • MEL2 cells: this cell line is supplied by the Australian Stem Cell Centre, http://www.stemcellcentre.edu.au/
  • Mitotically inactivated mouse embryonic fibroblasts (MEFs)15
  • PBS, without CaCl2 and MgCl2 (Gibco, cat. no. 14190-144)
  • Agarose (Promega, cat. no. V3125)
  • MassRuler DNA Ladder (Fermentas, cat. no. SM0403)

Equipment

  • Cesium source
  • 26G × 1/2″ (0.45 × 13 mm) needles (Terumo, Code NN*2613R)
  • 1 ml syringe (Terumo, Code SS+01T)
  • Center-well organ culture dishes, 60 × 15 mm (Falcon, cat. no. 353037)
  • 60 × 15 mm tissue culture dishes (Falcon, cat. no. 353002)
  • 150 cm2 tissue culture flask with vented cap (Iwaki, 3133-150)
  • 75 cm2 tissue culture flask with vented cap (Iwaki, 3123-075)
  • 48–well plates (Falcon, cat. no. 353078)
  • 15 ml centrifuge tube (Iwaki, cat. no. 2325-015)
  • Tissue culture incubator at 5% CO2
  • Electroporator ( Gene Pulser II System; Bio-Rad)
  • Gene pulser cuvette 0.4 cm electrode gap (Bio-Rad, cat. no. 165-2088)
  • Centrifuge (Sigma, 4K15)
  • Stereomicroscope (Leica, MZ6)
  • TrypLE select (Gibco, cat. no. 12563-029)

Reagent setup

  • HESC media DMEM/F12 (Gibco, cat. no. 11320-033), 20% (v/v) knockout serum replacer (Gibco, cat. no. 10828-028), 10 mM non-essential amino acids (Gibco, cat. no. 11140-050), 2 mM L-glutamine (Gibco, cat. no. 25030-081), penicillin/streptomycin (Gibco, cat. no. 15070-063), 50 mM 2β-mercaptoethanol (Gibco, cat. no. 21985-023), 8 ng ml−1 bFGF (R&D, cat. no. 233-FB-025)16.
  • MEF media DMEM (Gibco, cat. no. 11960-044), 10% (v/v) heat-inactivated fetal calf serum (JRH Biosciences, cat. no. 12003), 2 mM L-glutamine (Gibco, cat. no. 25030-081), penicillin/streptomycin (Gibco, cat. no. 15070-063).
  • Trypsin Trypsin–EDTA (0.25% (w/v) trypsin, EDTA·4Na) (Gibco, cat. no. 25200056) with 2% (v/v) chicken serum (Hunter Antisera, item no. 110). Diluted 1:2 with PBS13.
  • DNA lysis buffer 100 mM Tris pH 8.0 (Amresco, 0497), 200 mM NaCl (Merck AnalaR, product no. 1.06404), 5 mM EDTA (Merck AnalaR, product no. 10093.5V), 0.2% (w/v) SDS (Sigma-Aldrich, cat. no. L4390), 200 μg ml−1 proteinase K (Sigma-Aldrich Pty Ltd, cat. no. P2308).
  • TE 10 mM Tris pH 8.0, 1 mM EDTA pH 8.0.
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Procedure

  1. During weeks before electroporation: cell expansionThis step and Step 2 pertain to cells that are maintained in organ culture using mechanical passaging. For cells already maintained using enzymatic passaging procedures, go to Step 3. To ensure their genetic integrity, stock cultures of HESCs should be maintained in organ culture using traditional cut-and-paste passaging17, 18, 19 (Fig. 1a–h). To prepare HESCs for electroporation, an expansion period involving single-cell disaggregation is required. Using a 23G needle, slice colonies from 18 organ culture dishes (containing 10 colonies per dish) into a grid motif containing a minimum of 25 small pieces, collect into a 15 ml centrifuge tube and pellet (480g, 3 min, 25 °C) (Fig. 1i–j).
    Figure 1: Photomicrographs of HESCs in organ culture.
    Figure 1 : Photomicrographs of HESCs in organ culture.

    (a) Day 5 HESC colonies grown on MEFs. (b and c) × 50 magnification of a single colony under bright-field and phase-contrast, respectively. The colony has a dense central area. (d) Day 5 HESC colonies grown on MEFs with the central area removed. (e and f) × 50 magnification of a single colony under bright-field and phase-contrast, respectively, with the central area removed. (g and h) × 50 magnification of an HESC colony 2 days after removal of central area (day 7) under bright-field and phase-contrast, respectively. (i and j) × 50 magnification of a day 7 colony under bright-field and phase-contrast, respectively, sliced into a grid motif before dislodgement and transfer to new organ culture dishes.

    Full size image (95 KB)

  2. Transfer the pelleted cells into a T75 containing MEFs seeded at a density of 4 × 104 per cm2 and maintain them in a 37 °C, 5% CO2 incubator. The viability of HESCs following this procedure should approximate that obtained during standard organ culture to organ culture passaging. After this step, which is designated as the first passage in bulk culture, the cells are still tightly bound together (Fig. 2a).
    Figure 2: Morphology of HESCs expanded in bulk culture, following electroporation and during selection in G418.
    Figure 2 : Morphology of HESCs expanded in bulk culture, following electroporation and during selection in G418.

    (a) × 50 magnification of HESC in bulk culture passage 1. (b) × 50 magnification of HESC in bulk culture passage 2 after treatment with TrypLE select. (c) × 50 magnification of HESC in bulk culture grown on feeders at reduced density on the day of application. (d) × 50 magnification of HESC 1 day after electroporation. (e) × 50 magnification of HESC on day 5 post-electroporation, immediately before selection. (f) × 50 magnification of an HESC colony on day 5 of selection. (g) × 50 magnification of HESC grown for 7 days after removal of selection. (h) Individual colonies from (g) were cut and passaged, in duplicate, into wells of a 48-well tray and allowed to grow for 7 days. (i) Colonies from (h) were crudely cut and passaged back into organ culture.

    Full size image (94 KB)

  3. Three days later, enzymatically disaggregate the cells to a suspension of small clumps and single cells using trypsin20 or TrypLE select, collect into a 15 ml centrifuge tube and pellet (480g, 3 min, 25 °C).
    Critical step Trypsin must be subsequently neutralized with the addition of serum-containing media before centrifugation.
  4. Following centrifugation (480g, 3 min), resuspend the cells in HESC media and replate onto a T75 flask containing MEFs seeded at 2 × 104 cells per cm2. A substantial proportion (up to 50%) of cells die during this first enzymatic passage (Fig. 2b). The extent of cell death accompanying enzymatic passaging diminishes with each passage, indicating the adaptation of cells to this method of passaging19.
  5. Enzymatically disaggregate HESCs to single cells at each passage, as described in Steps 3 and 4, biweekly, and split no more than one in two on each occasion. By passages 4 and 5 (11–14 days in bulk culture), the number of cells will have increased to approximately 2 × 107, sufficient for a single electroporation.
  6. Day 1: day before electroporationEnzymatically passage HESCs using trypsin/TrypLE select, as described in Steps 3 and 4, onto flasks containing MEFs seeded at a density of 1 × 104 per cm2 (Fig. 2c). As a rule, on the day of electroporation, HESCs should be semi-confluent, that is, approximately 8 × 106 hES cells per 150 cm2. A single electroporation requires 1 × 107 cells, that is, 2 × T150 flasks in which HESCs are semi-confluent.
  7. Day 2: day of electroporationHarvest cells using trypsin/TrypLE select, as described in Steps 3 and 4. Perform cell count and subtract total MEF number from the count. For example, 2 × T150 flasks will contain approximately 3 × 106 MEFs. The MEFs will account for less than 15% of the total cell count and will not hinder the electroporation. Because MEFs have been mitotically inactivated (irradation), they do not divide to generate colonies.
  8. Resuspend 1 × 107 HESCs (together with MEFs) in ice-cold PBS containing 10–100 μg linearized DNA in a total volume of 800 μl.Troubleshooting
  9. Transfer the cell/DNA mix into 0.4 cm electroporation cuvettes.
  10. Keep cuvette on ice for a 5 min incubation.
  11. Electroporate cells using the parameters 250 V and 500 μF. These parameters were found to be optimal following testing a range of conditions, which addressed variables such as voltage, temperature, capacitance and buffer composition. The time constant should be between 9.0 and 13.0, although this may vary depending on the electoporator21.
  12. Transfer the contents of the cuvette (using a 1 ml glass Pasteur pipette) into a 15 ml centrifuge tube containing 2 ml of prewarmed HESC media.
    Critical step Electroporation results in extensive mortality of HESCs. This wash step removes cellular debris that negatively impacts on the viability of the MEFs and the surviving HESCs.
  13. Gently pellet cells (480g, 3 min, 25 °C).
  14. Gently resuspend the pellet into prewarmed 5–10 ml HESC media.
  15. Plate cells onto 5–10 × 6 cm tissue culture dishes seeded with 2 × 104 MEFs per cm2. The number of 6 cm dishes used for replating after electroporation depends on the proportion of HESCs surviving electroporation, which can be related to the particular HESC line used (Fig. 2d). The HESC density needs to be sparse enough to allow cells to proliferate for 5 days before the commencement of G418 selection (Fig. 2e).
  16. Change HESC media daily.Troubleshooting
  17. Day 7: selection of clonesApply selection 5 days after electroporation. By this stage, each HESC colony should contain 30–60 cells (Fig. 2e). It is recommended that 2 × 6 cm plates containing HESCs that have not been electroporated be placed under antibiotic selection as controls. The recommended final concentrations of antibiotics are as follows: G418 (Gibco, cat. no. 10131-027), use at 50 μg ml−1; puromycin (Sigma, cat. no. P9620), use at 0.75 μg ml−1; or hygromycin (Invitrogen, cat. no. 10687-010), use at 50 μg ml−1.
    Critical step The antibiotic may need to be optimized for HESCs of different origin. A titration is recommended.
  18. Maintain selection for 5–7 days and change media daily (Fig. 2f). MEFs can support HESC growth for up to 1 week. For this reason, supplement dishes with 1 × 104 to 2 × 104 per cm2 MEFs toward the end of the selection period (5 days after electroporation).
    Critical step The amount of MEFs to be added can be determined empirically. The aim is to maintain an MEF density of 2 × 104 per cm2. If the MEFs are sensitive to the chosen antibiotic, the dishes need to be supplemented with MEFs sooner.
  19. Allow the colonies to grow for an additional 7 days (approximately), after which time they should reach organ culture size, that is, 2 mm in diameter (Fig. 2g).
  20. Days 19–21: colony transferUsing a stereomicroscope and a 23G needle, cut colonies into a grid motif (a minimum of 16 pieces) and transfer pieces, in duplicate, into 48-well plates containing MEFs seeded at a density of 2 × 104 per cm2. Transfer pieces unevenly so that 2/3 of the colony is transferred onto one plate and 1/3 of the colony onto the other. The plate with the highest density of HESCs is then used for DNA preparation approximately 1 week later (Fig. 2h). Up to 200 clones can be isolated by one person in an 8-h day.
  21. Days 26–28: screening clones and DNA preparationAspirate media and add 100 μl DNA lysis buffer containing proteinase K. Incubate at 55 °C from 3 h to overnight in a humidified chamber13.
  22. Add 2.5 × volume of 100% ethanol and vortex.
  23. Spin the plates at 1,910g for 15 min. Gently decant the supernatant.
  24. Wash each well with 100 μl of 70% ethanol. Blot dry plates by placing them upside down on blotting paper and then allow plate(s) to air-dry.
  25. Resuspend each clone in 100 μl TE and incubate at 55 °C from 2 h to overnight to resuspend DNA13.
  26. Analyze the DNA. This involves a PCR screen using primers specifically designed to amplify a segment of DNA spanning the junction between the targeting vector and the specific locus. Clones positive at this point are then confirmed via Southern blot analysis13.
  27. In the event that clones containing the desired genetic modification are identified, these should be transferred (from the duplicate tray) onto an organ culture dish containing MEFs seeded at a density of 6 × 104 per cm2. This transfer is accomplished by scraping the colonies using a 200 μl tip until they have fragmented. The colony pieces can then be collected and transferred into an organ culture dish. The new lines should be maintained in organ culture as described above (Fig. 2i).
    Critical step All targeted clones should go through a thorough screening procedure to ensure they have maintained stem cell characteristics and a normal karyotype. These tests should include analysis of stem cell marker gene expression and teratoma formation.
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Troubleshooting

Troubleshooting advice can be found in Table 1.


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Anticipated results

This protocol should yield stably transfected clones at a frequency of 20–500 per 107 input HESCs (Table 2 and 3), depending on the vector used and the HESC line. The protocol was used to generate the Envy hES3 cell line12 except that no G418 selection was applied; rather, GFP+ cells were identified using fluorescence microscopy and manually isolated. In addition, it has been successfully used in our laboratory to generate karyotypically normal targeted cell lines that have retained characteristic stem cell properties (Table 3).





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Acknowledgments

We thank Elizabeth Ng for her valuable contribution to the development of the organ culture protocol described in this report.

Competing interests statement: 

The authors declare no competing financial interests.

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References

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  1. Monash Immunology and Stem Cell Laboratories, STRIP 1, West Ring Road, Monash University Campus, Clayton, Victoria 3800, Australia.
  2. Monash Institute of Medical Research and the Australian Stem Cell Centre, Clayton, Victoria, Australia.
  3. Center for Stem Cell and Regenerative Medicine, Keck School of Medicine, University of Southern California, California 90089–9112, USA.
  4. These authors contributed equally to this work.

Correspondence to: Edouard G Stanley1 e-mail: ed.stanley@med.monash.edu.au