| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
EXPERIMENTAL PROTOCOLS FOR EMBRYONIC STEM CELL RESEARCH |
University of Pennsylvania, Department of Cell and Developmental Biology, Philadelphia, Pennsylvania, USA
Key Words. Embryonic stem cells • Mouse • Electroporation • Homologous recombination
Patricia A. Labosky, Ph.D., University of Pennsylvania, Dept. of Cell and Developmental Biology, 1109 BRBII/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6058, USA. Telephone: 215-573-7547; Fax: 215-898-9871; e-mail: plabosky{at}mail.med.upenn.edu
ABSTRACT
The manipulation of embryonic stem (ES) cells to generate targeted mutations via homologous recombination has proved an invaluable resource for researchers from fields as diverse as embryology, immunology, physiology, and biophysics. The derivation and culture of these cells has been described in many places, but there is no substitute for hands-on experience. In lieu of that, here we provide a step-by-step guide for the technique of electroporation, complete with micrographs of the cells at each step of the process. This is intended to allow the first time ES cell worker, as well as the more experienced researcher, to view the morphology of ES cells under ideal conditions as well as when the cells are suboptimal and should be discarded.
Murine embryonic stem (ES) cells are derived from the inner cell mass of the 3.5-days post-coitum mouse embryo. These cell lines were first described by Evans and Martin, independently, in 1981 [1, 2]. In 1984, studies on chimeras made with ES cells demonstrated that ES cells could contribute to all tissues of an adult mouse and, most importantly for the field, they have the potential to contribute to the germline and therefore to offspring of chimeras [3]. Several years later, Capecchi’s laboratory showed that an individual gene could be repaired using homologous recombination in ES cells [4], and reports of the first genetically engineered mutant mouse with a mutation in the Int-1 proto-oncogene (now called Wnt1) were published in 1990 [5, 6]. The generation of mice carrying targeted mutations has grown exponentially since, and this technology has been used to produce mice harboring mutations in thousands of different genes.
Laboratories across the world are working with ES cells, and there is a constant demand for details about the cells and their culture conditions. Culture and derivation of the cells, electroporation, DNA extraction, and the design and construction of targeting vectors have been published in many other places [7–10]. Here we provide a detailed technical report of ES cell electroporation complete with micrographs documenting the appearance of the cells through all steps of the procedure. This review is intended only to supplement existing work, not to replace it. Although this method is certainly not the only way to electroporate ES cells, it is consistently successful in our hands. Whatever conditions are used, it is crucial that the cells are cultured optimally. There is no substitute for looking through the microscope at the cells every day. The authors urge that this constant monitoring is the key to consistent germline-transmission of stem cells as described in this review.
CULTURING ES CELLS
ES cell cultures to be used in the genetic engineering of mice should be subconfluent (~70%), well established, and contain colonies that maintain well-defined edges and have not yet begun to differentiate. If you are unfamiliar with the ES cell line with which you are starting, it is advisable to test the parental ES cell line for germline transmission before going through the effort of generating a targeted ES cell line. To consistently maintain ES cells in an undifferentiated state, we use "complete ES medium" consisting of Dulbecco’s modified Eagle’s medium (DMEM; 414 ml) with 4,500 mg/l D-glucose and L-glutamine (Cat. #11965-084; GIBCO; http://www.lifetech.com), 15% fetal calf serum (75 ml) tested for ES cells (HyClone; Grand Island, NY; http://www.hyclone.com; Sigma; St. Louis, MO; http://www.sigmaaldrich.com; and Summit Biotechnology, all carry good serum), 2 mM glutamine (5 ml, GIBCO; Cat. #25030-081), 0.1 mM MEM nonessential amino acid solution (5 ml, GIBCO; Cat. #11140-050), 0.1 mM ß-mercaptoethanol (4 µl, Sigma; Cat. #M7522), 50 µg/ml gentamicin (0.5 ml, GIBCO; Cat. #15750-060), and 1,000 U/ml leukemia inhibitory factor (LIF) (also called ESGRO; Chemicon International; Temecula, CA; http://www.chemicon.com; Cat. #ESG1107). When diluted to 1,000 units/ml, one vial of LIF will be enough for 10 liters of ES cell medium. Prior to use, LIF should be diluted in ES medium and aliquoted to a convenient concentration, then stored at 4°C. ES cell medium lasts for about 2 weeks stored at 4°C. This and all other medium used throughout should be prewarmed to 37°C before use. There are variations on this medium that contain sodium pyruvate, or use penicillin instead of gentamicin that also are very reliable [10].
In Figure 1A
, a 1-day old culture of TL1 ES cells is shown. All cultures shown here and in subsequent figures were imaged on a Nikon TMS microscope (http://www.nikon.com), and images were captured with a Micropublisher digital camera (http://www.qimaging.com). Note the small round clumps of cells evenly spaced on the dish. One day later (Fig. 1B), the colonies of cells are larger but still maintain defined edges. On the third day (Fig. 1C) the culture is overgrown and should not be used. Although some patches of undifferentiated cells are seen (arrow), most of the cells are beginning to differentiate as noted by their flattened appearance and loss of defined boundaries (arrowheads) and would not be optimal for further manipulations. Eight to ten 6-cm plates of ES cells at optimal density are required per electroporation, approximately 2–5 x 107 cells. Before any manipulations (electroporation, blastocyst injection, etc.), ES cells should be subcultured at least twice after thawing. If the cells are not used after 2 weeks of culturing, they should be discarded. Our laboratory uses the ES cell line TL1 originally derived in 1993 [11], but other cell lines (D3, R1, RW4, E14) have been used with similar results. A catalog of ES cell lines with sources can be found in [10]. It is sometimes important to know the genetic background of the blastocyst that gave rise to the ES cell line you are using, and much of this information can be found in [12, 13] and from The Jackson Laboratories (http://jaxmice.jax.org/library/notes/483.pdf). Most ES cells are 129 and there are several strains of 129 mice available for purchase from The Jackson Laboratories (Bar Harbor, ME) and Taconic Farms (Germantown, NY; http://www.taconic.com) in the U.S.. It is convenient if mice of the same genetic background can be purchased so that any mice produced from ES lines can be bred to isogenic strains if desired. Strain differences have proved informative in developmental studies and in cancer models [14–16].
|
illustrates feeder cells at optimal density for supporting ES cell cultures. Alternatively, STO fibroblasts are often used, and some researchers use a combination of STOs and primary cells. STO cells are available from American Type Culture Collection (ATCC; http://www.atcc.org; Cat. #CRL-1503). Feeder cells are plated at the following densities: 10-cm dish, 5 x 106 MEFs, 3 x 106 STOs; 5-cm dish, 1.5 x 106 MEFs, 1 x 106 STOs; 1-cm well of a 24-well dish, 2.5 x 105 MEFs, 1.5 x 105 STOs; 1 well of a 96-well dish, 1 x 105 MEFs, 6 x 104 STOs. The major benefit of using STO cells is that it is not necessary to generate new feeder lines from mouse embryos, something that is laborious and limited by the availability of a mouse colony. However, the drawback of using STO cells is that the cultures can be allowed to overgrow and the properties of the feeders might change unbeknownst to the user. ELECTROPORATION
For each electroporation, approximately 100 µg of double-stranded DNA are used, prepared as described in [17]. DNA must be clean and free from organic compounds and salt. We have used CsCl purified DNA and Qiagen Maxi-prep purified DNA side by side with no difference in colony number or targeting frequency.
Eight to ten 6-cm plates of ES cells at optimal density (Fig. 1B), approximately 2–5 x 107 cells, are washed once with phosphate-buffered saline (PBS; GIBCO, Cat. #14190-136), and a single cell suspension is generated as follows: Add 1 ml per plate of 0.25% trypsin/EDTA (GIBCO, Cat. #25200-072) and incubate at 37°C and 5% CO2 for 5 minutes or until the color of the trypsin/EDTA begins to yellow. Eight to ten plates can be trypsinized in two or three batches depending on experience and should be agitated once or twice during incubation. Add 3 ml ES medium to each dish to stop the trypsin reaction. Using a 5-ml pipette, pipette up and down vigorously to obtain a single cell suspension. Pool and count cells, and spin them down in a clinical centrifuge such as an IEC Centra® CL2 (Thermo Electron Corp.; Waltham, MA; http://www.thermo.com) for 3 minutes at 1,000 rpm. Wash the pellet twice in 10 ml PBS to remove all serum to avoid altering the electroporation conditions.
Resuspend the DNA pellet in approximately 0.6 ml PBS and transfer to the tube containing the pellet of ES cells. Resuspend the cell pellet in the DNA solution adding more PBS to a total volume of 0.8 ml. Transfer this solution to an electroporation cuvette (Bio-Rad; Munich, Germany; http://www.biorad.com; Cat. #165-2088) and electroporate. Many different electroporation conditions have been published, including 300 V, 500 µF, 1 pulse with the cells placed on ice before and after; 230 V, 25 µF, two pulses with cells on ice before and after; and 250 V, 500 µF, 6–7 seconds, with cells at room temperature [10]. In our hands, one 800 V pulse, 3 µF at room temperature using a Gene Pulser II (Bio-Rad) with a Capacitance Extender II, time constant approximately 0.4 seconds, has consistently given more colonies than some harsher conditions. The frequency of gene targeting, however, has not been compared directly between the different electroporation conditions although in many instances these differences might not matter. If the researcher is consistently getting very few colonies, they might wish to change the electroporation conditions.
After electroporation, incubate the cells at room temperature for 5 minutes, then transfer into 125 ml of ES medium. Rinse the cuvette twice with medium to recover all the cells. Place the electroporated cells onto five 15-cm dishes preseeded (6 x 106 cells/plate) with mitotically inactivated primary embryonic feeder cells (25 ml medium/plate). Grow the electroporated cells in ES medium without selection agent for 24 hours. It is crucial that the feeder cells used are resistant to the antibiotic of choice, usually neomycin, as the next step will be antibiotic selection of the transfected cells. Mice carrying a neomycin-resistant gene can be purchased from the Jackson Laboratories (strain: C57BL/6J-TgN(pPGKneobpA)3Ems, stock # 002356), and STO cells carry a neomycin-resistant gene [10].
SELECTION
After allowing the electroporated cells to grow for 24 hours in ES medium, begin selection. ES selection medium contains antibiotic in doses lethal to all ES cells except those that are expressing the gene providing resistance to your selection agent. For TL1 cells we use 300 µg/ml G-418 (GIBCO, Cat. #11811-023) or 1.5 ml of a 100-mg/ml stock to 500 ml ES medium. To make a 100-mg/ml stock, solubilize 1 g G418 in 9.8 ml PBS or DMEM, add 180 µl 10N NaOH, filter sterilize, and store at 4°C. Selection of the cells usually takes 7–10 days and is portrayed in Figure 2A–2D. At days 1 and 2, the cultures look like fairly dense ES cell cultures (Fig. 2A and 2B); very little cell death is observed until day 3–5 (Fig. 2C and 2D). After 5–6 days of selection, small round colonies will become visible in the dish (Fig. 3A and 3B). Different cell lines may vary in response to G418 concentration, and the time course of selection will vary with different targeting vectors.
|
|
Several 96-well feeder plates should be prepared 1 day before the selection process is complete. The number of required feeder plates can be estimated by circling colonies on the bottom of the 15-cm dishes (pen marks in Fig. 3 and Fig. 4). It is easiest to do this when feeding the ES cell cultures near the end of the selection period (day 5 or 6) by removing the culture medium and tilting the dish at a 30–45 degree angle so that the three-dimensional colonies are clearly visible. Use a fine point Sharpie pen to mark the bottom of the dish.
|
Only ideal colonies should be isolated for screening and future manipulation. Colonies should be slightly smaller than the end of a 20-µl pipette tip. Edges should be clearly defined and there should be no cells flattening and differentiating along the outside edges. Figure 4 provides a comparison of good and bad colonies. If nothing is known about the targeting frequency of the locus, we usually select a maximum of 500 colonies and try for a minimum of 200 colonies. If over 1,000 colonies are screened in multiple electroporations and no correctly targeted clones are identified, it is advisable to re-engineer the targeting vector.
Once identified, ideal colonies are isolated with a micropipette, dissociated in 96-well flat-bottom dishes, and moved onto the previously prepared 96-well feeder dishes as follows. The 15-cm dish is washed once with Ca2+- and Mg2+-free PBS (GIBCO, Cat. #14190-136), and 25 ml PBS is added to the dish. Under a microscope, nudge the colony loose from the bottom of the dish with the tip of a pipette set to 10 µl. Aspirate the colony into the tip in one motion, steadying the pipette with your opposite hand as shown in Figure 4A. Place the colony into one well of a clean (no feeders) 96-well dish, matching the 8 x 12 grid of the pipette tip box to the 8 x 12 grid of the 96-well dish to keep the dish orientated. Filling a 96-well dish should take approximately 30 minutes to 1 hour. We pick colonies in the open air (Fig. 4A), but some investigators place the microscope in a laminar flow hood. Others pick colonies without the use of a microscope, but this approach requires identification of optimal colonies in the microscope before picking. It is impossible to distinguish subtle differences in morphology of colonies without a microscope. For example, the suboptimal colonies in Figure 4F and 4G cannot be distinguished from optimal colonies in Figure 4B–4E with the naked eye.
Once all the wells of a 96-well dish contain colonies, trypsinize the colonies with 50 µl trypsin/EDTA for 5 minutes at 37°C and 5% CO2. During incubation, remove the fibroblast culture medium from one of the 96-well dishes containing feeder cells. Stop the trypsinization by adding 100 µl ES cell medium (without selection) to each well of the dish containing trypsin/EDTA. Pipette up and down to mechanically dissociate colonies, monitoring the process under the microscope at first to ascertain that single cell suspensions are obtained. Transfer all 150 µl of single cell suspension into the 96-well dish containing fresh feeders and feed daily with ES cell medium. A 12- or 8-multichannel pipettor and an 8-channel aspirator are recommended for all manipulations involving 96-well dishes.
EXPANSION AND CRYOPRESERVATION OF COLONIES
Although all the colonies picked survived selection, homologous recombination of the construct is rare and must be ascertained via Southern analysis of extracted DNA. Careful expansion and cryopreservation of cells during this period is crucial, and ES cells growing in 96-well dishes should be treated as carefully as any culture of ES cells. On the first day after plating, the ES cells may not be visible in the dish (Fig. 5A), but over time colonies will appear (Fig. 5B). The curved appearance of the 96-well flat bottom dishes make ES cells difficult to image, but because ES cell colonies are three dimensional, they are easily distinguished from feeder cells in the dish.
|
), the culture should be split in half. Half of the culture stays in the current dish and will be lysed for DNA extraction and Southern analysis. Polymerase chain reaction can be used as an initial screen, but positive clones should be confirmed on a Southern blot before blastocyst injection. The other half of the culture is plated into a new dish of feeders and will be cryopreserved. To subculture the ES cell cultures, wash with 100 µl PBS, add 50 µl trypsin/EDTA, and incubate 5 minutes at 37°C in a 5% CO2 incubator. Add 50 µl ES medium and pipette to obtain a single cell suspension. Place 50 µl into new wells of a 96-well dish preplated with feeders, and the remaining 50 µl stays in the old dish. It is absolutely critical to orient the old and new dishes accurately so they are exact duplicates. Add 100 µl medium to all the wells; each well should have a total volume of 150 µl. The plate of cells for cryopreservation should be grown 2–3 days and then frozen down. Aspirate off medium and rinse each well with 100 µl PBS. Trypsinize by adding 50 µl trypsin/EDTA and incubating 5 minutes at 37°C 5% CO2. Add 50 µl 2x freezing medium and resuspend the cells gently to a single cell suspension. 2x freezing medium is 20% dimethylsulfoxide (Sigma, Cat. #D5879), 60% fetal calf serum, and 20% ES medium, filter sterilize after mixing. Layer 100 µl mineral oil on the top of each well to prevent evaporation. Wrap the edge of each plate with parafilm, place the plates into a well-insulated styrofoam box, and store for no more than 3 months at –80°C.
Leave the remaining 96-well plates designated "for DNA" to grow to high density (Fig. 5D) before lysing for DNA extraction. It is advisable to subculture these cells after 2 or 3 days to make a duplicate "back-up" plate in case something unforeseen should happen in the screening process or more than one Southern blot is desired to identify clones.
Lysing Cells for DNA Extraction
Culture the cells for DNA until they are very dense to the point of the medium turning bright yellow (Fig. 5D). It is useful to mark wells that do not have colonies growing in them, so if an error is made it will be easy to catch. For example, if well F6 on plate 3 did not have any cells growing in it yet, DNA extracted from the well results in a strong signal on a Southern blot, which could be problematic. To prepare DNA, rinse wells twice with 100 µl PBS. At this point the plates do not need to be sterile and washes of PBS can be changed by discarding the solution directly into the sink. Place 96-well dishes for DNA extraction at -80°C for at least 1 hour. Wrap back-up plates in parafilm and store indefinitely at -80°C.
Remove plates for DNA extraction from the freezer and allow them to reach room temperature. Lyse the cells with 50 µl lysis buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5% SDS with 1.0 mg/ml Proteinase K) per well. Parafilm the plates closed, place in a sealed container lined with wet paper towels to prevent drying, and incubate at 60°C overnight.
The next morning, spin down any moisture from lid (1,000 rpm for 5 minutes in a centrifuge such as the Joan CR4-12 with an adaptor for 96-well plates) before extracting DNA from the lysed cells by adding 50 µl isopropanol to each well; tap to mix. After 1 hour of incubation at room temperature you should see a stringy white DNA precipitate. The DNA will precipitate faster if you use a nutator to agitate the plate gently during the incubation. Spin the plate to attach the DNA firmly to the bottom (1,500 rpm for 5 minutes) and gently invert onto a stack of paper towels to drain liquid; blot gently. Wash the wells with cold 70% ethanol (using a squirt bottle), and spin the plate again as above to make sure DNA is attached. Invert the plate and drain liquid onto paper towels, blotting gently. Repeat ethanol wash two additional times and air dry DNA for 30 minutes or longer. If wells are not completely dry, samples will float up out of the agarose gels when you load them. However, do not dry them overnight since this will inhibit complete resuspension of the DNA.
When DNA is dry, add the appropriate restriction enzyme mixture for your Southern assay directly to well, mix well, and digest overnight at 37°C. Use an excess of enzyme (5 µl of enzyme in a 50-µl reaction/well). Perform the Southern analysis to identify the clones that have undergone homologous recombination of the targeting vector.
THAWING CELLS
Once correctly targeted clones have been identified from Southern analysis, individual colonies are thawed and plated onto a 24-well dish for expansion. To thaw cells, place entire 96-well dish into 37°C 5% CO2 incubator. Allow 10–15 minutes for an entire plate to thaw; less is needed if desired clones are in outer rows. Gently pipette lower (aqueous) phase of well into one well of a 24-well dish, avoiding mineral oil. Do this early in the morning, and then feed the wells late in the day to remove the DMSO from the freezing medium.
Colonies will become apparent in 3–4 days and will be ready for subculturing in 5–7 days. Expand colonies to freeze down several (3–5) vials of cells. In addition, prepare more genomic DNA to repeat the Southern analysis with probes from each end of the targeting vector before injecting the cells to make chimeras. ES cell lines can also be karyotyped. Only cells with 40 chromosomes should be used to generate mice.
PREPLATING CELLS FOR MICROINJECTION
When preparing ES cells for blastocyst injection or morula aggregation, it is helpful to remove excess feeder cells by taking advantage of the different adhesive properties of ES cells and fibroblasts. Using one subconfluent 6-cm dish (~80% confluent), feed the culture 1–2 hours before harvesting (2.5–3.5 hours before microinjecting). One hour before microinjection, rinse with PBS and trypsinize ES cells for 5 minutes as usual in 1 ml trypsin/EDTA. Add 4 ml ES medium to inhibit the enzymatic reaction, and pipette up and down vigorously until a single cell suspension is obtained (Fig. 6A). Place the 5 ml into a fresh 6-cm gelatinized (no feeders) dish. It is not necessary to use all of these cells: 1/10 or 1/4 can be plated onto fresh feeders to continue culturing the line. Place the dish into the 37°C 5% CO2 incubator for 1–1.5 hours. Feeder cells will adhere tightly in this time, while the best ES cells will adhere weakly. Differentiated cells and dead feeders will remain afloat (Fig. 6B). Immediately prior to microinjection, aspirate medium and all the floating cells. Add 5 ml ES medium and blow the loosely adhering ES cells off the dish by vigorously pipetting with a 5-ml pipette at an angle (do not touch the bottom of the plate with pipette tip). Spin down this 5 ml (1,000 rpm for 3 minutes) and resuspend the pellet in 2 ml of medium. These are the cells for microinjection (Fig. 6C). Cells are stored at 4°C until microinjection is complete.
|
ACKNOWLEDGMENT
We thank Dr. Joanne Thorvaldsen for her careful reading of this manuscript and LiLi Tu, Shing Jen Tai, and Kristin Sinclair for their ongoing and observant work in the cell culture room. Funding in the laboratory is provided by an American Heart Association Established Investigator Award and the NIH, Grant No. HD36720.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  L. Teng, N. A. Mundell, A. Y. Frist, Q. Wang, and P. A. Labosky Requirement for Foxd3 in the maintenance of neural crest progenitors Development, May 1, 2008; 135(9): 1615 - 1624. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Ai, T. Kitazawa, A.-T. Do, M. Kusche-Gullberg, P. A. Labosky, and C. P. Emerson Jr SULF1 and SULF2 regulate heparan sulfate-mediated GDNF signaling for esophageal innervation Development, September 15, 2007; 134(18): 3327 - 3338. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Suter, L. Cartier, E. Bettiol, D. Tirefort, M. E. Jaconi, M. Dubois-Dauphin, and K.-H. Krause Rapid Generation of Stable Transgenic Embryonic Stem Cell Lines Using Modular Lentivectors Stem Cells, March 1, 2006; 24(3): 615 - 623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. I. Civin, A. M. Gewirtz, R. G. Hawley, and M. A. Goodell Advancing the Fast-Paced Field Of Stem Cell Research: STEM CELLS Increases from 6 to 10 Issues in Its 23rd Year of Publication Stem Cells, January 1, 2005; 23(1): 1 - 2. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
