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The production of mouse chimeras is a common step in the establishment of genetically modified animal strains. Chimeras also provide a powerful experimental tool for following cell behavior during both prenatal and postnatal development. This protocol outlines a simple and economical technique for the production of large numbers of mouse chimeras using traditional diploid morula ↔ diploid embryonic stem (ES) cell aggregations. Additional steps are included to describe the procedures necessary to produce specialized tetraploid chimeras using tetraploid morula ↔ diploid ES cell aggregations. This increasingly popular form of chimera produces embryos of nearly complete ES cell derivation that can be used to speed transgenic production or ask developmental questions. Using this protocol, mouse chimeras can be generated and transferred to pseudopregnant surrogate mothers in a 5-d period.
The mouse, Mus musculus, is the preeminent mammalian genetic model system. This is due, in part, to the relative ease with which preimplantation embryos can be manipulated in vitro and later returned to pseudopregnant foster females. One of the more powerful manipulations is the generation of chimeric mice by combining a host embryo with genetically dissimilar, or modified, embryo-derived cells. In many cases, the germ line in these chimeras will contain cells derived from both the host and transplanted cell genomes. Thus, these chimeras can pass either genome to their progeny. Although this represents the traditional manner by which genetically modified ES cells are used to establish transgenic mice1,2, chimeric animals may also be used directly for phenotypic analyses to determine the developmental potential or fate of wild-type and mutant embryonic cells3, as well as the developmental potential of other embryo-derived stem cells4,5. Over the past decade, chimeras have undergone a renaissance in their use and now, in combination with improved techniques for manipulating the mouse genome and for deriving nuclear transfer ES cells, provide a powerful way to study mouse development6–8.
Chimeric embryos can be produced by a number of techniques, including simply culturing embryos on a lawn of ES cells9. The two most common methods of producing chimeric mice from ES cells are by the injection of cells into the blastocoel cavity of a blastocyststage host embryo6,10 or by the aggregation of ES cells with a morula-stage host embryo6,11. Although blastocyst injection allows the investigator to scrutinize each cell that will be injected and select only those cell morphologies that are most likely to colonize the chimeric animal efficiently, it requires access to specialized equipment and is considered by some to be technically more demanding. Aggregation, on the other hand, requires no specialized equipment and is inexpensive and relatively easily learned, but it sacrifices the ability to select ES cells individually. This can result in the reduction of chimera quality, particularly for higher-passage or otherwise problematic ES cells12. These differences aside, the choice of technique is largely a matter of individual preference.
In the case of certain hybrid ES cell lines such as R1, a 129/Sv×129/J F1 hybrid line12, and various other hybrid ES cell lines13,14 (predominantly 129×C57BL/6 Fls hybrids), the contribution of the ES cells to embryonic regions15 can be elevated by the use of tetraploid (4n) host embryos and the generation of tetraploid embryo ↔ diploid ES cell chimeras16 (reviewed in ref. 17). In these chimeras, the tetraploid cells become principally restricted to the extraembryonic membranes with minimal contribution to the embryo proper18,19. This results in almost completely ES cell–derived embryos, which give rise to animals that are viable and fertile. This technique, which was developed for mice, has been adapted and used in other species, such as cattle20.
In diploid embryo ↔ diploid ES cell chimeras, the fetus will be comprised of a mixture of ES-derived cells along with a high proportion of host embryo–derived cells. The use of tetraploid ↔ diploid chimeras can allow for the analysis of experiments in which the host diploid embryo derivatives of diploid ↔ diploid chimeras might otherwise mask phenotypes in the ES-derived cell populations. In addition to producing high-percentage chimeras, this technique can also be used to determine embryonic versus extraembryonic phenotypes experimentally or to analyze embryos derived from ES cells produced by other manipulations including lentiviral or RNAi modifications.
Tetraploidy may be induced by a number of methods (reviewed in ref. 17). The most common being by electrofusing the plasma membranes of a two-cell diploid embryo to produce a temporarily binucleate one-cell embryo. An alternative protocol is to inhibit the first zygotic division with cytochalasin21,22. A limitation of this latter approach is that there is no visual confirmation that the procedure has worked, whereas successful electrofusion may be readily confirmed.
The method described below requires that several individual protocols be coordinated. Figure 1 presents a general timeline for the procedures described in this protocol. The protocol describes procedures that are common to the production of either diploid ↔ diploid or diploid ↔ tetraploid chimeras. Box 1 describes steps that are specific to the production of diploid ↔ tetraploid chimeras. Typically, this protocol will take less than 3 h each day over the course of the experiment.
The techniques described here have existed for many years, and variations may be found in several original publications, review papers and book chapters. The protocols described in most publications, including the one here, are derived in large part from methods12,23,24 that are themselves derivates of an array of experimental embryological protocols documented in earlier works (e.g., ref. 25). The reader is referred in particular to two sources6,11 for alternative descriptions of these and other techniques that pertain to mouse experimental embryology. Additional information regarding extensions of these techniques as well as alternative protocols describing zona pellucida removal or ES cell and embryo culture can be found26,27. A methodical description of the generation, troubleshooting and subsequent analyses of mouse mutations is also available7. Although this protocol is focused on making mouse chimeras, many of these techniques can be adapted to other mammalian species. As a final note, although these techniques are commonly used in a variety of laboratories around the world, special permission to use these protocols may be required by institutional animal care and use committees. All experiments with live mice should be carried out in accordance with relevant guidelines and regulations.
On day 3 (5–24 h before aggregation), place 14 drops of KSOMon a 3.5-cmtissue culture dish. The drops should be 2–3 mm in diameter and should be covered with a layer of mineral oil. Use the pattern indicated (Fig. 2, part 3). Sterilize the aggregation needle with 70% (v/v) ethanol and rinse in sterile dH2O. While viewing the plate through a stereo dissecting microscope, press the needle firmly into the plastic, making six depressions per drop (Fig. 2, part 3). Place aggregation plates in the 37 °C, 5% CO2 incubator 5–24 h before aggregation to equilibrate.
KSOM typically contains penicillin and streptomycin. Simple ethanol sterilization of the aggregation needle in combination with the use of antibiotics is generally sufficient to maintain an aseptic culture.
Larger drops will be more affected by vibration and movement. This will lead to greater fluid movement in the depression wells. Generally, six embryos will be placed per drop, with six drops per plate maintained as ES cell or embryo reservoirs.
The malleability of the plastic directly influences the depth of the depression one can make in a dish. This, in turn, affects the probability that cells and embryos will come into contact in the well. Falcon 35-3001 3.5-cm tissue culture dishes have proven to be sufficiently malleable. Pressing too hard will crack the plastic, but gentle pressure will produce shallow depressions that will not result in efficient juxtaposition of the embryos and ES cells. The goal is to form the deepest depression possible, without cracking the plastic. In practice, one will find that placing the depressions away from the center of the drop facilitates manipulation of embryos and ES cells in later steps.
Although usually made on day 3, aggregation plates can be made on the day of aggregation (day 4). Although at least 5 h of equilibration is typical, the minimum time required for maximum efficiency has not been tested. Plates over 24 h old should not be used.
ES cells should be thawed approximately 5 d before beginning the protocol and passaged once (Fig. 1) ES cells are often grown on mitotically inactive fibroblast feeder cell layers. The two most commonly used feeder cells are primary mouse embryo fibroblasts (MEFs) and STO cells, a thioguanine- and ouabain-resistant subline of SIM mouse fibroblasts28. In many cases, the addition of leukemia inhibitory factor (LIF) to the ES cell medium and the plating of cells on a gelatin substrate alleviate the requirement of the feeder cell layer6. To retain full developmental potential, especially of hybrid cell lines used for generating completely ES cell–derived embryos through the generation of tetraploid chimeras, we would, however, recommend the use of both feeders and medium supplemented with LIF. If you are generating conditional alleles, it is recommended that you test the loxP or FRT sites using transient transfection of the appropriate site-specific recombinase7.
Prepare a solution of the following: 15% (v/v) FCS (e.g., Hyclone, cat. no. A-1115-L; we routinely batch test for optimum performance), 2 mM glutamine (GIBCO, cat. no. 35050-61), 0.1 mM MEM non-essential amino acids (GIBCO, cat. no. 11140-050), 0.1 mM β-mercaptoethanol (Sigma, cat. no. M7522), 1 mM sodium pyruvate (GIBCO, cat. no. 11360-070), 50 U ml–1 penicillin, 50 µg ml–1 streptomycin (GIBCO, cat. no. 15070-063), 1000 U ml–1 LIF/ESGRO (Chemicon, cat. no. ESG-1107). Media should be prepared in a tissue culture hood and should require no further sterilization. Store up to 1 month at 4 °C.
Preparation of ES cell medium may present exposure to ingestion or absorption hazards.
The choice of strain used as a host embryo is important7. Generally, one chooses a strain with a genetic reporter that is different from the ES cell background. This will most typically be a coat color polymorphism or an allele that encodes a ubiquitously expressed fluorescent protein in either the host embryo or donor ES cell7,29,30. Thus, individual cells that arise from either the ES or host embryo compartment of the chimera can be distinguished. In addition, the offspring of the chimera can be rapidly identified as progeny derived from either the host embryo or ES cell genome. It has been shown in chimeras produced by morula ↔ morula aggregation that, in some strain combinations, one strain will be represented in a higher percentage of cells than the other31. Although this has not been rigorously tested, it is commonly believed that ES cell ↔ morula aggregation chimeras will have similar behaviors. In recent years, the use of 129B6 hybrid ES cells has proven particularly efficient for the production of tetraploid ↔ diploid chimeras6,32,33. Pups derived from this technique show essentially wild-type characteristics, although they tend to be slightly larger and have an elevated hematocrit when compared with diploid ↔ diploid chimeras34. For tetraploid hosts, it should be noted that although the embryonic portion of a midgestation tetraploid ↔ diploid chimera will be >99% diploid, a few tetraploid cells do persist throughout the fetus, especially in tissues such as the heart18. If the study requires that the chimeras themselves be analyzed, it is recommended that the embryonic stem cells or host embryo be marked by a ubiquitously expressed, genetically encoded reporter. The most commonly used reporters are fluorescent proteins or chromogenic substrates such as alkaline phosphatase or lacZ (reviewed in ref. 35). This allows the investigator to distinguish between the two populations of cells and therefore to measure the extent of (tetraploid) host embryo contribution to the tissue of interest. F1 CBA/B6 and outbred stocks have commonly been used for induction of tetraploidy by electrofusion (reviewed in ref. 17).
Although the procedure must be coordinated over several days, the timing of individual steps is as follows. The preparation of aggregation plates on day 3 requires 10 min. Preparation of ES cells on days 1–4 requires the following time: 15 min on days 1 and 3 but 5 min on day 2 for Step 1, 5 min for Steps 2–3, 10 min for Steps 4–9 and 10 min for Steps 11–13. Host embryo preparation (on day 3 or 4, depending on whether tetraploid or diploid host embryos are to be used) requires 20 min to 1 h for Steps 14–17 and 30 min to 1.5 h for Steps 18–25. Aggregation on day 4 requires 30 min to 1 h for Steps 26–30. Transferring embryos to recipient pseudopregnant mice on day 5 requires 40 min to 1.5 h for Step 31. If the induction of tetraploidy by electrofusion is carried out on day 3, this will require 10 min for Steps 1–5, 20 min for Steps 6–13 and 1–2 h for Steps 14–16.
In Step 24, if the blastomeres of the embryo separate, simply pool the four (diploid) or two (tetraploid) blastomeres into a single depression well; there is a good chance they will re-adhere.
By careful breath control, one can guide the ES clump to rest against the embryo in Steps 27 and 28. This is done by gently expelling and aspirating fluid against the ES cell clump during its descent. If few embryos are available, it is possible to aggregate the ES cells with only one zona pellucida–free embryo each in Steps 28 and 29, but this will lead to a reduced efficiency.
Although the greatest blastocyst transfer efficiencies are obtained with 2.5-dpc pseudopregnant females (Step 31), a 0.5-dpc pseudopregnant female can be used in an emergency40. In this case, blastocysts should be transferred to the infundibulum of the oviduct rather than to the uterus. Transfer of blastocysts to 3.5-dpc uteri is also possible but results in the lowest efficiency.
Chimeras derived from tetraploid embryos (Box 1) can be delivered vaginally, but they are at risk for complications. Many investigators choose to deliver by caesarian section. This requires that a pregnant foster mother of the same gestational age as the pseudopregnant female be available to raise the pups.
During induction of tetraploidy by electrofusion (Box 1), if there is protein buildup on the electrode (Step 3), it can be removed by digestion with trypsin. If the embryos lyse in Step 11, check to make sure the attenuator (Apl./10) function is on. Because of variability in the apparatus, it may be necessary to optimize the voltages on a given piece of equipment. Do not let the embryos remain in mannitol longer than the minimum required time to equilibrate and transfer them. Use fresh mannitol after every one or two batches of embryos. If only a few embryos fuse (Step 14), try decreasing the number of embryos placed between the two electrodes at any one time. Unfused embryos can be subjected to electrofusion additional times.
The successful production of blastocysts depends to the greatest degree on the quality of the culture medium. Under optimal conditions, almost all aggregation wells should produce late-stage morula or blastocysts after overnight culture (Fig. 4b). For the tetraploidy protocol (Box 1), the efficiency of electrofusion is also variable. Electrofusion should occur in over 50–95% of embryos. Unfused embryos may be subjected to additional pulses as needed.
We thank R.R. Behringer, E.H. Lacy and V.E. Papaioannou for advice, discussions and support.
COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests.
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