The primary purpose of the Core is to assist Cancer Center investigators interested in generating mutant mice using conventional transgenic or embryonic stem cell technology. Core personnel will perform gene-targeting experiments in ES cells and inject DNA or ES cells into fertilized mouse oocytes or blastocysts respectively. Investigators must provide their own targeting vectors for gene-targeting experiments and their own transgenes for transgenic experiments. The Core will perform all cell culture work involved in gene-targeting experiments and will provide investigators with genomic DNA to conduct screening assays for correctly targeted clones. Mice generated by the Core will be returned to investigators at weaning for them to perform their own screening and breeding procedures. Core personnel are available to provide advice on gene-targeting and transgenic experiments.
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Gene Targeting in Embryonic Stem Cells
Embryonic stem (ES) cells will be electroporated with targeting vectors provided by investigators. Transfected cells will be plated and subjected to drug selection. Clones of cells will be picked and replica-plated in 96-well format. One replica plate of cells will be frozen when confluent, while DNA will be prepared from the other plate and given to the investigator for analysis. Each gene-targeting experiment will provide the investigator with DNA from 100-500 clones of cells for analysis, depending on the nature of the construct, drug selection strategy, and the efficiency with which clones are generated. The DNA purification procedure employed by the Core is best suited to analysis by Southern blot but can also be used for PCR screens.
The Core has experience using a variety of different ES cells for gene-targeting experiments. The preferred choice at present is the E14Tg2A.4 feeder cell-independent line from 129/Ola mice prepared by Dr. William Skarnes (The Wellcome Trust Sanger Institute, Cambridge, England). C57BL/6 ES cells are available for use in gene-targeting experiments.
Expansion of Individual Targeted Clones of ES Cells
ES cell clones identified as interesting by the investigator will be thawed, expanded, and refrozen. DNA will also be extracted from the thawed clones, and this will be provided to the investigator for confirmatory analysis prior to microinjection. Up to 12 clones will be expanded for secondary analysis in each gene-targeting experiment.
Secondary Transfection of Gene-Targeted Clones
The Core can retransfect gene-targeted ES cells with Cre or Flp recombinase vectors as required by the investigator. The cells will be replated and subjected to drug selection if necessary. Clones of cells will be picked, expanded, and frozen down. 96-well plates of DNA will be returned to the investigator for analysis.
Microinjection of Embryonic Stem Cells into C57BL/6 Blastocysts
Two or more days of microinjection are required to complete the injection of up to 100 blastocysts with 2-3 clones of ES cells. The injected embryos will be transferred to pseudo-pregnant recipient mice. Implanted dams will be maintained in the facility until their pups can be weaned and delivered to the relevant investigators. 30-50 injected embryos are usually sufficient to generate chimeric mice that are representative of what can be expected for a given ES cell clone. Details concerning the preparation of ES cell suspensions to be used for microinjection are provided below. The Core will microinject ES cells from the gene-targeting experiments it performs, or ES cells provided by investigators.
Microinjection of DNA into Fertilized FVB/N or C57BL/6xDBA/2 F2 Oocytes
Two or more days of microinjection are required to complete the injection of up to 200 embryos with DNA. The injected embryos will be transferred to pseudo-pregnant recipient mice. Implanted dams will be maintained in the facility until their pups can be weaned and delivered to the relevant investigators. Approximately 30% of the transferred embryos give rise to viable mice, and of these typically 20% carry the transgenic DNA in their germlines (i.e., about 10 transgenic founders should be expected). Details concerning the preparation of DNA for microinjection are provided below.
Microinjection of DNA into Fertilized C57BL/6 and Other Oocytes
Essentially as described above, but note that the efficiency of generating transgenic mice using C57BL/6 mice is lower than when using FVB/N or hybrid mice, thus the costs are higher for the C57BL/6 experiments. Transgenic mice can also be generated using embryos from several other strains, though again costs are likely to be higher. Investigators with special needs are encouraged to contact the Core manager to discuss them.
Embryo Transfer for Rederivation of Mouse Strains within the Barrier Facility
Core personnel will perform embryo transfers to facilitate the introduction of mouse strains into the barrier facility. Personnel will recover embryos from timed matings set up by the investigator. The embryos will be transferred at the one-cell or blastocyst stage to pseudo-pregnant recipients maintained within the barrier facility. Pups from the implanted dams will be delivered to the investigators after weaning. Interested investigators should contact the Core manager for details.Consulting Services Advice about the design of transgenic or gene-targeting experiments can be obtained from the Core manager or director.
Additional Services to be Added in the Future
- BAC mutagenesis by recE/recT or reda/redb recombination in E. coli
- Gene targeting in ES cells using modified BACs as vectors
- Screening for homologous recombination in ES cells by real-time PCRPreparations
Preparation of ES Cells for Microinjection
What to submit:
A vial of frozen ES cells with instructions on how to expand them. Alternatively, if the Core has generated the cells, it will expand and inject them according to standard procedures. Investigators may also expand their own cells and provide a healthy single-cell suspension of them to the Core on the day of injection. The cells should be plated the day before microinjection using healthy confluent cultures as the source (e.g., thaw the vial of cells several days in advance, trypsinize and replate them at least once but, of course, try to avoid extended culture). On the day prior to injection, you should plate several dilutions (e.g., 1:2, 1:3, 1:4) and you may also want to plate the cells with and without feeder cells (using gelatin-coated plastic for the latter). A convenient option is to use a 6-well plate with different plating dilutions and conditions in each well.
On the day of injection, change the medium 2 hours before trypsinization. Choose the well that has the nicest looking culture and trypsinize these as follows:
- Wash the well with PBS
- Add tryspin for 5-7 minutes
- Add medium, THEN pipet (using a short-form pasteur pipet or a 5ml pipet) to get single cells (but avoid pipeting so vigorously that you start killing lots of cells)
- Add more medium and pellet the cells by centrifugation
- Remove the supe and add a small amount (e.g., 0.5-1ml) of fresh medium (avoid old alkaline medium)
- Pipet briefly but effectively to resuspend the cells, and place the suspension in a freezing vial or an eppendorf microcentrifuge tube on ice
It would be wise to double-check with the Core manager to confirm details about preparing the cells and when they should be trypsinized on the day of injection. Try to avoid letting the cells sit on ice for any longer than necessary (ideally, they should be brought to the Core facility just before they are ready to be microinjected). Avoid anything that might reduce the viability of the cells, e.g., pipetting too much, or having the cells in really pink medium, or failing to feed the cultures 2 hours before trypsinization. Avoid an overabundance of feeder cells in the cell preparation, i.e., have the cultures at a good healthy density. If possible, it makes sense to have two ES cell lines ready to go on the day of injection; that way if one of the cultures does not look good, you will have a fallback. Also, there are times when we recover larger numbers of embryos than normal; if this happens, and if the injections are going well, you may be able to get two or more clones injected on the same day.
Note: it is the responsibility of the investigator to ensure that the cell suspension will be ready on time. The success of the microinjection experiment depends critically on the health of the ES cell suspension (assuming that the ES cells are otherwise proficient at colonizing the germline).
Preparation of DNA for Microinjection
What to submit:
Digest 100 micrograms of the transgene plasmid with restriction enzymes that will separate the vector from the transgene. Use Qiagen or CsCl-purified DNA for this digestion; sequencing-grade purity of DNA should be sufficient (i.e., unusual purification steps are not required). Digest the DNA in a volume of 100-200ml. DO NOT purify the transgenic fragment; the Core will perform this purification. Provide the Core with the digested DNA along with an image of an Ethidium bromide-stained agarose gel showing which bands should be purified for microinjection. BAC DNA should be provided to the Core in supercoiled or linearized form at high concentration. Core personnel will dilute the BAC DNA with an appropriate buffer before microinjection.
Note carefully: By far the most robust way to screen initially for transgenic founders is to perform a Southern blot using DNA prepared from tail biopsies of mice. Southern blots are preferable to PCR-based assays at this stage because they unequivocally identify founders and are much less prone to false-negative or false-positive results than other assays. When designed appropriately, the Southern blot can readily provide information about transgene copy number. It can also help to make clear when a founder is a mosaic (with only some of its cells carrying the transgene) or when a founder carries independent chromosomal integrations of the transgene. (In such instances, more than one transgenic line can be produced by mating the founder, and these lines may differ in expression characteristics.) Screens for founders based on transgene expression can accompany Southern blot assays, but ideally should not replace them. Accurate information concerning the success of a microinjection experiment can best be obtained from a Southern blot.
Following the identification of founders (and ideally also their F1 progeny), PCR assays can be established for routine screening for transgenic mice. Ideally, such assays should include internal control reactions. Quantitative real-time PCR assays can also be developed to distinguish heterozygous from homozygous transgenic mice. The Cancer CenterŐs Genome Analysis Core has experience with the use of such assays for this purpose and can provide assistance in their design, development, and use. The Transgenic/Targeted Mutagenesis Core will take every opportunity to keep investigators informed concerning progress on their microinjection experiments. In turn, the facility depends heavily on the investigators for accurate feedback concerning transgenic founder frequencies and germline transmission from ES cell-derived chimeric mice.
Preparation of DNA for Gene Targeting
What to submit:
Linearize 100-200 micrograms of the targeting vector using whatever enzymes are required for the targeting strategy. Precipitate the DNA and wash the pellet with 70% ethanol. Work under sterile conditions from the 70% ethanol wash stage onwards. Air-dry the pellet and resuspend it in sterile TE or sterile PBS at approximately 1 microgram per microliter. Run 0.2 micrograms of the DNA on an analytical agarose gel with appropriate standards to confirm that it has the required form and is at the right concentration. Provide this DNA, a picture of the gel, and ideally a rudimentary map of the construct noting drug resistance elements, to Core personnel. Ensure that you have made clear the nature of the drug selection strategy and the type of embryonic stem cells to be used.
Note that many gene-targeting experiments stall at the screening stage because insufficient prior effort has been invested in ensuring that the screening strategy is robust and effective. If using a Southern blot strategy, it is essential to test the probes being used in advance to make certain that they hybridize to bands of the expected size in the absence of excessive background hybridization. A Southern blot strategy usually requires that the probe and the targeting vector should not share sequence in common. The wild-type band detected by the probe should be clearly separated from the mutant band, and the choice of enzyme used for digestion of the genomic DNA should be based on cost and the efficiency of cutting DNA that is not highly purified. Differences of 10 vs.11Kb or 20 vs. 18Kb may be difficult to detect when working with small amounts of DNA from hundreds of clones. A PCR strategy should be tested using appropriate control plasmids and ideally should involve small amplified regions and highly optimized reactions.
Additional Resources
A searchable database of core facilities at all UCSF campus locations, provided by the Clinical and Translational Science Institute at UCSF, is available here.
