Mouse Embryonic Stem Cell Core
The main focus of the Mouse Embryonic Stem Cell core is to facilitate the creation or generation of genetically modified mice for BCM-IDDRC investigators. The core has several missions, including:
- Providing expert advice on targeting vector design
- Generating targeted ES cells
- Maintaining quality controlled ES cell lines
- Teaching methods relating to embryonic stem cell manipulation
- Creating genetically engineered mice (GEM) for investigators
The procedures for the manipulation of ES cells and the construction of GEM carry a significant cost in both resources and time. The Mouse Embryonic Stem Cell core provides a centralized service for GEM mice production for the BCM-IDDRC community and makes it possible for those investigators not familiar or not equipped with ES cell manipulation to obtain GEM mice. It offers a wide range of services for investigators such as electroporation of targeting vectors into ES cells, mutant ES cell injection into blastocysts, clonal expansion and injection of Gene Trap ES cell lines obtained from international Mouse Knockout Consortiums, quality control testing of targeted ES cell lines, in addition to the care and housing of the mice until they are transferred to the participating investigator.
The ES Core also generates a wide range of “mutations” or alleles for investigators ranging from "simple" knockouts to more sophisticated genetic alterations obtained through successive manipulations of ES cells. For example, this might include the generation of chromosomal deficiencies, the generation of conditional alleles such as those flanking an exon of a gene with loxP sites. A summary of the types of alleles that will be generated by the Core is presented in Figure 1.
The Core will be set up to assist in a wide-range of projects relating to basic mental retardation research modeling as well as other developmental disabilities projects. The availability of mouse models as disease models is an essential step to understand the mechanism of pathogenesis and molecular causes of disease progression in live mice.
Investigators will be able to receive the assistance of the Core at several phases in their experiments:
Targeting vector design assistance, project consultation, supply of vectors and genomic libraries:
Initially the Core will provide advice on construct design to assist investigators with selecting the best approach to mutate genes by homologous recombination in ES cells. Since homologous recombination efficiencies are directly related to the identity between the input DNA and the genome, the core will provide phage genomic libraries or ES cell DNA for PCR that is matched to the ES cell lines used in gene targeting experiments. In gene targeting experiments different selectable markers are used for a variety of different purposes. Currently we routinely use neo, puro , and Hprt for positive selection and HSVtk and Hprt for negative selection as well as ready-optimized targeting vectors suitable for particular applications.
Electroporation and Gene Targeting ES cells:
The Core will provide the technical assistance necessary for embryonic stem cell targeting. The laboratory pursuing individual targeting experiments is expected to provide pure linearized DNA for electroporation into ES cells. The ES Core will provide the following services:
- The preparation of the 'non-standard' media required by ES cells
- The preparation of feeder cells in a ready to use state of the required genotype for selection, this will depend on the selection marker in the targeting vector.
- Expansion of an appropriate ES cell line for a specific gene targeting experiment.
- Electroporation of the ES cells and positive/negative selection of the transfected clones.
- Picking and arraying the ES cell clones in a 96 well format.
- Replica plating the 96 well master plate to provide duplicates for freezing and DNA extraction.
- The retrieval of targeted clones for more detailed analysis and blastocyst microinjection.
The 96 well plates with lysed ES cells for DNA isolation are passed back to the laboratory which supplied the targeting vector. Clones are screened by the individual laboratory for gene targeting events. Once targeted clones are identified, they are retrieved and expanded and frozen back pending more detailed analysis to confirm the fidelity of the recombination event. The Core will provide a maximum of 300 arrayed clones per targeting vector and will advise on alternative strategies if the desired targeting events are not obtained at a reasonable frequency. It is anticipated that this Core will be able to handle 20-25 targeting experiments per year (at a cost of about $3900 per experiment and includes a single ES cell electroporation and expansion of five clones).
Figure 1. Types of mutant alleles which can be generated by the Core.
A. A hypothetical three exon gene with a non-coding first exon.
B. A simple knockout where the Hprt selection cassette deletes the second exon thereby mutating the gene.
C. A Cre knockin into the hypothetical gene. Cre is brought under the transcriptional control of the target locus by virtue of a splice acceptor site (SA). This allele also contains Hprt for positive selection, but this could be removed if necessary. D1 & D2 represent the two stages in the generation of a floxed allele.
D1. In the first step the locus is targeted using Hprt for positive selection. Hprt is placed in the intron so that after removal by Cre-excision the remaining loxP site in the intron is unlikely to interfere with the function of the locus.
D2. The final locus is similar to the wild type locus except one of the exons (exon 2) is flanked by loxP sites. Tissue specific expression of Cre will delete the DNA flanking these sites resulting in tissue specific mutation of the gene of interest. E1, E2, & E3 depict the 3 stages in the generation of a deletion allele.
E1. A region of the genome with 4 genes, V, W, X & Z or a cluster of miRNAs.
E2. Targeting of the deletion cassettes containing the portions of the split Hprt minigene cassette to the deletion end points.
E3. The final product after Cre expression and HAT selection. In this case the genes W and X have been deleted. Clones with the desired recombinant allele will either be microinjected (in the case of a simple gene targeting event) or they will be used for subsequent recombination events such as targeting the second allele, removing the selectable marker to generate a floxed allele, or they will be targeted at a second locus to make deletions.
Germline transmission of targeted alleles:
Once targeted ES clones have been identified they will be microinjected into C57 blastocysts. Injected embryos will be briefly cultured and transferred back to foster mothers. The Core will inject sufficient C57 embryos to reasonably test 2-3 clones per targeting experiment. Once the chimaeras are born, the degree of coat color chimaerism and the sex ratio will be assessed to determine whether it may be necessary to repeat the injection of any particular clone. Ideally the core aims to pass on to the investigator 4 to 5 high percentage (greater than 50% coat color contribution from the ES cells) male chimaeras. It is anticipated that 2 to 3 clones per targeting experiment will be injected. The cost for the microinjection component totals $1,900 per allele.
Monica Justice, Ph.D.
Isabel Lorenzo, B.S.