Gene Targeting Outline

1. Introduction. This is a brief outline of the steps necessary to produce mice with a mutation targeted to a specific gene. These animals are referred to as "knock-out" mice or "gene targeted" mice. The broad outline of the experiment includes:

1) the investigator constructs a gene targeting vector containing a mutation in the target gene;

2) the targeting vector is introduced into ES cells;

3) ES cell clones which undergo homologous recombination with the targeting vector are identified;

4) ES cell-mouse chimeras are produced;

5) the chimeras breed and transmit the chromosome with the targeted gene to their offspring;

6) homozygous animals are produced from the mating of hemizygous chimera offspring; 

7) the phenotype resulting from the genetic mutation is characterized. Core personnel are available for consultation on all aspects of gene targeting research. Contact Thom Saunders with any questions or Meet the Staff. The Transgenic Core prioritizes all requests for service on a "first-come, first-serve" basis. Your requests for electroporation, clone expansion, and blastocyst microinjection will be added to the Transgenic Core work queue in the order that they are received.

2. Plan the experiment. What is the purpose of your experiment? Do you want to completely ablate gene expression in all tissues? Do you want to analyze tissue specific regulatory sequences? Do you want to produce a model for tissue-specific gene deletions? You will need to obtain or clone the structural gene that you want to mutate. The source of the DNA is important since published studies demonstrate genomic DNA isogenic to the embryonic stem cell line give higher gene targeting efficiencies in ES cells (see Key Gene Targeting Papers). While complete sequence information is not necessary, a detailed restriction map of the gene is an irreplaceable tool. During the planning stages the material transfer agreements for R1 ES cells and other reagents should be initiated so that they are ready in time. Once a genomic clone of the gene is in hand and restriction mapped, then it is time to...

3. Clone and the Verify the Targeting Vector. The Core has cloning vectors for investigators to use in gene targeting research. They include pPNT, a basic targeting vector, pNZTK2, designed to insert the lacZ gene behind the promoter of the targeted gene, and pflox, which is designed to introduce loxP sites into the gene. A complete materials transfer agreement is required before they can be distributed. You will need to determine which 5' and 3' fragments of the gene will be used to construct the targeting vector. Generally speaking, a couple of Kb on either end should give a reasonable gene targeting frequency. You will also need to consider the methods and probes that you will use to identify ES cell clones that have undergone homologous recombination with the targeting vector. This means a Southern blot approach with probes outside of the targeting vector or a PCR based assay with primers outside of the targeting vector. Once the orientation of the fragments in the targeting vector are confirmed and the probes are tested then it is time to...

4. Electroporate ES Cells and Select Recombinants. ES cells are a very sensitive reagent, and most often the weakest link in the process of producing gene targeted mice. Fastidious tissue culture technique is necessary to maintain the totipotent state of the cells during electroporation, selection, and expansion. In order to maximize successful outcomes, the Transgenic Core maintains ES cells and companion reagents that are quality tested by producing ES cell-mouse chimeras that transmit the ES cell genome to offspring. Investigators may choose to use the Core's Gene Targeting Service (we do all of the tissue culture work, you do all of the molecular biology), or to use space in the Core lab (rent space in our completely equipped laboratory for ES cell culture work), or to do the tissue culture work in their own space. After the cells are electroporated with the targeting vector and put under drug selection, resistant clones are picked and cultured. DNA prepared from the clones is screened and ES cell clones which have undergone homologous recombination with the targeting vector are identified. During screening the clones are cryopreserved in 96 well plates at -80 degrees C. Since these conditions are not optimal for long term storage of cells, it is important to complete the screen in a timely fashion.

After clones are identified, they are brought out of the freeze and expanded. During expansion, additional DNA is prepared, if necessary, to confirm recombination with the targeting vector, clone morphology is examined, chromosome counts are used to see if the clone is euploid, and a mycoplasma test is done to see if the cells are infected. Investigators may choose to have the Core carry out these tasks, perform them in the Core stem cell laboratory, or to do them in their own space. Once targeted clones have been verified the next step is to...

5. Inject Blastocysts with ES Cells. The Core will microinject ES cells into blastocysts. See the service description for more information. The goal is to produce chimeric animals with high contribution from the ES cell clone and low contribution from the host embryo. This is typically assessed by coat color contribution. ES cells produce agouti fur and the C57BL/6 host cells produce black fur. Another sign that the ES cells have successfully colonized the host embryo is a distorted male:female sex ratio in the chimeras. Since ES cells are X:Y, a good clone will convert female host embryos to phenotypic males. Thus, the desired outcome is for 75% or more of the chimeras to be male and to include animals that have 90% or more agouti coat color. Unfortunately, we can not guarantee that all ES cell clones will produce germline chimeras. In our experience we have seen very good clones that produce high contribution chimeras that transmit to 100% of their offspring and we have also seen apparently "lethal" clones in which zero animals were born after transferring 50 injected embryos, we have seen "bad" clones in which only animals with black coat color were produced, we have seen "bad" clones which produced low agouti coat color contribution chimeras. Unfortunately there are no in vitro tests that will differentiate among these outcomes, the only way to discover the germline potential of an ES cell is to inject it. In general, we suggest that you inject at least three ES cell clones to assure transmission from one, and possibly two clones.

6. Chimera Breeding and Germline Transmission. Once the chimeras are produced, breeding is carried out to obtain offspring that carry the targeted gene. Male ES cell-mouse chimeras are mated with C57BL/6 females (also see Breeding Suggestions). The first sign of germline transmission is the appearance of pups with agouti coat color. Pups produced from sperm derived from ES cells will have agouti coats and half of these animals should carry the targeted gene. Pups that are produced from sperm derived from the C57BL/6 host embryo will have black coats. Tail biopsies from the agouti pups are screened for the presence of the targeted gene in the same way that you screen for the presence of a conventional transgene.

7. Breeding and Analysis. Once hemizygous mutant mice are identified they are mated to produce homozygous mice. The final stage is to study the animals to determine the consequences of the mutation introduced by homologous recombination in ES cells.