ES Cell-Mediated Gene Targeting
Note: If you want a transgene targeted to the ROSA26 or H11 loci, please see our Targeted Transgenesis service description.
Gene targeting is the process of disrupting or mutating a specific genetic locus in embryonic stem (ES) cells, usually with the intention of making knock-out or knock-in mice. Other methods of mutating the mouse genome include chemical mutagenesis, insertion of viral vectors or transposons, and random integration of gene-trap or promoter-trap vectors. Use of Cas9 nuclease is probably the most popular method to mediate genome engineering directly in mouse embryos, without the need to use ES cells. Note, CRISPR engineering can also be used in ES cells to engineer changes in the genome, including insertion or substitution of relatively large regions of DNA.
The targeting construct is usually a plasmid that contains two long stretches of genomic DNA, called homology arms, which are designed to match as closely as possible the genomic DNA of the ES cell line being targeted. These arms drive the homologous recombination event that results in insertion of the construct into the desired locus. Bacterial artificial chromosomes (BACs) can also be used as targeting vectors.
It is the client’s responsibility to design and engineer the targeting construct. See our tips on targeting construct design. We can recommend outside sources of construct engineering services, including recombineering. Please feel free to contact the TMF for advice on the design of targeting constructs for use in ES cells.
The entire gene targeting process consists of the following major steps. Each of these steps can be ordered as a separate service from our facility.
1) Linearize the targeting construct; introduce it into ES cells by electroporation; grow clones under positive selection; pick several hundred resistant clones into 96-well plates; split clones into duplicate sets; freeze one set and extract DNA from the other set.
2) Genotype all clones by Southern blot using a probe specific for one end of the inserted DNA. PCR is usually less suitable for this step. The probe sequence must lie outside of the homology arms, and probes must be tested by the client prior to electroporation of the targeting construct.
3) Expand clones that have the correct genotype (heterozygous for the targeted allele), freeze back multiple vials of each clone, and prepare about 50 micrograms of genomic DNA from each clone for a second Southern blot to characterize the other end of the inserted DNA. We can perform the second Southern blot for an additional fee.
We recommend doing a third Southern blot with a probe specific for the selection cassette in the targeting construct, to rule out the possibility that multiple copies of the construct were inserted. We can do this Southern blot for an additional fee. Clones that pass all 3 assays should have their average chromosome number determined and, if they are at least 70% euploid, they may then be used for injection into blastocysts to make chimeric mice (see our ES cell injection service).
Additional in vitro manipulations may be desired if a conditional targeting construct is being used (see below). We offer a secondary electroporation service in which we transfect an ES cell clone with a recombinase-expressing plasmid, pick two 96-well plates of clones, and extract DNA from them, which the client then analyzes for the desired recombination event. However, we recommend that all Cre or Flp recombinations be done in vivo to avoid the additional time in culture that may compromise the ability of the ES cells to "go germline."
We normally produce chimeric mice by injecting an ES cell clone into mouse blastocysts. We also have the capability of making chimeras by aggregation, but this will not work for some ES cell lines. A chimera is said to "go germline" when some of its offspring arise from ES cell-derived gametes. The ability of ES cells to contribute to the germline of a chimeric mouse is highly dependent on culture conditions, the total number of passages (cycles of splitting or freezing), and the degree of aneuploidy. Most targeted clones are heterozygous for the targeted allele and therefore ES cell-derived gametes have only a 50% chance of carrying the targeted allele. Clones with <50% euploid cells can produce chimeras but will most likely NOT go germline.
In addition to the Service Request Form, you must submit the following:
- At least 200 micrograms of your targeting construct plasmid, purified using the Qiagen Endo-Free Plasmid Kit, and dissolved in TE.
- A map of the construct showing all relevant restriction enzyme sites, homology arms, selection cassette(s), intron-exon boundaries, and recombinase recognition sites (for conditional targeting).
- A gel picture of uncut plasmid and plasmid cut with the restriction enzyme that we will use to linearize the plasmid.
It is the client’s responsibility to make sure the targeting construct has been engineered correctly, mapped, sequenced, and tested.
The minimum time needed to go from electroporation of the targeting construct to clone DNA ready for genotyping is about 4-5 weeks. Factors that affect this time include growth rates of different ES cell lines, selection times of different antibiotics, and the number of clones picked.
Southern blotting turnaround times can be quite variable, depending on the status of concurrent projects and the number of clones picked. The DNA of clones from two 96-well plates can be digested, electrophoresed, and blotted by one person in one week. Hybridization and autoradiography require several more days.
Expansion of positive clones from 96-well plates, freezing back multiple vials, and extracting more DNA for a second genotyping assay require about 2 weeks.
Currently we do not offer any performance guarantees for our gene targeting services, because the outcomes are too dependent on the exact locus being targeted and on the design of the targeting construct. However, we will repeat the services at no additional cost if technical errors on our part are responsible for a failure to produce any targeted clones.
- Electroporation and clone-picking service includes:
- Linearization and purification of the targeting construct DNA
- Electroporation into ES cells
- Growth of cells under selection
- Picking up to 384 drug-resistant colonies into 96-well plates
- Splitting the plates into 2 sets
- Freezing one set and extracting DNA from the other set
- Southern blotting service includes:
- Digestion of ES cell colony DNA from all 96-well plates with one restriction enzyme
- Agarose gel electrophoresis and gel photography
- Capillary transfer of digested DNAs to nylon membranes
- Hybridization of membranes with a radioactive probe (probe DNA supplied and tested by client, or by TMF for an additional fee)
- Washing blots and imaging on phosphorimager
- Analysis and annotation of images
- Clone expansion service includes:
- Thawing from 96-well plates of colonies chosen based on Southern blot results
- Expansion to 24-well and then 6-well plates
- Freezing 6-9 vials of cells per clone and storing in liquid nitrogen
- Extraction of about 50 micrograms of genomic DNA for further genotyping
- Chromosome counting service includes:
- Preparing metaphase spreads
- Counting chromosome numbers for 20 spreads/clone
- Secondary electroporation service includes:
- Electroporation of cells from a single ES cell clone with a Cre or Flp-expressing plasmid to obtain transient transfection
- Growing cells without selection
- Picking up to 192 colonies into 96-well plates
- Making duplicate sets of plates
- Freezing one set of plates and extracting DNA from the other set for analysis by the client
Mouse and ES Cell Strain Considerations
The first mouse ES cell lines were derived from one of the many so-called "129" strains of mice. There is a great deal of genetic variation among these strains (see, for example, Simpson, et al, Nature Genetics 16:19-27, 1997), and it is important to know exactly which one gave rise to the ES cell line being targeted. For a list of 129 strains and their most current nomenclature, see this Jackson Laboratory web page.
Techniques for deriving new ES cell lines have improved dramatically in the last few years, and lines from many genetic backgrounds are available. The TMF now performs electroporations on a routine basis with an ES cell line (JM8.N4) derived from C57BL/6NTac mice. JM8.N4 and other lines derived from C57BL/6 substrains offer several advantages over 129-derived lines. Foremost is the ability to generate an inbred gene-targeted strain in one generation simply by crossing your chimeras with C57BL/6N mice. (Be careful to match the substrain!) While this can also be done for 129 lines, many of these lines can be quite difficult to breed (or even to obtain), and their experimental history is not nearly as extensive as that of the venerable C57BL/6 substrains. Additionally, available genetic resources such as BAC libraries are still predominantly based on the C57BL/6J substrain, and this was the first strain to have its genome sequenced.
The distinction between C57BL/6J and C57BL/6N substrains is important because the C57BL/6J substrain carries a homozygous null mutation in the Nnt gene (nicotinamide nucleotide transhydrogenase) that is not present in the C57BL/6N substrains. By contrast, the C57BL/6N substrains from the various vendors (C57BL/6NTac = Taconic, NCrl = Charles River, NHsd = Harlan, NJ = Jax) differ by only a few SNPs located outside of known coding regions.
In addition to the JM8.N4 ES cells, our facility offers the E14TG2a line of ES cells, derived from 129P2/OlaHsd mice. Like JM8.N4, the E14TG2a cell line is grown without feeder cells, which reduces the time and cost of tissue culture and simplifies genotyping. Both lines were acquired as generous gifts from Dr. William Skarnes.
If possible, the genomic DNA used for the homology arms of a targeting construct should come from the same strain of mice as the ES cells being targeted, or from the ES cells themselves. This ensures the best sequence match between the construct and the locus of interest. Although a small number of mismatches can be tolerated, the frequency of homologous recombination will go down as the extent of mismatch increases. If you have a targeting construct made from genomic DNA from 129 and want to know if there is significant sequence difference between this and another strain (e.g. C57BL/6J), you can look for differences using the genomic DNA resequencing information available at Ensembl. The frequency of homologous recombination can be improved by using longer homology arms, but traditional DNA cloning methods are limited to using arms of ~10kb or less. Recombineering techniques allow one to overcome this limitation and use entire BACs as targeting constructs.
See the introduction to our ES cell injection service for a brief discussion of breeding considerations for chimeric mice made from targeted ES cells.