UCI > Research > TMF > Gene Targeting

Gene Targeting

• Introduction
• Ordering
• Turnaround Times
• Performance Guarantees
• Service Description
• Mouse Strain Considerations

Introduction:

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 by injecting those ES cells into blastocysts.

The targeting construct is a plasmid that contains two long stretches of genomic DNA, called homology arms, which must 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.

It is the client’s responsibility to design and engineer the targeting construct. See our tips on targeting construct design.

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 and purify the targeting construct; introduce it into ES cells by electroporation; grow clones under positive selection by antibiotics; pick several hundred resistant clones into 96-well plates; split clones into duplicate sets; freeze one set and isolate 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 not suitable for this step.)

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 genotyping assay (Southern blot or PCR) to characterize the other end of the inserted DNA. This assay is usually performed by the client.

Clones that are positive for both genotyping assays may then be used for injection into blastocysts to make chimeric mice (see our ES cell injection service).

Additional in vitro manipulations may be needed 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 2 96-well plates of clones, and extract DNA from them, which the client then analyzes for the desired recombination event.

Ordering:

To order this service, go to our on-line order form. After filling out the on-line Service Request Form, print a hard copy, have it signed by the Principle Investigator, and mail or fax it to Tom Fielder.

In addition to the Service Request Form, you must submit the following:

1. at least 100 micrograms of your targeting construct plasmid, purified using cesium chloride density gradient centrifugation or the Qiagen Endo-Free Plasmid Kit, and dissolved in TE;
2. 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);
3. 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.

Tips and protocols for designing and preparing your targeting construct are also available.

Turnaround Times:

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.

Performance Guarantees:

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 lack of results.

Service Description:

1. 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 3 sets
   • freezing one set and extracting DNA from the other two
2. Southern blotting service includes
   • digestion of ES cell colony DNA 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)
   • washing blots and imaging on phosphorimager
   • analysis and annotation of images
3. 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
   • aliquotting cells and freezing medium into about 8-9 cryovials and storing in liquid nitrogen
   • extraction of about 50 micrograms of genomic DNA for further genotyping
4. Karyotyping service includes
   • Giemsa staining of mitotic cells
   • Counting chromosome numbers for 20 spreads
5. 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 Strain Considerations:

Most ES cell lines are 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), so 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.

ES cell lines derived from C57BL/6 mice are now available. These lines offer several advantages over 129-derived lines. Foremost is the ability to create an inbred gene-targeted strain simply by crossing your chimeras with C57BL/6 mice. While this can also be done for 129 lines, many of these lines can be quite difficult to breed, and their experimental history is not nearly as extensive as that of the venerable C57BL/6 line. Secondly, far more genetic resources, including the entire genome sequence, are available for the C57BL/6 line.

Our facility prefers to do gene targeting with the E14TG2a line of ES cells, derived from 129P2/OlaHsd mice. This cell line is grown without feeder cells, which reduces the time and cost of tissue culture and simplifies genotyping. It was derived by William Skarnes and was a generous gift from BayGenomics. We also offer the HGTC8 line, derived from C57BL/6 mice at NIH.

If possible, you should always use genomic DNA for the homology arms of your targeting construct from the same strain of mice the ES cells were derived from, 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 does go down as the number increases.

See the introduction to our ES cell injection service for a brief discussion of breeding considerations for chimeric mice made from targeted ES cells.

 

University of California, Irvine
Transgenic Mouse Facility
B123 Gillespie Bldg. • Irvine, CA 92697-1140
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Last Updated: 02.08.2007

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