Gene-targeting in embryonic stem (ES) cells is a powerful technique to introduce mutations at specific chromosomal loci. This technology is based upon experiments which show that foreign DNA can integrate precisely in the genome through homologous recombination in cultured cells. With the advent of ES cell technology, methodologies have been developed to alter target genes through homologous recombination, while still maintaining the pluripotent state of the cells. Genetic alterations introduced into ES cells can be transmitted to the mouse germline by injecting the gene-altered ES cells into mouse early embryos to form chimeras. Together, gene-targeting and ES cell technology allow one to study the in vivo function of a gene by the modification of the endogenous gene.
A targeting vector, supplied by the investigator, will be introduced into ES cells via electroporation. These cells will then be grown on appropriate selection media. DNA from surviving cell clones will be provided to the investigator for Southern or PCR analysis. Positive clones will be expanded and frozen, and will be maintained within the GEMF for subsequent blastocyst injection or given to the investigator (upon request).
$4000/construct for MD Anderson investigators; $10,900/construct for external investigators
Time Line for Gene-Targeting in ES Cells
- Day 1: Initiate ES cell culture for electroporation
- Day 5: Electroporate targeting vector
- Day 6: Start drug selection
- Day 16: Pick all surviving clones, or up to 3 x 96-well plates (288 total) single or double resistant colonies (additional plates can be picked. Contact facility manager for current pricing.)
- Day 20: Split ES cell culture on 4 x 96-well plates (for each single 96-well plate picked), two duplicates to freeze temporarily at -80°C and the remaining two to generate DNA for the investigator for duplicate Southern analysis
- Day 23: Extract DNA from 96-well plates and provide to the investigator for screening.
Time Line for Cell Expansion
- Day 1: Expand three targeted clones according to results from Southern hybridization (additional clones can be expanded. Contact facility manager for current pricing.)
- Day 6: Large scale DNA provided to investigator for re-confirmation
- Day 21: Freeze cells in liquid nitrogen
Points to Consider
Gene-targeting experiments are very time consuming and labor intensive. Below are some points to consider when designing a gene-targeting experiment to avoid costly delays.
Source of Genomic DNA
When constructing a targeting vector, it is very important to use genomic DNA that is isogenic with the ES cell line to insure homologous recombination. We presently have 129SvEv (S6), 129/SV/J (X1) and hybrid 129SvEv/C57BL/6 ES cell lines that we use routinely in our facility. Other genetic backgrounds may be used if prior arrangements have been made with the facility to insure the availability of ES cell lines of the same background.
Strategy for Identifying Targeted ES Cell Clones
Identifying a scheme to determine targeted clones is the most important part of the gene-targeting experiment. The strategy has to be rigorously tested and proven to be both reliable and reproducible. Since this aspect of the experiment is so crucial and has often proven to be problematic, the GEMF requires each investigator to submit data demonstrating the feasibility of their strategy, and a Southern analysis demonstrating the utility of the probe prior to the initiation of any ES cell work.
Aspects to consider when designing a strategy are listed below:
- Southern analysis is highly recommended for detection of homologous recombination
- Diagnostic restriction enzymes should be chosen to give obvious size differences between targeted and non-targeted cell clones
- Through experience, we have been able to categorize enzymes according to their performance on mini-Southerns (see below). Try to adhere to this list as much as possible
- Identify 5’ and 3’ external probes for Southern analysis that give good signals on genomic Southerns. Both probes should be outside of the regions of homology used in the targeting vector
List of Recommended Enzymes for Mini-Southern
To Obtain Service from the GEMF
- Schedule an appointment with Jan Parker-Thornburg (713-745-2654 or e-mail firstname.lastname@example.org) on North Campus, or Chen Dong (713-563-3263 or email email@example.com) on South Campus, to review targeting strategy. We will cover experimental design, from construction of the targeting vector to the method of diagnostic analysis for targeting events
- Supply the GEMF with the Institutional Biosafety Committee (IBC) approval number for DNA recombinant research
- Complete and submit a service request form for electroporation of ES cells
Service request form for MD Anderson investigators (pdf)
Service request form for external investigators (pdf)
- Provide the facility with a targeting vector prepared according to the specified protocol. A detailed map of the targeting vector and the strategy for identifying targeted clones along with results from a pilot experiment for analysis is to be submitted at the same time
- When electroporation and selection is completed, the facility will provide the investigator with duplicate 96-well plates containing uncut genomic DNA from selected ES cell clones. The investigator should perform a genomic Southern analysis on these DNAs and bring the resulting film to the facility for review. Three positive clones will be expanded, and an aliquot of DNA from each of the selected clones will be provided for confirmation by a genomic Southern analysis
- Frozen aliquots of targeted ES cells will be maintained in the facility for subsequent blastocyst injection or can be provided to the investigator upon request
Read the Policies and Procedures section for important information regarding services rendered by the GEMF.
Note: Gene-targeting is an experiment that is both costly and time consuming. It is, therefore, essential that the targeting strategy be well thought through and methodically planned. By investing time in the thoughtful design of a gene targeting strategy, you can save time, money and research animal lives.