Transgenics - Generation of KO / KI mice
The technique of gene targeting has proved to be very powerful. As opposed to pronuclear microinjection, which is the random integration of the gene of interest into the genome, gene targeting in ES cells, followed by the generation of KO/KI mice allows precise modification in the mouse genome, including: loss of function (knockout), replacement (knockin), subtle mutation, and conditional mutation of a target gene. It involves a homologous recombination reaction between the targeted genomic sequences and an exogenous targeting vector.

This service is divided into two steps. The first, gene targeting in ES cells, consisting of the introduction of the targeting vector into ES cells by electroporation followed by the selection and identification of correctly mutated ES cell clones. Homologous fragments included in the construct are able to promote homologous recombination events at the target gene in ES cells generating ES cell clones with targeted mutations. The second step, ES-cell mouse chimera production, consists of the injection of correctly mutated ES cells in blastocystes, which are subsequently reimplanted in pseudo pregnant females, where they will contribute to the development of the embryo.

Depending on the type of service required a combination of either both steps 1 and 2, or only step1 or step 2 is offered. If researchers require the generation of targeted ES cells and the subsequent microinjection for the generation of a chimeric mouse, we require a detailed description of the targeting vector and demonstration of the feasibility of the targeting method used. If the service only requires the blastocyst injection, two frozen vials need to be provided, one of which will be screened for mycoplasma, at the cost of the researcher. Only once the test for mycoplasma is negative will the second vial be thawed and used.

Timelines:



 

i. Gene Targeting in embryonic stem (ES) cells

This service will produce embryonic stem (ES) cells with mutations induced by homologous recombination with a targeting vector.

Service Description:

The center will perform the following tasks:

• Thaw and place in culture the centers proven ES cells ;
• Electroporate wt ES cells with the investigators targeting construct ;
• Select the transfected clones based on resistance to antibiotic ;
• Pick clones in up to five 96-well plate and further expand each clone into three ;
• One plate is cryopreserved and one plate grown and splited into two replicates for DNA preparation*. The cells will be maintained at -80^o C until we receive the results. The result (photocopy) of the Southern blot or PCR must be submitted before any clone is thawed ;
  * some centers will perform the DNA analysis for the investigator
• Once ES cell clones with targeted mutations are identified, the center will thaw and expand the ES cell clones until 5 vials of 5 X 10⁶ cells/ml is produced for storage in liquid nitrogen ;
• Each positive clone is then tested for mycoplasma and its morphology evaluated by the center.

Responsibility of the Investigator

• Preparation of the targeting construct for ES cell electroporation according to a proven protocol.
• DNA preparation and subsequent screening by Southern Blot of potentially positive clones.
• Chromosome count and confirmation of gene targeting in thawed ES clones
• Testing for euploidy.
• Download the request form here and e-mail to (M. Chagnon) or fax to (514) 398-6769.

Guarantees
We cannot guarantee the generation of a targeted clone as this depends on the targeting vector.

ii. ES Cell-Mouse Chimera Production



Service Description:
The Center will inject mouse blastocystes with gene targeted ES cells for the generation of chimeric mice. The center will perform the following tasks:

Service includes:

• Microinjection of 2 positive ES cells into mouse blastocystes. Approximately 60 blastocystes are injected (~30 blastocystes/clone);
• Transfer of injected blastocystes into pseudo pregnant females ;
• Two weeks after birth, a first selection is made. Mice with the highest contribution of mutated ES cells are selected. Their recognition is based on coat color (ES cell contributes to an agouti color) ;
• Once the pups are 3 weeks old they are transferred and billed to the investigator.

Responsibility of the Investigator

• The investigator must provide their targeted ES cells.
• The cells must have been screened for micro organisms (e.g. mycoplasma).
• Download the request form here and e-mail to (M. Chagnon) or fax to (514) 398-6769.

NOTE: Only clones that meet the criteria for microinjection (euploidy, goodmorphology, clear of infection, correctly targeted) can be scheduled for microinjection into blastocystes for chimera production.

Guarantees
If the targeted ES cells were generated by the center, we guarantee a minimum of 2 chimeras. Please note that the level of chimerism (percentage of contribution of ES line compared to the wild type phenotype) varies considerably and it is possible that there will be no germ line transmission. If the targeted ES cells were generated by the investigator, we cannot guarantee the generation of chimeric mice since we have no control over the quality of the ES cells.