Once we have the digested DNA in hand, we should produce founder mice within two months.  This is dependent on the number of transgenic requests currently underway in the LRTGT.

 

2. Why is a description of the potential phenotype required?

 

We need to be aware of the possibility that the transgene may cause embryonic or neonatal mortality.  This will allow us to plan accordingly.  If there is a distinct possibility for embryonic lethality, we can collect various embryonic stages for analysis.  Likely this will increase the costs associated with the service.

 

3. How many founder lines should I expect?

 

It has been our experience that injection of 150 embryos will result in 3-5 founder mice.  This will be affected by the quality of DNA.

 

4. Why can mice not be maintained longer than two weeks after wearing?

 

The LRTGT has limited capacity for both space and manpower.  For London-based researchers, we can arrange for Vivarium staff to handle your mice after we take them out of the Facility.  Both the LHSC and UWO Vivariums have competitive rates for maintaining colonies and providing biopsies for genotyping.

 

5. What is the price for transgenics?

 

Currently, the price to for generating transgenics for a single transgenic construct is $2500 internally (London researchers) and $3500 externally.

 

6. What, if any, are the guarantees?

 

We will make every effort to generate 3 founder mice for each transgenic construct.  In the event that we do not produce 3 founder mice (and embryonic lethality has been excluded), we will reimburse the investigator %50 of the cost ($1250 internally, $1750 externally)

 

7.  Why do I need to provide evidence that I can detect a single copy of the transgene in genomic DNA?

 

To provide any guarantees on our work, we need to be assured that your laboratory has a genotype protocol that is sensitive enough to detect the insertion of a single copy of the transgene.  In addition, from experience we have found that working out the genotyping protocol before hand greatly increases the efficiency of identifying founder mice and saves significant costs in mouse handling charges.

1. What if the first 192 clones does not produce a targeted ES cell line?

The expected frequency of homologous recombination in ES cells varies dramatically depending on the genomic region to be targeted.  Typically, 192 selected colonies should identify at least 2-3 targeted events.  In the event that a positive 192 clones  

2. Will the TGT perform genotype analysis?  Why or why not?

Due to the variability for each genotype assay and the time demands on the staff of the LRTGT, we do not offer genotyping services.  However, the LRTGT does offer support in the way of laboratory space and equipment to perform to analysis and will provide technical support in this area.

3. Can you guarantee correctly targeted clones?

Several factors affect the integration of targeting vectors that are beyond our control, most especially the genomic locus that is being targeting.  While we will make every effort to get a targeted clone and will work with each laboratory to increase the chances of success, we can not guarantee that a targeted ES clone will be obtained.

4. What type of analysis is required for genotyping?

Each laboratory should work out a strategy to identify targeted integration of the transgene prior to proceeding with the project.  This would involve Southern blotting with a probe external to the targeting vector and a genomic DNA digestion that would delineate the endogenous locus from the targeted locus.  While PCR-based strategies can be used for genotyping mice for hetero and homozygosity, we strongly discourage the use of this strategy for screening ES cell clones.

Typically, we inject up to three different clones of targeted ES cells clones into 45 blastocysts each.  If desired, we can inject additional clones, but this will be done at an additional cost to the investigator.

Currently, we do not offer this service.

The LRTGT will perform blastocyst injection of any ES cell type, however; we are currently only doing gene targeting in R1 (SvJ129 background) ES cells.

8. What is the price for gene targeting?

The standard rates for all service can be found here.  For specialized services, please contact the Scientific Director.

We guarantee the best possible effort in obtaining chimeric mice from gene targeted ES cells.  However, given the variability of targeting specific loci within the genome, we do not make any guarantee of success on this aspect of the work.

1. What type of cryopreservation is offered?

 

2. Why are their different packages for cryopreservation?

 

The package prices are based on the number of embryos to be cryopreserved, and the rationale for freezing the embryos.  The minimal package is for 150-200 embryos and is used specifically as an emergency backup for a colony.  The standard package is for the freezing of 350-400 embryos and can be used to distribute embryos for internal use.  Finally, the largest package is for distribution outside of the London area and requires the freezing of 750-800 embryos.  This last package results in 32 vials of embryos and should be good for shipping to 15 different locations.

 

3. How many mating pairs do I need to set up?

 

 

4. When should I consider doing cryopreservation?

 

There are three different occasions for cryopreserving embryos.  The first is to preserve an important line of mice that is not easily replaced.  The second is when the line is no longer useful, but may be required in the future.  Finally, if the line is of significant value to other investigators.  Transfer of mouse lines from one Facility to another is more efficient when shipping embryos.  This method of shipping provides some evidence that the line is pathogen free, and there is limited concern regarding the effects of stress from shipping on the mouse line.  However, YOU NEED TO BE SURE THAT THE RECIPIENT FACILITY HAS REDERIVATION SERVICES.

 

5. What are the MTA forms for?

 

 

6. How many embryos should I freeze down?

 

It depends on the reason for cryopreserving.

 

7. How many embryos do you get per session?

 

 

8. What happens when the stock of embryos gets low?

 

 

 

9. Who is responsible for rederivation or shipping of embryos?

 

 

 

10.  What guarantees are there for the cryopreservation package?