The outcome of any given experiment is not guaranteed. The staff of this Resource has years of experience and routinely perform the services offered with success. However, variability can be due to the nature of the materials originating from Investigator's laboratories, biological variability of the mice, or sometimes simply bad luck. Thus, experiments do not always provide the desired results. The Resource will not be obliged to provide service when the PI fails to provide materials or results that are of judged to be of insufficient quality. Any deviation from the routine fee scheduled will be reviewed and approved by the director of this resource.
The Transgenic Mouse/ESC Shared Resource will repeat specific experiments as necessary to achieve the deliverables that have been specified for each service type. However, any additional service beyond that specified for each service type must be approved by the Manager and Director of this Resource and will usually require an additional charge.
Working with Preexisting Mutations for Microinjection
The TMESCSR is willing to utilize a mouse line with preexisting mutations for microinjections. However, this does generate additional complexities and effort to perform this type of experiment. Such experiments will require additional communication with the Manager of this resource and the Investigator’s lab. The Investigator’s lab will be responsible for a number of aspects of such a project including, but not limited to the following:
The investigator’s lab will need to maintain 8 to 10 stud males which must be singly housed during the experiment timeline. Scheduling of such experiments will be done so that it is least disruptive to the TMESCSR routine schedule. The TMESCSR will superovulate wildtype females and submit a transfer request for the DAC to move to the superovulated females to the investigator’s room on a specific date and time. Once received, the Investigator’s lab will be responsible for mating the females with the stud males. This will need to be completed by 2 pm on the day the females are moved to the Investigator’s room. The following morning, someone from the Investigator’s lab will meet the TMESCSR to retrieve the females. A minimum of 50 fertile embryos will be necessary for microinjection to occur. If fewer than 50 fertile embryos are obtained, the Investigator will be responsible for the cost of ½ of an injection day. Due to the intricacy of such experiments, no guarantees are provided for an injection day to be completed due to low number of fertile embryos. The TMESCSR also does not guarantee that the desired mutation will be achieved. The Investigator will likely need to schedule multiple injection days for this type of procedure to be successful.
If your experiment does not allow for the use of wildtype females mated with the stud males that contain the preexisting mutation, please contact the Manager of this resource to discuss. This is usually a much more intensive mating scheme for the Investigator’s lab and would require 8 to 10 females at 3 to 5 weeks of age for each injection day planned in addition to the 8 to 10 stud males for mating. The Investigator’s animal protocol would also need modification to allow for the superovulation of the females to be used for microinjection.
Authentication of Key Biological and/or Chemical Resources
The responsibility for authenticating the mice generated by the Vanderbilt Transgenic Mouse/ES Cell Shared Resource (TMESCSR) lies principally with the users of the resource. However, we strive to the extent possible to assure that all genetically-altered mice developed through this resource have exactly the changes in DNA content that the investigator wants. To help assure this, all projects are reviewed by the Director of the TMESCSR to ensure that they have been properly designed and are feasible. Instructions are provided as part of the service forms for the acquisition and preparation of the reagents that are used in the blastocyst or pronuclear microinjection procedures. Authentication of the mice that are obtained is typically done in two stages. The first step is to confirm that the genetic alteration is present and can be routinely identified by standard genotyping and/or DNA sequencing methods. The second step is to determine whether the genetic manuscript functions as intended. Validation of functionality may involve multiple assays, depending on the nature of the genetic mutation.