Yale University

Targeting Constructs

Gene targeting constructs must be approved before electroporation. A map of the construct and a strategy for screening the clones for the correct targeting event must be presented. We recommend that you meet with us before or during targeting construction in order to solicit our input and to notify us of any unusual circumstances or strategies you plan to use.

Scheduling

Constructs will be microinjected or electroporated in the order they are submitted. The appropriate on-line form, including charging information, shall be sent prior to or at the time of submission. Non-Yale investigators may call or e-mail with billing information.

DNA Submission

DNA submitted for gene targeting or pronuclear microinjection should be of high quality, prepared as per protocols or equivalent methods. Transgenic constructs (1.5 µg) should be purified and resuspended at 5 ng/µl (300 µl at 5 ng/µl). BAC constructs should be submitted at 0.5-1 ng/µl preferably in polyamine buffer. For BAC constructs, please refer to the protocols on the website of the University of Michigan Transgenic Animal Model Core, especially for the composition of microinjection buffer. Gene targeting constructs (50 µg) should be linearized with an appropriate restriction enzyme, extracted, precipitated, and resuspended at 1 µg/µl in TE or similar buffer.

Animal Use Approval

Investigators must have an IACUC-approved protocol for use of mice prior to relocation of mice to the investigator’s animal room(s).

Success of Experiments

Due to the inherent variability in gene targeting experiments, and the variety of constructs submitted, we cannot guarantee the success of any particular experiment. However, through consultations and troubleshooting, we will make a good faith attempt to achieve correct gene targeting.