The Transgenic Core Facility will make every effort to make transgenic mice with your construct, but cannot guarantee success with every construct, since a variety of factors can affect the production of transgenic pups. Some of the most crucial factors under the control of individual investigators are the design of the injected construct, and the production of properly prepared, highly purified linearized DNA containing no vector sequences (see details below).

The TCF also provides injection of embryonic stem cells (ES cells) into blastocysts and implantation into pseudopregnant females. Because of the nature of this work, investigators should contact the TCF well in advance of these studies to discuss their project.

Mouse Strains: Ten B6D2F1 (C57BL/6 x DBA from Jackson Labs) or 15 C57BL/6 females or 10 FVB females are superovulated and mated to B6D2F1or C57BL/6 males or FVB males to produce the fertilized eggs for injection. Investigator provided mice will be dependent on the number of females and males provided. (Other strain combinations can be requested, but at additional cost and without the standard guarantees. Such strains must be requested at least 3 weeks before injection to allow time to obtain the necessary mice and have them pass through quarantine procedures).

Mice are housed in the barrier wing , ARF 215 in microisolator cages in specific pathogen free conditions.

To keep costs down for all investigators, these set-up fees are non-refundable once animals are ordered. This charge will be applied to the injection's cost if the injection days are used as scheduled. This fee covers the set up costs of purchasing mice, housing, reagents, etc., which the TCF incurs whether or not the injections take place. Should an investigator inform the TCF ahead of time for the need to reschedule injection days, and should DNA from other investigators be available to use on that specific scheduled injection day, then the fee can be refunded; however, there will be no refund of set up fees for "wasted" injection days.Also note that if a properly completed Request Form for Transgenic Productionis not received before the 1st injection day, the injections will be postponed until the completed form is received, and an additional fee for housing mice may be incurred.

Costs for Blastocyst Injections (Embryonic Stem Cell Gene Targeting/KnockOuts):

The investigator is responsible for all ES cell work and the Transgenic Core Facility will inject the cells provided into blastocysts obtained by superovulating 10 C57BL/6 females per injection day.

Guarantee: 150 embryos transferred, 20 candidate pups or 2 transgenic founders produced (whichever criterion is met first). If none of these are met after 2 injection days, an additional day or days will be scheduled as soon as possible at no additional charge. (No additional injection days will be scheduled until Southern analysis of potential founder DNAs, including appropriate positive control, is provided by the investigator).

1. File an approved Animal Protocol with the Department of Comparative Medicine (531-8460, Room CG721, Mailcode H054). This protocol number must be included on the Request for Transgenic Mouse Production form.

2. Design and produce appropriate plasmid constructs for injection. (For blastocyst injection for gene-targetting/knockout production, all ES cell work is the investigators responsibility)

3. Arrange space for housing any transgenic mice produced (Doug Ednie, Dept. of Comparative Medicine 531-8465)

4. Fill out the Request for Transgenic Mouse Production form (available in Room C1735 or on-line at (http://www.hmc.psu.edu/core/Transgenics/transreq.htm), and schedule injection days with:

• Alane Seidel, Research Support Associate
  Phone: 531-4714 FAX: 531-4715
  email: aks2@psu.edu
  Rm. ARF173, College of Medicine

(NOTE: An approved animal protocol must be on file in the Department of Comparative Medicine before injections are scheduled to begin, and this protocol number must be filled in on the injection request sheet.)

5.         By Friday at noon before the first scheduled injection day, deliver purified linearized DNA devoid of any vector sequences to the second shelf down of the freezer straight back from the door in C1732 (Anne Stanley's Macromolecular Core lab). Proper preparation, purification, and concentration of this DNA is crucial to successful production of transgenic pups.

         Please carefully follow the Approved DNA Preparation procedures, available either in Room C1735 or on-line at: (http://www.hmc.psu.edu/core//Transgenics/DNAprep.htm). We also require a photo of a EtBr-stained gel (with markers of known concentration) or preferably an Agilent Bioanalyzer 2100 analysis (available in our Functional Genomics Facility, x 5823) to document DNA concentration and purity, and a brief description of the construct and its expected phenotype are requested as well.

(Superovulations are schduled for Saturdays and Mondays, and will not be done until the DNA and gel photo and Request for Transgenic Mouse Production form are in the possession of the TCF!)

6.              Approximately 2-3 weeks after pups are born (i.e., 5-6 weeks after initial transgene injections), be prepared to promptly analyze the tailclips of candidate transgenic pups to verify the presence or absence of the transgene. The TCF will supply the tail clips after scheduling with the investigator, but it is crucial that these be rapidly and correctly analyzed (correct positive controls such as endogenous locus haploid or diploid, or negative sample spiked with 1 copy of your construct per cell, etc. See additional details below) For additional tailclips requested by the investigatorës lab there will be an additional $25 charge for up to 20 tail clips and an additional $0.40 for each tail clipped over 20.

7.            Maintain, breed, and further analyze any transgenic founders produced.  

If an investigator needs to cancel or delay their DNA injections, they should contact the TCF lab as soon as possible to allow time to arrange other DNA constructs from other investigators to be injected on those days.