The Yale University and Yale Cancer Center Transgenic Mouse and Gene Targeting Resource (TMGTR) produces transgenic and knock out mice for Cancer Center members and members of the Yale community. The Resource has the expertise and equipment to produce transgenic mice from cloned DNA provided by users. It also offers mouse embryo cryopreservation as an economical alternative to maintaining genetically unique colonies of mice.

Transgenic Mouse Service

The creation of transgenic mouse lines have wide-ranging utility in biological experimentation. Investigators wishing to create novel transgenic lines may do so using the Transgenic Mouse Service (TMS). After the desired transgene construct has been created, DNA will be linearized and purified by the investigator according to standard recommendations listed below. The targeting construct is given to the TMS where it is microinjected into zygote pronuclei. Injected embryos are transferred into pseudopregnant females to await delivery of potential founder animals. Following their birth the investigator is notified of the number of mice born and is given a tail clipping of each at the time of weaning. After positives are identified an animal transfer request form is completed so that animals may be transferred to the investigators animal room(s). Because the TMS is partially subsidized by the Yale Cancer Center (YCC), YCC members are charged a reduced fee.  You may order this service on-line via the Transgenic Mouse Service Order Form.  Also see the DNA Purification Protocols section.

Interested in learning more about the Transgenic Mouse Service? Click HERE.

Targeted Mutagenesis Laboratory

The more recent development of mouse "knockout" technology has also created new opportunities in many research fields. The Targeted Mutagenesis Laboratory (TML) offers several services for investigators. Each individual component is described below.

Electroporation of Embryonic Stem (ES) Cells

After appropriate targeting construct design, the TML lab will accept appropriately purified and concentrated targeting construct DNA for subsequent electroporation. Following electroporation ES cells will be selected on embryonic fibroblasts that contain the appropriate selection cassette resistance gene (typically Neomycin). Colonies of ES cells that survive selection will be picked 10-14 days after electroporation and expanded in non-selective medium. After clones have increased in size an aliquot is frozen in liquid N₂ and a second sample is pelleted from which the investigator can extract DNA for screening. After the investigator completes screening positive clones will be regrown and the screening process repeated to provide additional verification.

ES Cell Karyotyping

Prior to blastocyst injection it is recommended that the ES cell line be analyzed for correct chromosome number (40).

Blastocyst Injection

Positive clones are injected into C57Bl6/J blastocysts at 3.5 days of gestation and transferred into pseudopregnant females. Chimerism in offspring will be evident 4 weeks later. Reasonably chimeric progeny will be mated with the appropriate tester stock when the chimeras reach 6 weeks of age. Germ line transmission of the ES cell lineage could first be detected 6 weeks later or beyond.

You may order these services on-line via the Targeted Mutagenesis Service Order Form.

Interested in learning more about Targeted Mutagenesis? Click HERE.

Mouse Embryo Cryopreservation

Due to the inherent value of genetically altered mouse strains, the Yale/YCC Core Services will offer cryopreservation of individual mouse strains effective November 1, 1999. The Cryopreservation Service is unable to accommodate more than 2 lines/month at the present time however this could change in the future. Strains will be cryopreserved in the order in which requests are submitted. Please read the following information carefully to understand the benefits and limitations of cryopreservation.

Investigators responsibilities:  After receiving approval, investigators will select 5 males, aged 1-4 months, for transfer to the YARC quarantine facility. If 5 appropriately aged males are not available it is recommended that the investigator postpone starting cryopreservation. Per Diem charges while the animals remain in quarantine and beyond are assumed by the investigator. If mice are genetically altered, investigators have the option of providing either heterozygous or homozygous males with an overall reduction in cost if homozygosity is chosen. After the mice are released from quarantine they are transferred to our facility where they will be mated to superovulated B6SJLF1(C57BL/6 X SJL) females. After cryopreservation is complete, investigators have the option of timely reclaiming their males if they choose to do so. If animals show poor fertility another 5 males will be requested. If fertility is again poor no further attempts will be made to cryopreserve the line.

Cryopreservation Service Responsibilities:  Gestational day 2.5 embryos are isolated and cryopreserved in straws and stored in liquid N₂. The first and last straw are subsequently thawed and transferred to foster females to demonstrate viability of the line with the assumption that all embryos frozen between the first and last straw will behave similarly. If viable progeny are not observed a second embryo transfer will be performed. After birth, the investigator has the option of transferring the mice to their own animal rooms or having them euthanized by YARC. There will be a yearly liquid N₂ fee to maintain the embryos in two separate liquid N₂ tanks in our facility. It should be noted that the strain background of the cryopreserved line will be altered using our mating regimen and this fact needs to be carefully considered by the investigator prior to making the decision to cryopreserve. The Cryopreservation Service is unable to accommodate alternate strain cryopreservation at the present time however this could change in the future. The number of embryos cryopreserved and the costs are listed separately.

Interested in learning more about Crypreservation? Click HERE.

This information adapted for the WEB and maintained by Greg Blasko
Last modified: November 22, 1999