Services

The Core laboratory offers an annual one-day course in Mouse Handling and Breeding. This course covers all phases of transgenic and ES work as well as useful information regarding analysis and handling of the transgenic mice. The Core encourages all DRTC investigators contemplating using its facilities to take the course.

Embryonic stem cell technology has revolutionized mouse genetics, enabling researchers to remove a specific gene either from the entire genome or from a particular tissue (using the Cre-Lox recombination system). Embryo freezing is required for those situations where valuable transgenic mouse lines have been developed and where investigators plan on studying these strains for extended periods of time. If sufficient numbers of embryos are frozen, rederivation of the mouse line can be attained from implantation of thawed embryos into a pseudopregnant female. Embryo rederivations are necessary to rescue valuable mouse lines from frozen embryos, or to rederive trangenic mice that have become infected or are being transferred from dirty to clean facilities into a disease-free strain.

The following services have and will be provided:

1. Advice on vector construction for transgenic projects:

It is provided as a free service to potential users of the Core to ensure that Core use is appropriate. The Investigator is responsible for all cloning and DNA preparation procedures in his/her own laboratory. DNA preparation for microinjection will be offered as a new service in the future

2. Preparation of transgenic mice, through the F1 generation:

If the criteria above are met, the Core Laboratory will accept the construct(s) for injection. DNAs provided to the Core Laboratory by a Friday are usually checked by agarose gel electrophoresis for concentration over the weekend, and then injected according to a first come first served priority list as long as there is no more than a two-week backlog of constructs. If an investigator wishes to have more than one construct injected, they are asked to prioritize the constructs for the injection schedule. For each transgenic construct, females are typically mated on Sunday, Tuesday, Wednesday and Thursday nights. On Monday, Wednesday, Thursday and Friday, ~fifty fertilized embryos are injected with the DNA construct and cultured. On Tuesdays, Thursdays and Fridays, these embryos are transferred to the oviduct of each pseudopregnant female. Each day, all mice in the colony are checked. Additional operations, e.g., taking mouse tail/ear samples for DNA isolation, conducting embryo freezings, rederivations, etc., are conducted as time permits during each week Pregnant mothers are monitored until birth. As soon as a female is noticeably pregnant, Investigators are notified of the anticipated birth dates, so they can be prepared to analyze their newborn mice to determine if any seriously aberrant phenotype occurs. They are also informed that, if no aberrations occur, ear or tail samples will be available as soon as 10 days after birth. Core Laboratory technicians provide them with these samples, and the user is responsible for DNA analyses and for informing the technicians as to how long to keep the mice and which ones should be bred. The technicians are responsible for breeding, sacrificing animals, and taking tail/ear biopsies at the instructions of each user. Users are expected to provide any special instructions and to conduct all analyses. The Core laboratory has the following strain available to produce transgenic animals: CD-1, C57BI6/J, C3H, C57/CBA F1, and has developed the ability to produce transgenic NOD mice. The use of NOD mice as recipients of transgenes has broad and important implications for the use of gene manipulation in this model of spontaneous diabetes. Currently, the use of outbred CD-1 or inbred (B6xSJL) F1 mice for the generation of transgenics demands at least 10 backcrosses to the NOD strain to ensure that the NOD background is essentially pure. This procedure can take years from the time the first F1 generation are produced. In the previous funding period, the Core laboratory has developed the ability to produce transgenic mice directly in the NOD background and potentially in other inbred strains. The procedure is identical to that used for outbred strains, although special care must be taken as the eggs are more fragile during the injection procedure and of course the mice are significantly more expensive. Although the number required injections is about twice that for conventional transgenics, the yield is equivalent and the germline transmission of the transgene is essentially 100% and the time and expense saved is significant.

3. Culture of feeder and embryonic stem cell lines:

Since 1996, the DRTC Core has been providing embryonic stem cell and gene knockout technology as a service. Lab members have evaluated three different embryonic stem cell lines: D3, CCE, and R1. When cultured on primary mouse embryo fibroblasts, the R1 line gave rise to germline transmission with high frequency, and it was therefore expanded and frozen for general use. The J1 line was added to the available cell lines in 2000. It also has a demonstrated record of high frequency germline transmission. Both lines were tested extensively for microbiological contamination prior to freezing. Presently, the Core Laboratory provides potential users with a vial of frozen R1 or J1 cells, and instructions for handling, passaging and culture. The Core also provides users with instructions for embryo fibroblast derivation and for fibroblast feeder layer preparation. Finally, the Core provides instructions on electroporation of constructs and positive/negative selection.

4. Embryonic stem cell transfer to mouse blastocysts, and subsequent generation of chimeric and F1 animals:

The Core Laboratory requires that users take responsibility to handle the cells properly, according to instruction/guidance, and to engineer and test for their knockout. Upon demonstration by Southern blot/PCR analysis that the ES cells have been successfully targeted, Investigators contact the Core Laboratory to arrange a time to provide a designated technician with targeted ES cells, which the technician then injects into prepared C57Bl6 mouse blastocysts. Injected blastocysts are then implanted in pseudopregnant females. If the cells have been properly handled, they are easily injected, and chimeric mice are readily generated. Typically, agouti (ES) represents >75% of the coat color and is a strong indication that germline transmission will be achieved. The Core Laboratory provides at least two chimeric mice with >75% coat color from at least two independently derived targeted This is usually obtained six weeks after blastocyst injection (four weeks gestation, two week to obtain coat color). The technicians will then breed male chimeric founder animals with black C57Bl6 female animals to obtain heterozygous F1 mice; the mice will be analyzed for coat color by the Core and for transmission of the targeted allele by the Investigator. Tail samples are provided to the investigators, and once transmission has been confirmed, mice will be bred to homozygosity. The Core breeds animals to the homozygote stage, and freezes heterozygote or homozygote embryos if desired. After this time, the mice are transferred from the Core laboratory to the Investigator, and he/she assumes responsibility.

5. Embryo freezing and storage:

For embryo freezing, Investigators indicate which mouse strains they wish to have preserved by embryo freezing. A technician mates the appropriate mice, and obtains and freezes at least 200 embryos per line. 8-32 Cell Embryos are frozen in a Planar Systems Embryo Freezer and stored in Liquid N2. Approximately 1800 vials of mouse embryos are currently stored in facility.

6. Rederivation of lines from either frozen embryos or embryos obtained from non S.P.F. mice:

For embryo freezing, Investigators indicate which mouse strains they wish to have rederived. A technician either mates the appropriate mice, or thaws and introduces the appropriate embryos into pseudopregnant females. For mouse rederivation from dirty mice, one technician is designated to obtain the embryos, and one then transfers the embryos to clean pseudopregnant females. The service is successful as lines of mice frozen over five years ago have been successfully rederived.

7. Timed-mouse pregnancies:

The final technical service conducted by the Core Laboratory is the timed pregnancy program. A number of faculty require mouse embryos at various ages in order to conduct developmental studies. In addition, pregnancy is a crucial time in the natural history of diabetes in view of the tendency for deterioration in glucose control. The Core laboratory makes arrangements with the Investigator to generate mice of a particular strain or construct and provide users with females at the desired stage of pregnancy.