The Core will microinject purified DNA into fertilized embryos harvested from either C57BL/6 x SJL hybrid strain mice or an inbred strain of the investigator's choosing (at an additional cost). Forty or more live offspring from this experiment will be generated. All pups resulting from this experiment will be weaned onto the investigator's IACUC docket, and tail biopsies will be given to the investigator to identify putative founder mice. Following identification, the transgenic founder mice will be turned over to the investigator for subsequent breeding and study. Depending upon the quality of DNA, the Core has an approximate 15% success rate of transgenesis (4 - 8 founder mice per construct).
The Gene Targeting Lab will assist in the design of replacement or insertion vectors for gene targeting in ES cells and has a variety of plasmids available for use in constructing targeting vectors. Upon construction and purification of the targeting vector by the investigator, the Core will electroporate the vector into mouse embryonic stem cells. Utilizing the appropriate drug selection, the Core will isolate and array between 300-400 individual ES cell clones in a 96-well format, and will isolate genomic DNA from the clones. Following identification of the correctly targeted clone(s) by the investigator via PCR of Southern analysis, the Core will expand the clone(s) for further analysis and/or for use in generating chimeric mice. For a description of the time involved for gene targeting, please see timetable.
The Core will microinject ES cells into a minimum of 20 (d3.5) mouse blastocysts in order to generate chimeric mice. This typically results in the production of 3 -7 chimeric mice. The percentage of chimericism and the germline transmission of modfied alleles is partly a function of the quality and pluripotency of the ES cells, thus the Core cannot guarantee germline transmission. Chimeric mice will be weaned onto the investigators docket number, and will be transferred to the investigator to assess germline transmission of coat color alleles derived from the ES cells.
Upon receiving 2-5 fertile male mice of a given genotype, the Core will mate the males with pseudo-ovulated, wt female mice of the appropriate genetic background and will recover compacted morula stage embryos at day 2.5 of gestation for cryopreservation. Between 80 -120 embryos will be vitrified in liquid nitrogen, and a test batch will be thawed and grown in vitro at one week post-vitrification to confirm an embryo viability rate of greater than 50%. The Core will store and maintain the frozen embryos in liquid nitrogen for the investigator, or provide the investigator with the frozen embryos. If maintained properly, the embryos should remain viable indefinitely. Upon request, (and at extra cost) the Core will thaw and implant the frozen embryos into foster host mothers to regenerate the line. Tail biopsies of the resulting mice as well as the mice themselves will be turned over to the investigator upon weaning.
The Core is able to perform mouse sperm isolation and in vitro fertilization (IVF). Most investigators using this Core service do so to overcome infertility problems that occasionally arise in genetically modified mice. In addition, the Core is able to cryopreserve lines of mice by isolating and freezing sperm, and to regenerate hybrid and inbred lines of mice using cryopreserved sperm. Although we have had success using sperm previously frozen by the Core or by select third-party facilities such as the Jackson Laboratory, it should be noted that many other facilities fail to freeze and/or ship cryopreserved sperm properly, and the Core strongly recommends that UMMS investigators wishing to import lines of mice by using frozen germplasm obtain cryopreseved embryos (in lieu of frozen sperm) from all third parties whenever possible. Given the large heterogeneity of the quality of cryopreserved mouse sperm we have observed, the UMMS Core cannot guarantee the successful regeneration of mouse lines from cryopreserved sperm if the UMMS Core did not previously perform the sperm cryopreservation.
For further information, please see notes on sperm cryopreservation & regeneration.
The Core will utilize embryo transfer technology in order to rederive contaminated lines of mice to specific pathogen free (spf) status. Mice placed in quarantine by Animal Medicine will be bred either to each other or to superovulated wildtype female mice. Following harvest of the uterine tissue, the zygotes will be recovered under aseptic conditions and implanted into spf foster host mothers. After birth and weaning, all offspring will be turned over to the investigator. The cost of this service does not include quarantine costs assessed by Animal Medicine.
The Core will perform backcross mating services in order to rederive the genetic background of a line of mice. Mice obtained from the investigator will be crossed to a specified strain of mice by natural mating. Offspring will be weaned onto the investigators' docket number, and tail biopsies will be provided by the Core to the investigator for analysis. Select offspring will be backcrossed to mice of the specified strain. By following this protocol, the starting line of mice will have been successfully transferred onto a new genetic background after 10 generations (time = prox 24 months to completion). A modified, faster version of this procedure utilizing embryo transfer technology is available at a higher fee (time = prox 16 months to completion).
The Core is willing and able to assist investigators with various procedures not listed above, consult with investigators regarding their mouse experiments, and provide limited training of individuals, as long as these services do not interfere with the ability of the Core to fulfill its primary mission of creating genetically modified mice for the UMASS community in a timely and efficient manner.