- Animal Modeling
- Gene Targeting & Stem Cells
- Service Forms
- Links Protocols Info
The Animal Modeling Facility of the TAMC will microinject purified DNA (purified plasmid or BAC) or RNA into fertilized oocytes of hybrid BL/6 x SJL strain mice, SD rats, or of an inbred mouse or rat strain chosen by the Investigator. The injected oocytes will be transferred into foster dams, resulting in the generation of thirty-five or more live mice or 20-25 live rats. Frozen tail biopsies of all weaned pups will be provided to the investigator to identify founder mice or rats incorporating the transgene. Following their identification, all transgenic animals will be turned over to the investigator for subsequent breeding and study. Depending upon the quality of the nucleic acid, the Core has an approximate 15-20% success rate of transgenesis in all tested strains of mice and rats (regardless of construct size), and most projects generate between 5 - 7 founder animals. The procedures, methodology, and associated costs are highly similar for standard DNA constructs, BAC transgenes, and for transgenesis mediated by retroviruses, lentiviruses, transposons, and nuclease targeting procedures. For further information on mouse and rat strains commonly used by the Core in these procedures, see rodent information. For information on pricing, please see service charges. For in-house ordering of transgenic mice or rats, please see service forms. Third parties, please contact TAMC for further information and to arrange ordering.
The Animal Modeling Facility of the TAMC has injected Zn-finger mRNA into fertilized oocytes of mice or rats to generate mutant alleles for UMMS Investigators. The requisite steps are very similar to those used in performing standard transgenesis in rodents. In addition, the TAMC has worked closely with the UMMS Mutagenesis Core to facilitate the use of TALENS and CRISPR/Cas9 nucleases to site-direct mutations (including recombination to generate conditional and knock-in alleles) into mice and rats. We have successfully undertaken 8 different nuclease-targeting projects in the past 10 months. As with standard pronuclear injections, the costs for these procedures are strain and species-dependent (see service charges). In addition, the Gene Targeting & Stem Cell Facility of the TAMC offers functional testing of nucleases (see below). Further information regarding nuclease concentrations can be obtained here, or by contacting the TAMC. For in-house orders, please see service forms. Third parties, please contact TAMC for further information and to arrange ordering.
The Animal Modeling Facility of the TAMC will microinject ES cells into a minimum of 20 (E3.5) mouse diploid 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. However, our success rate for a project when injecting two individual clones is 100% for chimericism, and 94% for subsequent germline transmission. Chimeric mice will be transferred to the Investigator for breeding to assess germline transmission of coat color alleles derived from the ES cells. Upon request, the TAMC can also perfrom ES cell injections into tetraploid embryos to assess mouse ES cell or iPS cell pluripotency. Typically, the injected embryos are implanted into foster moms, which are transferred over to the Investigator prior to delivery of the pups. In some instances and with previous notice, the TAMC can assist in the recovery of such transient transgenics or chimerics. For in-house orders, please see service charges and service forms. Third parties, please contact TAMC for further information and to arrange ordering.
The TAMC can assist Investigators in growing orthotopic or ectopic tumors by transplanting transformed cells subcutaneously, or into the mammary fat pad or testis capsule of Nude-Scid or NSG mice. Typically, cells are provided to the Gene Targeting & Stem Cell Facility of the TAMC for requisite MAP testing prior to transplantation. Cells are then expanded, transplanted by the Animal Modeling Facility into mice, and the mice turned over to the Investigator. Alternatively, the mice remain in the TAMC and tumors are harvested and fixed by the Animal Modeling Facility, with a portion of each tumor flash-frozen. Fixed samples and frozen tissues are then given to the Investigator. For in-house orders, please see service charges and service forms. Third parties, please contact TAMC for further information and to arrange ordering.
The TAMC will utilize embryo transfer technology in order to rederive contaminated lines of mice to specific pathogen free (spf) status. Mice placed in quarantine by UMMS Animal Medicine Department will be bred either to each other or to super-ovulated 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. For in-house orders, please see service charges and service forms. Third parties, please contact TAMC for further information and to arrange ordering.
Upon receiving 2-4 fertile male mice or rats of a given genotype, the Animal Modeling Facility of the TAMC will mate the males with pseudo-ovulated, wildtype female rodents 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 in cryo-straws. A test straw will be sampled one-week post-vitrification to confirm an embryo viability rate of greater than 50%. The TAMC 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, and the TAMC has successfully thawed mouse embryos cryo-preserved by the TAMC after 12 years in storage. Upon request, (and at extra cost) the TAMC will thaw and implant the frozen embryos into foster host mothers to regenerate the line. Tail biopsies of the resulting mice or rats and those rodents scoring positive for the gene of interest will be turned over to the Investigator upon weaning. For in-house orders, please see service charges and service forms. Third parties, please contact TAMC for further information and to arrange ordering.
The Animal Modeling Facility of the TAMC offers mouse or rat 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 rodents. In addition, the TAMC is able to cryopreserve lines of rodents 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 TAMC or by select third-party facilities such as the Jackson Laboratory, it should be noted that some external facilities fail to freeze and/or ship cryopreserved sperm properly. Thus, the TAMC strongly recommends that UMMS investigators wishing to import lines of mice by using frozen germplasm obtain cryopreseved embryos (in lieu of frozen sperm) from 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 frozen sperm if the UMMS Core did not previously perform the sperm cryopreservation. For in-house orders, please see service charges and service forms. Third parties, please contact TAMC for further information and to arrange ordering.
For further information, please see notes on sperm cryopreservation & regeneration.