Gene Targeting and Transgenic Core
Director
Lin Gan, Ph.D.
Mission Statement
The mission of the Gene Targeting and Transgenic Core is to produce genetically modified mice for the investigators in the University of Rochester in a timely and cost-effective manner. The Core will also provide consultations on gene modification projects and a full range of services including animal breeding, traditional and speed congenics (backcross), genotyping analysis, rederivation, and cryopreservation.
Core Services Provided
Gene Targeting in Mouse ES cells
The Core will assist in the design and construction of gene targeting vectors. Upon the construction and purification of the targeting vector, the Core will electroporate the vector into mouse 129S6 (129SvEv) embryonic stem (ES) cells. After the appropriate drug selection, the Core will array approximate 192 individual ES cell clones in the 96-well format and will isolate genomic DNA from the clones. Following Southern blotting identification of the correctly targeted clones, the Core will expand and freeze the clones for further analysis and/or for generating chimeric mice.
Production of Chimeric Mice Using ES cells
The Core will microinject ES cells into mouse blastocysts in order to generate chimeric mice. Chimeric mice will be weaned onto the investigators UCAR protocol and will be transferred to the investigator to assess germline transmission of coat color alleles derived from the ES cells. Typically, a minimal of 50 C57BL/6 blastocysts will be injected or 5 chimeric mice will be delivered. The percentage of chimericism and the germline transmission depend on the quality and pluripotency of the ES cells as well as the effect of genetically modified alleles. Thus, the Core cannot guarantee germline transmission.
Transgenic Mice
The Core will microinject purified DNA into fertilized embryos from either C57BL/6 x SJL hybrid strain or an inbred strain of the investigator's choosing. All pups generated will be weaned onto the investigator's UCAR protocol and tail biopsies will be given to the investigator to identify putative founder mice. Following identification, the founder mice will be turned over to the investigator for subsequent breeding and study. A minimal of 150 embryos will be injected or 3 transgenic founder mice will be delivered per DNA construct.
Mouse Embryo and Sperm Cryopreservation
The Core will mate the male mice provided by the investigator with wild type female mice of the appropriate genetic background and will recover compacted morula stage embryos for cryopreservation. Between 80-100 embryos will be frozen in liquid nitrogen, and a test batch will be thawed and grown in vitro at one week post-cryoperservation 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. Upon request, the Core will thaw and implant the frozen embryos into foster host mothers to regenerate the line. The resulting mice will be transferred to the investigator upon weaning. The Core will also to perform sperm cryopreservation and in vitro fertilization of mice without a guarantee. This service will be provided in order to overcome infertility problems associated with certain genetically modified mice.
Rederivation of Mice to Obtain Specific Pathogen Free (SPF) Status
The Core will use the embryo transfer approach to rederive contaminated mouse lines to SPF status. Mice placed in quarantine will be bred either to each other or to wild type mice. The blastocysts will be harvested under aseptic conditions and implanted into SPF foster host mothers. After birth and weaning, all offspring will be genotyped and transferred to the investigator.
Mice Congenics (Backcross)
The Core will provide backcrossing services to rederive a genetically modified mouse line in a selected genetic background. Mice obtained from the investigators will be crossed to a specified mouse strain by natural mating. Offspring will be weaned and genotyped. Select offspring will be backcrossed to the specified mouse strain. By following this protocol, the starting line of mice will have been successfully transferred onto a new genetic background after 10 generations. A modified, faster version of this procedure utilizing the Marker Assisted Accelerated Backcrossing method is available at a higher fee to rederive the mice onto a new genetic background after 5 generations.
Other services
In addition to the services listed above, the Core will 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 generating genetically modified mice in a timely and efficient manner. The Core will also provide common mouse strains, timed pregnant female mice for staged embryo harvesting, and Cre- and Flp-expressing mouse strains for generating conditional activation or deletion mice.
Specific Instrumentation
The Core consists of a barrier facility for procedures involving production and maintenance of genetically modified mouse strains, a tissue culture facility for ES cells, and a molecular laboratory for generating gene targeting constructs and genotyping experiments. The Core is equipped to perform all procedures involving production and maintenance of genetically modified mice. Instrumentation includes two injection workstations, Nikon stereoscope, CO2-incubator, laminar flow hood for surgical procedures, liquid nitrogen tanks and a freezer for cryopreservation.
Faculty User Group
The following faculty members have been invited to serve on a faculty user group for this core:
Rulang Jiang
Joe Miano
Hucky Land
Andrea Bottaro
Kerry O'Banion
