Transgenic and Embryonic Stem Cell Core Services :: The Research Institute at Nationwide Children's Hospital

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Transgenic and Embryonic Stem Cell Services

  • ES Cell Targeting In Vitro
  • Chimeras for germline transmission of targeted ES cells
  • Conditional Knockout Mice (Cre or FLP Electroporation)
  • Embryonic Stem Cell Line Derivation
  • Pronuclear Injection
  • Rederivation
  • Inter-Vivarium Transfer
  • Embryo Cryopreservation
  • Sperm Cryopreservation
  • Training


ES Cell Targeting In Vitro

This service produces embryonic stem cells (ES) cell lines with mutations introduced by homologous recombination using a targeting vector provided by the investigator. The targeted ES cells can be used for in vitro assays and to generate germline ES cell-mouse chimeras that can be bred to generate mouse lines. We require the construct DNA prepared for electroporation and will return selected clones for genotyping. We have several standard ES cell lines that can be used for targeting that have been tested for germ line transmission in our facility.

Chimeras for germline transmission of targeted ES cells

The Core performs microinjection of blastocysts with targeted ES cells prepared by the core, or in the investigator's laboratory. Typically, a minimum of 48 blastocysts will be injected with up to two clones. The Core will purchase the required mice and pay for their housing, and also provides females (mated to vasectomized males) for foster mothers. Blastocysts for ES cell injection are obtained by mating C57BL/6 female mice with C57BL/6 male mice, but blastocysts of other mouse strains can be provided upon request. Embryos will be collected, injected with ES cells, and transferred to pseudo pregnant recipients.

The core can also perform tetraploid aggregation and most other chimera approaches used to manipulate lineage contribution. By using tetraploid aggregation the bias to embryonic lineages can be directed. Alternatively, asynchronous aggregation of different stage diploid embryos can be used to bias embryonic versus extraembryonic contribution. The injection of ES cells into morula stage embryos can also increase the contribution of the ES cell component using fewer cells.

Conditional Knockout Mice (Cre or FLP Electroporation)

Investigators can choose to make a conditional knockout line of mice when tissue- or developmental stage specific mutations need to be studied in vivo. As required for some construct designs the Core can perform a second, transient transfection with a plasmid containing the gene for the Cre or Flp recombinase under the control of a ubiquitously expressed promoter. For in vivo removal of the conditional cassette, several strains of mice with Cre or Flp recombinase under the control of selected promoters are available in the Core.

Embryonic Stem Cell Line Derivation

Embryonic stem cells can be derived from several strains including 129 variants, C57Bl6, CBA and various hybrid strains. This approach is useful for generating homozygous ES cell lines from mutant mouse strains with embryonic lethality, as an alternative to performing second targeting events. Typically we require 5-10 breeding age females to produce several male ES cell lines with a normal chromosome number.

Pronuclear Injection

The Transgenic Core assists investigators with the production of transgenic mice whereby additional DNA of interest is integrated (usually randomly) to the mouse genome. The core injects sufficient zygotes to generate enough offspring to produce at least 3 transgenic (not necessarily expressing) founder animals

Rederivation

This service makes it possible for an investigator to rederive mice from unclean sources into the vivarium. The investigator will provide the core with one or both gender animals of the appropriate breeding age of the desired strain. The core will super-ovulate the females of interest and mate them to the males or use natural matings if appropriate. For normally breeding mouse strains the core will produce offspring sufficient for at least one mating pair.

Inter-Vivarium Transfer

As part of the process to move or quickly establish a colony within a facility we can transport and embryos rather than mice, to and from other vivariums. This facilitates not only minimal quarantine requirements but can be used to rapidly expand a new genotype colony. The core staff can perform the collection and embryo implantations at both local and source/destination vivariums.

Embryo Cryopreservation

The Core offers cryopreservation of cleavage-stage embryos on these backgrounds: C57BL/6J, FVB/NJ, DBA/2J, BALBcByJ, and NOD/ShiltJ. The core will freeze up to ten vials, each sufficient to produce one litter. One vial will be thawed after freezing to confirm viability. For normally fertile animals, we require five males (preferably not more than 16 weeks old) from your colony, and the same number of four week old females. For cryopreservation of heterzygous embryos, either male or female mice are required along with a preference of the strain which to mate with.

Sperm Cryopreservation

The protocol associated with the recently available JAX kit for sperm cryopreservation now permits male germline preservation of several strains not previously recommended. With purchase of one JAX kit, three mouse strains can be preserved and sperm stored at JAX for three years at the initial charge. Rederivation can be performed either by JAX or by the transgenic core. Several pricing incentives are provided for volume freezing. We are also developing sperm freezing coupled with in vitro fertilization independently to help reduce costs for investigators and reduce vivarium usage.

Nationwide Children's Hospital
700 Children's Drive Columbus, Ohio 43205 614.722.2000