Transgenic Core

Transgenic Mouse Production

  • Three transgenic mice are guaranteed.
  • Mice are produced in the (C57BL/6 X SJL)F2 genetic background.
  • Other genetic backgrounds may be specified by investigators (additional cost).
  • Large DNAs (BACs, YACs, P1s) may be specified at no additional cost.

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Transgenic RAT Production

  • Three transgenic rats are guaranteed.
  • Rats are produced in the Sprague-Dawley genetic background  (Crl:CD (SD)IGS BR).
  • Other genetic backgrounds may be specified by investigators (additional cost).
  • Large DNAs (BACs, YACs, P1s) may be specified at no additional cost.

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Gene Targeting in Mouse ES Cells

Purpose: to produce embryonic stem (ES) cells with mutations induced by homologous recombination with a targeting vector.

Phase I:

The investigator demonstrates a sensitive screen for homologous recombination and clones the targeting vector. Core staff electroporate ES cells, isolate clones, and prepare DNA from clones.

Phase II:

The investigator screens ES cell DNA and identifies recombinants. Core staff expand ES clones from frozen stocks, count chromosomes, and the investigator confirms gene targeting in thawed ES clones.

Phase III:

The investigator and Core staff select ES cell clones for recombination. Core staff microinject ES cells into C57BL/6 blastocysts to produce chimeras.

Phase IV:

The investigator breeds the chimeras to obtain germline transmission and analyzes the resulting animals.

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ES Cell-Mouse Chimera Production

  • Microinjection of embryonic stem (ES) cells into C57BL/6 blastocysts.
  • Microinjection of C57BL/6 derived ES cells into albino C57BL/6 blastoycsts.
  • We guarantee to inject 60 blastocysts with each ES cell clone.
  • The production of germline chimeras depends on the quality of the ES cells.

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De Novo Mouse ES Cell Line Derivation

  • Preparation of ES cell lines from mouse blastocysts.
  • We will collect blastocysts from your genetically engineered mouse model and culture them under conditions that are conducive to establishing ES cell lines.
  • The number of ES cell lines produced is propotional to the number blastoycsts available for culture.
  • The success of the Transgenic Core in de novo ES cell line derivation meets or exceeds published standards.

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.Bacterial Artificial Chromosome Recombineering

  • Acquistion of BACs from BAC library resources.
  • Purification of BAC DNA suitable for pronuclear microinjection (transgenic mice or transgenic rats).
  • Pulsed field gel mapping of BAC DNA to confirm identiy of BAC.
  • Genetic modification of BACs by recombineering approaches to produce gene knockins, knockouts.
  • Prepareation of gene targeting vectors for the modifcation of genes in embyronic stem cells.
  • Discuss structure of the desired BAC with the investigator and propose a recombineering path.
  • Plasmids for knockins of fluorescent reporter molecules and Cre recombinase are available.
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Reagents

Access to cell lines and plasmids is only provided to investigators who have complete, approved material transfer agreements.

Other Services

  • Space in the ES cell lab can be provided to labs that want to produce targeted ES cells.
  • Microinjection workstations are available for labs that want to do their own injections.
  • Adherent cell microinjection workstation equipped with Eppendorf Injectman System for Microinjection of Adherent Cells. This consists of a Model 5179 Micromanipulator and a FemtoJet 5247 Microinjector.
  • Conversion of Mice to Specific Pathogen Free Status by embryo transfer.
  • Mouse strain cryopreservation.
  • Recovery of Cryopreserved Mouse Embryos.
  • Consulting on transgene design, targeting vector design, and experimental plan.
  • Specialized research projects that take advantage of our equipment and expertise such as intraplacental microinjection.

Provisos

  • We guarantee the production of transgenic mice, but can not guarantee that any given transgene will be expressed in transgenic mice.
  • We guarantee a minimum number of blastocysts will be injected with ES cells, but can not guarantee that any given ES cell clone will form germline mouse chimeras.
  • We guarantee that the gene targeting reagents are germline competent.
  • The Genetically engineered animals provided by this facility can only be used for research purposes.
  • Access to cell lines and plasmids is only provided to investigators who have complete, approved material transfer agreements.
  • Investigators must have approval to use animals in research and teaching from the Institutional Animal Care & Use Committee.