This Includes:

• 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

• 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

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

• 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

• 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.
  

• 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.
  

• Pluripotent ES cell lines tested for germline chimera formation (see list of ES cell lines)
• Fetal bovine serum tested for ES cell proliferation with minimal differentiation
• Feeder cells (neor) for ES cell culture (5X10⁶ cells per vial)
• Plasmids for gene targeting, Cre/loxP technology, FLP/FRT technology, reporters, etc. are available.
• Genomic lambda library prepared from 129X1/SvJ mice.
• Genomic DNA isolated from ES cells in 96-well plate format so that you can test your screening method.

Embryonic Stem Cell Culture Training

Participants will learn all techniques necessary to produce and identify embryonic stem (ES) cells that have undergone homologous recombination with a targeting vector. Research papers providing the experimental background underpinning methods used to engineer gene targeted mice will be presented and discussed daily. Laboratory training includes setting up a lab for ES cell culture, media and reagent preparation, normal ES cell culture, preparation of feeder cells, freezing and thawing of ES cells, identification of differentiated and undifferentiated ES cell morphologies, ES cell electroporation, picking ES cell clones, DNA preparation from ES cell clones in 96 well plates, and counting chromosomes in ES cell spreads.  Download a Syllabus.

Pronuclear Microinjection Training

Participants will learn all techniques necessary to produce transgenic mice. This includes superovulation, glass microinstrument fabrication, egg collection, identification of pseudopregnant females, surgical transfer of eggs to pseudopregnant recipients, and microinjection of DNA into the pronuclei of fertilized mouse eggs. A majority of trainees produce transgenic mice during training. Download a Syllabus.

Rat transgenic production training is also available. Download a Syllabus.

Blastocyst Microinjection

Participants will learn all techniques necessary to produce embryonic stem cell-mouse chimeras. This includes superovulation, glass microinstrument fabrication, blastocyst collection, identification of pseudopregnant females, surgical transfer of blastocysts to pseudopregnant recipients, and microinjection of ES cells into the blastocoels of C57BL/6 blastocysts. A minority of trainees produce chimeric mice during training. Download a Syllabus.

Mouse Embryo Transfer

Participants will learn all techniques necessary to transfer mouse embryos to the reproductive tract of recipients. This includes superovulation, isolation of pathogen free fertilized eggs from infected stocks, transfer pipette fabrication, identification of pseudopregnant females, surgical transfer of eggs to pseudopregnant recipients. All trainees produce offspring from transferred eggs during training.

Consult on design and construction of transgenes and targeting vectors; identification of transgenic founders; and breeding.

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

• 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 University Committee on the Care and Use of Animals.