Immunology, DRTC - Washington University

DRTC Washington University Immunology :

Immunology Core

The Immunology Core consists of two branches, a transgenic facility and a diabetic mice component.

Transgenic Component - Instructs and helps investigators in the production of transgenic mouse strains. Instructions are given for the preparation of DNA. Technicians are responsible for the injection and production of the transgenic mice. The facility is also capable of producing mice with ablation of genes by homologous recombination. (details)

Animal Component - The facility maintains a standardized colony of NOD mice which it supplies to investigators carrying out research in the immunology of insulin-dependent diabetes mellitus. The facility also maintains NOD mice on the SCID background. Such mice are used for transplantation of genetic strains, including gene null mice. Finally, the facility maintains SCID colony in the H-2d background (CH.17 SCID) which is used by investigators for islet transplantation experiments. (details)

Services :

Transgenic Component:

Services provided: The four genetic manipulations that the core provides include:

1) the creation of transgenic lines. The investigator prepares the DNA construct following advice given by the Director and the Technical Director, Michael White; the facility proceeds with the injections into the pronuclei of fertilized eggs, which are then planted into pseudopregnant females, prepared at the facility.

2) the targeted mutation of a gene by homologous recombination. The investigator produces the gene construct, which is then transfected into ES cells maintained by the Core. The facility is in charge of the injection into blastocysts and their implantation into pseudopregnant females. ES lines are derived from Bl/6 and 129 mice. An ES line derived from NOD has just been obtained from T. Watanabe at Kysuhu University; we are currently examining this cell line which will be extremely useful for investigations using the diabetogenic NOD mice. The targeted mutation approaches have now been extended to use gene constructs containing lox P sites and deleting it by expression of the Cre recombinase. This method allows us to generate genetically pure mutant mouse lines lacking the Neomycin-resistant gene used for selection of transfectants. Furthermore, this method combined with tissue specific expression of Cre gene allows us to generate mouse lines that lack the targeted gene function in a tissue-specific fashion.

3) the generation of mice using the blastocyst complementation procedure. These are particularly useful in studies on lymphocytes; ES cells are produced containing a mutated gene and are then injected into blastocysts derived from RAG-2 deficient mice, which do not generate lymphocytes.

4) cryopreservation of embryos. A protocol has been developed using a control rate freezer: we harvest morulas at day 2.5, and place 20-40 of them into Nunc cryo tubes mixing them with Gibco's Freezing media and DMSO.

In brief, the staff is responsible for the entire process described above, from the time it receives the DNA preparation to the production of the mice. The protocols for isolation of the eggs, their injection and reimplantation have been standardized and now followed for several years. The staff also helps the faculty with the analysis of the lines and the identification of the transgenic founders.

Personnel, management, facilities and space: The transgenic facility is directed by Robert Schreiber, a senior outstanding immunologist with extensive experience in cytokine research. He has overseen the operation of the facility since its inception 7 years ago. It is part of a larger facility run jointly by the Departments of Pathology and Immunology and Medicine. The technical coordinator is Michael White. He has extensive experience, having operated our facility since its inception seven years ago. He has been responsible for producing the 163 unique transgenic mouse lines and 50 targeted mutants in the facility. We also have a technician who helps in the microinjection (Kara Parton), and another responsible for the mouse work (Darren Kreamalmayer). The Genetics facility is located in the Specialized Research Animal Facility, a building of about 95.000 NSF of space recently constructed to hold inbred mice. This is a pathogen-free, barrier facility, AALAC accredited. We have a laboratory of 400NSF dedicated to the Genetics Core.

Contact Information: The transgenic facility director, Robert Schreiber, may be contacted at: phone: (314)-362-8747, fax: (314) 747-4888, and e-mail: Schreiber@immunology.wustl.edu.

Animal Component:

Personnel: The diabetic mice component is staffed by Osami Kanagawa and Katherine Frederick. The director, Dr. Kanagawa, has extensive experience in mouse genetics through his research on NOD diabetes and in T cell tolerance. Katherine Frederick has twenty years of experience in mouse breeding. You may contact Dr. Kanagawa at: email: kanagawa@pathology.wustl.edu, phone: (314) 362-8679, fax: (314) 362-4096.

List of mice available (italicized are new lines being generated):

NOD.SCID: NOD mouse lacking T and B lymphocytes due to the SCID mutation. Has been used extensively as a recipient for transfer of lymphocytes. The line was originally obtained from E. Leiter at Jackson Labs.

IFN-gamma receptor negative NOD: The line was produced here by Robert Schreiber and backcrossed to NOD for 15 generations. Has been used extensively by two of us, OK and RDS, to study role of IFN-gamma in autoimmunity. A new line of Stat-1 knockout mice is being backcrossed to NOD.

IFN-gamma negative NOD: The line was obtained from the Jackson Laboratory. Has been used in combination with IFN-gamma receptor negative mouse to study role of IFN-gamma in autoimmunity.

IL-4 negative NOD: The line was obtained from Dr. M. Kopf and backcrossed to NOD for 15 generations. Has been used to study role of IL-4 and Th2 T cells in autoimmunity.

I-Ea transgenic NOD: NOD mouse expressing class II I-E molecule was originally obtained from the Jackson Laboratory. Has been used to study the protective effect of non I-Ag7 class II MHC in NOD diabetes.

BDC 2.5 TCR transgenic NOD: There are two TCR transgenic mice that we maintain: i) the BDC 2.5 line was created by Mathis and Benoist. We have the TCR expressed in NOD. Kanagawa has recently made a specific monoclonal antibody to the TCR, to use for marking the cells and for biological studies; ii) a new TCR for a diabetogenic CD8 T cells was created and also maintained in NOD mice. The BDC TCR transgenic NOD on the SCID mutation has also been established in our mouse colony. The original TCR transgenic mice do not develop diabetes despite harboring a large number of potential pathogenic T cells due to the specific inhibition of disease development by regulatory T cells expressing endogenous TCR.

9.33 TcR transgenic NOD: NOD strain expressing islet antigen specific and class I MHC restricted TcR transgene. This line was developed by Kanagawa in our facility. It has been used to study role of class I MHC restricted cytolytic T cells in diabetes development.

NOD.GD: NOD line expressing I-Ad class II molecule instead of diabetogenic I-Ag7. The B10.GD mice was obtained from the laboratory of Ted Hansen and backcrossed to NOD for 13 generations. Has been used to study protective effect of non I-Ag7 class II MHC in NOD diabetes.

B6.g7: C57BL mouse carrying H-2g7 MHC. Has been used to study role of non-MHC genes in the regulation of diabetes development. (Line was originally obtained from E. Leiter at Jackson Labs.)

BALB/g7PD: BALB/c mouse expressing mutant I-Ag7. This line was established in our transgenic core facility. Has been used to study protective effect of non I-Ag7 class II MHC in NOD diabetes.)

NOD g7/PD: NOD strain expressing both wild type and mutant I-Ag7 class II MHC molecules. Has been used to study protective effect of non I-Ag7 class II MHC in NOD diabetes. The line was originally obtained from H.O. McDevitt at Stanford University).

RIP-HEL B10.BR: A new series of transgenic mice expressing the well-known protein Hen Egg-White Lysozyme (HEL) under the Rat Insulin Promoter (RIP) control. The first RIP-HEL was obtained from Christopher Goodnow: it expresses about 106 molecules of a membrane-form of HEL per beta cell. The RIP-HEL is on the B10.BR, i.e. (H-2k) background. Bred to a TCR for an HEL epitope, it develops diabetes. New lines are now being generated, including one with a cytosolic form of HEL; a new genetic construct is being prepared in which the HEL gene is flanked by lox sites; this will represent an attempt to regulate the amounts of HEL expressed after addition of the Cre recombinase.

Removable RIP-SV40 TG B6: A line now being developed by the Kanagawa laboratory--B6 mice carrying RIP driven SV40 Tag flanked with two lox-P sites. This allows us to remove the oncogenic capacity of pancreatic beta cell tumor by Cre/Lox-P mediated deletion of the transgene. After deletion of the transgene, the insulinomas and the cell lines derived from them can be used for transplantation to treat diabetic mice.

People :

Robert D. Schreiber, Ph.D. : Co-Investigator; Immunology Core, . Phone: 314-362-8747

Emil R. Unanue, MD : Director, Immunology Core, Campus Box 8108. Phone: 314-362-7440