Identification of immunological relevant phenotypes in ENU
Martin Hrabe´ de Angelis,
Institute for Medical Microbiology, Immunology and Hygiene, Technische Universita¨t Mu¨nchen, Troger Straße 4a, 81675 Mu¨nchen, Germany
Division of Environmental Dermatology and Allergology, Technische Universita¨t Mu¨nchen/GSF Research Center for Environment and Health,
Ingolsta¨dter Landstr. 1, 85764 Neuherberg, Germany
Institute of Molecular Animal Breeding, Gene Center, Feodor-Lynen-Str. 25, 81377 Mu¨nchen, Germany
Institute of Molecular Immunology GSF Research Center for Environment and Health Marchionistr. 25, 81377 Mu¨nchen, Germany
Institute of Mammalian Genetics, GSF Research Center for Environment and Health, Ingolsta¨dter Landstr. 1, 85764 Neuherberg, Germany
Received: 16 December 1999 / Accepted: 16 December 1999
Abstract. The immunology screen focuses on the identification of
novel gene products involved in the mammalian immune response
and on the establishment of mouse models for immunological
disorders. For this purpose, high throughput and semi-automated
techniques were developed and optimized for low cost per sample
and reproducibility. All assays are designed to be nonconsumptive
and are based on peripheral blood or direct PCR amplification.
Despite tremendous progress in the understanding of the immune
system on the basis of gene targeted and transgenic mice (Pfeffer
and Mak 1994; Rajewsky et al. 1996), the genetic basis of the
majority of primary immunodeficiency disorders and autoimmune
diseases remains elusive.
The “phenotype”-driven approach (Brown and Nolan 1998;
Hrabe´ de Angelis and Balling 1998) of the ENU mouse mutagen-
esis screen is expected to detect novel receptors, ligands, modu-
lators, and signaling pathways not discovered with the “gene”-
driven approach. F
offspring and G3 pedigrees of ENU-mutagen-
ized mice (see Hrabe´ de Angelis, this issue) are analyzed in a
nonconsumptive fashion by flow cytometry analysis (FACS) and
enzyme-linked immunosorbance assay (ELISA).
FACS analysis allows the rapid detection of the presence of
immunologically relevant cell populations as well as quantification
of the expression levels of selected cell surface proteins judged
important for immune function.
Employing an ELISA screen for basal immunoglobulin levels
enables the detection of a broad range of mutations which directly
or indirectly interfere with the induction and the maintenance of
the humoral immunocompetence (Sites et al. 1997; Bacharier et al.
1998). In addition, an ELISA-based assay for anti-DNA autoreac-
tive antibodies is included for the detection of autoimmune disor-
Materials and methods
Heparinized peripheral blood (600 l) is centrifuged
for 5 min at 4500 g, and the plasma is recovered and distributed to sub-
sequent screens (Fig. 1). Peripheral blood cells are resuspended in 300 l
PBS, and erythrocytes are removed by incubation in 10 ml lysis buffer (140
Tris, pH 7.2) for 15 min, followed by two washing steps
in FACS buffer (PBS/2% FCS/0.01% NaN
). The nucleated cells of each
sample are distributed into ten 1.4-ml linbro tubes (Integra Biosciences,
Fernwald) pre-racked in a 96-linbro rack. All subsequent pipetting steps are
performed with 12-channel pipettes, minimizing labor and pipetting errors.
Fc receptors are blocked by incubation with 10 g/ml 4G8 rat anti-
mouse Fc receptor for 5 min. After being washed with 300 l FACS buffer,
cells are incubated at 4°C for 20 min with 30 l of the respective antibody
combination. The antibody-labeling panel is flexible and depends on the
interest of the investigators. The current labeling panel consists of a num-
ber of antibody combinations (Table 1).
Correspondence to: H. Flaswinkel Fig. 1. Sample and data handling in the immunology screen.
Mammalian Genome 11, 526–527 (2000).
© Springer-Verlag New York Inc. 2000