NGFN-PLUS
Steroid Screen
| Coordinator: | Prof. Dr. Jerzy Adamski | |
| Institution: | Institut für Experimentelle Genetik, Helmholtz Zentrum München | |
| Homepage: | www.helmholtz-muenchen.de/en/ieg/group-molecular-endocrinology/index.html |
Steroids control the differentiation and proliferation processes of cells and tissues. They further participate in the regulation of apoptosis and neuroregeneration. Defects in steroid metabolism contribute as well to the pathogenesis of many different complex diseases like cancer, polycystic ovary syndrome, diseases of cartilage and bone or neurological diseases. Other hereditary diseases are caused by disorders in biosynthesis of the steroid precursor cholesterol (e.g. CHILD syndrome, Chondrodysplasia punctata and Smith-Lemli-Opitz syndrome). Additionally, corticosterone is an important marker for stress and for metabolic disturbances such as diabetes.
Up to now, only few animal models for the study of steroid-related disorders exist. The main focus of the steroid screen is the identification of new animal models for human steroid-related diseases therewith supporting the development of their future medical treatment.
We intend to detect mutant mouse lines with altered steroid metabolism in the primary screen. To accomplish this we focus on the detection of aberrations in the steroid concentrations in plasma of the mice. Adult male and female mice are screened for alterations in plasma steroid concentrations. Blood is collected retro bulbar from narcotized mice and plasma prepared by centrifugation.
The key steroids corticosterone, testosterone and andreostenedione are extracted from plasma and quantified by mass spectrometric methods. Mice with dwarfism, cataracts, skin defects, limb malformations and distorted reproduction biology or behaviour are the most interesting candidates for the screen since such phenotypic defects are observed in human steroid-related diseases.
Figure 2: Chromatogram of the key steroids
In our assay, the steroids corticosterone, testosterone, and androstenedione are quantified simultaneous by high-throughput HPLC-MS/MS technology in 50 microliter of mouse plasma (Haller et al, 2010).
Figure 3: HPLC-MS/MS technology in the GAC
We provide an option for secondary screen: Quantification of 186 endogenous metabolites (amino acids, hexose, glycerophospholipides, sphingomyelins and acylcarnitines) out of 10 microliter mouse plasma at the Metabolomic Platform of the Genome Analysis Center (GAC, http://www.gac-munich.de). An assays for dehydroepiandrosterone (DHEA) can be further applied by ELISA on request for mouse plasma.
Selected published results:
Testosterone levels have been decreased in male Srgap3-/- mice, but the phenotype was not associated with the morphological changes in the mutant mice. Results habe been published in FASEB (Waltereit et al, 2012).
Testosterone levels have been reduced in male mice with inflammatory arthritis, results have been published in Arthritis Rheum (Abe et al, 2011).
Results of line Cox4 (no change in DHEA and testosterone) have been published in FASEB (Huttemann et al, 2012).
Genotype specific changed corticosterone concentrations have been found the primary screening of the lines HMGN1, 3 and 5. The manuscript have been published in Nucleic Acids Research (Kugler et al, 2013).
Primary screening results of line ADAR2 – no change in DHEA and testosterone – have been published (Horsch et al, 2011).
Literatur/References:
Abe K, Fuchs H, Boersma A, Hans W, Yu P, Kalaydjiev S, Klaften M, Adler T, Calzada-Wack J, Mossbrugger I, Rathkolb B, Rozman J, Prehn C, Maraslioglu M, Kametani Y, Shimada S, Adamski J, Busch DH, Esposito I, Klingenspor M, Wolf E, Wurst W, Gailus-Durner V, Katan M, Marschall S, Soewarto D, Wagner S, de Angelis MH (2011) A novel N-ethyl-N-nitrosourea-induced mutation in phospholipase Cgamma2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model. Arthritis Rheum 63: 1301-1311
Haller F, Prehn C, Adamski J (2010) Quantification of steroids in human and mouse plasma using online solid phase extraction coupled to liquid chromatography tandem mass spectrometry Nature Protocols 10.1038/nprot.2010.22
Horsch M, Seeburg PH, Adler T, Aguilar-Pimentel JA, Becker L, Calzada-Wack J, Garrett L, Gotz A, Hans W, Higuchi M, Holter SM, Naton B, Prehn C, Puk O, Racz I, Rathkolb B, Rozman J, Schrewe A, Adamski J, Busch DH, Esposito I, Graw J, Ivandic B, Klingenspor M, Klopstock T, Mempel M, Ollert M, Schulz H, Wolf E, Wurst W, Zimmer A, Gailus-Durner V, Fuchs H, de Angelis MH, Beckers J (2011) Requirement of the RNA-editing Enzyme ADAR2 for Normal Physiology in Mice. J Biol Chem 286: 18614-18622
Huttemann M, Lee I, Gao X, Pecina P, Pecinova A, Liu J, Aras S, Sommer N, Sanderson TH, Tost M, Neff F, Aguilar-Pimentel JA, Becker L, Naton B, Rathkolb B, Rozman J, Favor J, Hans W, Prehn C, Puk O, Schrewe A, Sun M, Hofler H, Adamski J, Bekeredjian R, Graw J, Adler T, Busch DH, Klingenspor M, Klopstock T, Ollert M, Wolf E, Fuchs H, Gailus-Durner V, Hrabe de Angelis M, Weissmann N, Doan JW, Bassett DJ, Grossman LI (2012) Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology. FASEB J 26: 3916-3930
Kugler JE, Horsch M, Huang D, Furusawa T, Rochman M, Garrett L, Becker L, Bohla A, Holter SM, Prehn C, Rathkolb B, Racz I, Aguilar-Pimentel JA, Adler T, Adamski J, Beckers J, Busch DH, Eickelberg O, Klopstock T, Ollert M, Stoger T, Wolf E, Wurst W, Yildirim AO, Zimmer A, Gailus-Durner V, Fuchs H, Hrabe de Angelis M, Garfinkel B, Orly J, Ovcharenko I, Bustin M (2013) High Mobility Group N Proteins Modulate the Fidelity of the Cellular Transcriptional Profile in a Tissue- and Variant-specific Manner. J Biol Chem 288: 16690-16703
Waltereit R, Leimer U, von Bohlen Und Halbach O, Panke J, Holter SM, Garrett L, Wittig K, Schneider M, Schmitt C, Calzada-Wack J, Neff F, Becker L, Prehn C, Kutscherjawy S, Endris V, Bacon C, Fuchs H, Gailus-Durner V, Berger S, Schonig K, Adamski J, Klopstock T, Esposito I, Wurst W, de Angelis MH, Rappold G, Wieland T, Bartsch D (2012) Srgap3(-)/(-) mice present a neurodevelopmental disorder with schizophrenia-related intermediate phenotypes. FASEB J 26: 4418-4428
Additional relevant Internet links:
The German Mouse Clinic
Helmholtz Zentrum München
Further Coordinators:
Up to now, only few animal models for the study of steroid-related disorders exist. The main focus of the steroid screen is the identification of new animal models for human steroid-related diseases therewith supporting the development of their future medical treatment.
Figure 1: Quantified key steroids
We intend to detect mutant mouse lines with altered steroid metabolism in the primary screen. To accomplish this we focus on the detection of aberrations in the steroid concentrations in plasma of the mice. Adult male and female mice are screened for alterations in plasma steroid concentrations. Blood is collected retro bulbar from narcotized mice and plasma prepared by centrifugation.
The key steroids corticosterone, testosterone and andreostenedione are extracted from plasma and quantified by mass spectrometric methods. Mice with dwarfism, cataracts, skin defects, limb malformations and distorted reproduction biology or behaviour are the most interesting candidates for the screen since such phenotypic defects are observed in human steroid-related diseases.
Figure 2: Chromatogram of the key steroids
In our assay, the steroids corticosterone, testosterone, and androstenedione are quantified simultaneous by high-throughput HPLC-MS/MS technology in 50 microliter of mouse plasma (Haller et al, 2010).
Figure 3: HPLC-MS/MS technology in the GAC
We provide an option for secondary screen: Quantification of 186 endogenous metabolites (amino acids, hexose, glycerophospholipides, sphingomyelins and acylcarnitines) out of 10 microliter mouse plasma at the Metabolomic Platform of the Genome Analysis Center (GAC, http://www.gac-munich.de). An assays for dehydroepiandrosterone (DHEA) can be further applied by ELISA on request for mouse plasma.
Selected published results:
Testosterone levels have been decreased in male Srgap3-/- mice, but the phenotype was not associated with the morphological changes in the mutant mice. Results habe been published in FASEB (Waltereit et al, 2012).
Testosterone levels have been reduced in male mice with inflammatory arthritis, results have been published in Arthritis Rheum (Abe et al, 2011).
Results of line Cox4 (no change in DHEA and testosterone) have been published in FASEB (Huttemann et al, 2012).
Genotype specific changed corticosterone concentrations have been found the primary screening of the lines HMGN1, 3 and 5. The manuscript have been published in Nucleic Acids Research (Kugler et al, 2013).
Primary screening results of line ADAR2 – no change in DHEA and testosterone – have been published (Horsch et al, 2011).
Literatur/References:
Abe K, Fuchs H, Boersma A, Hans W, Yu P, Kalaydjiev S, Klaften M, Adler T, Calzada-Wack J, Mossbrugger I, Rathkolb B, Rozman J, Prehn C, Maraslioglu M, Kametani Y, Shimada S, Adamski J, Busch DH, Esposito I, Klingenspor M, Wolf E, Wurst W, Gailus-Durner V, Katan M, Marschall S, Soewarto D, Wagner S, de Angelis MH (2011) A novel N-ethyl-N-nitrosourea-induced mutation in phospholipase Cgamma2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model. Arthritis Rheum 63: 1301-1311
Haller F, Prehn C, Adamski J (2010) Quantification of steroids in human and mouse plasma using online solid phase extraction coupled to liquid chromatography tandem mass spectrometry Nature Protocols 10.1038/nprot.2010.22
Horsch M, Seeburg PH, Adler T, Aguilar-Pimentel JA, Becker L, Calzada-Wack J, Garrett L, Gotz A, Hans W, Higuchi M, Holter SM, Naton B, Prehn C, Puk O, Racz I, Rathkolb B, Rozman J, Schrewe A, Adamski J, Busch DH, Esposito I, Graw J, Ivandic B, Klingenspor M, Klopstock T, Mempel M, Ollert M, Schulz H, Wolf E, Wurst W, Zimmer A, Gailus-Durner V, Fuchs H, de Angelis MH, Beckers J (2011) Requirement of the RNA-editing Enzyme ADAR2 for Normal Physiology in Mice. J Biol Chem 286: 18614-18622
Huttemann M, Lee I, Gao X, Pecina P, Pecinova A, Liu J, Aras S, Sommer N, Sanderson TH, Tost M, Neff F, Aguilar-Pimentel JA, Becker L, Naton B, Rathkolb B, Rozman J, Favor J, Hans W, Prehn C, Puk O, Schrewe A, Sun M, Hofler H, Adamski J, Bekeredjian R, Graw J, Adler T, Busch DH, Klingenspor M, Klopstock T, Ollert M, Wolf E, Fuchs H, Gailus-Durner V, Hrabe de Angelis M, Weissmann N, Doan JW, Bassett DJ, Grossman LI (2012) Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology. FASEB J 26: 3916-3930
Kugler JE, Horsch M, Huang D, Furusawa T, Rochman M, Garrett L, Becker L, Bohla A, Holter SM, Prehn C, Rathkolb B, Racz I, Aguilar-Pimentel JA, Adler T, Adamski J, Beckers J, Busch DH, Eickelberg O, Klopstock T, Ollert M, Stoger T, Wolf E, Wurst W, Yildirim AO, Zimmer A, Gailus-Durner V, Fuchs H, Hrabe de Angelis M, Garfinkel B, Orly J, Ovcharenko I, Bustin M (2013) High Mobility Group N Proteins Modulate the Fidelity of the Cellular Transcriptional Profile in a Tissue- and Variant-specific Manner. J Biol Chem 288: 16690-16703
Waltereit R, Leimer U, von Bohlen Und Halbach O, Panke J, Holter SM, Garrett L, Wittig K, Schneider M, Schmitt C, Calzada-Wack J, Neff F, Becker L, Prehn C, Kutscherjawy S, Endris V, Bacon C, Fuchs H, Gailus-Durner V, Berger S, Schonig K, Adamski J, Klopstock T, Esposito I, Wurst W, de Angelis MH, Rappold G, Wieland T, Bartsch D (2012) Srgap3(-)/(-) mice present a neurodevelopmental disorder with schizophrenia-related intermediate phenotypes. FASEB J 26: 4418-4428
Additional relevant Internet links:
The German Mouse Clinic
Helmholtz Zentrum München
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