DE10352510A1 - Method for identifying active ingredients for influencing body mass and fat intake - Google Patents

Abstract

More particularly, the present invention relates to methods for identifying agents for affecting body mass and fat accumulation.

Description

The in particular, this invention relates to methods of identification of active ingredients for influencing the body mass and the fat deposit.

Obesity is a major health risk factor for type II diabetes, coronary heart disease and metabolic diseases. According to World Health Organization (WHO) surveys, about 8% of adults worldwide (about 300 million people) are extremely obese, measured by the body mass index. (BMI), which is more than 30 kg / m ² In developed countries with Western lifestyles, about one in four children and adolescents are overweight, so a broad area of research focuses on deciphering the genetic and physiological factors that cause obesity.

The Obesity results from the storage of surplus, food-derived Energy. The physiological causes of obesity can be one or several changes in appetite behavior, in nutrient metabolism, the heat development or the energy distribution. Body weight and appetite are essentially regulated by neuropeptides derived from the hypothalamus in response to signals from fat, muscle, liver and gastrointestinal tract be released. The answers of the hypothalamus can do that activate sympathetic nervous system that innervates the internal organs. The brain also controls energy expenditure via the hypothalamic-hypohyseal-thyroid axis. Further is the extremely obese phenotype often by a fault accompanied by the homeostasis related to obesity Hormone is regulated, which makes it for example to hyperleptinemia and hyperinsulinemia can come.

The Obesity is a complex trait that has the effect of a gene network reflected, and that by food, age, sex and movement affected becomes. A proof of a genetic influence on the Obesity came from twin studies. The heredity of Fat mass was reduced to about 40 - 70% estimated, and environmental factors contribute about 26% to phenotype variance.

Even though rare cases monogenic obesity have mostly been observed in children (e.g. Disruption of the single-minded homolog 1 gene or loss of the Function of for the leptin receptor independent genealogy studies give the clear genetic proof for oligogenic or polygenic predisposition obesity. These studies also show genetic differences regarding the prevalence of Obesity between ethnic groups. Further, the study showed Swedish Obese Subjects that different Genes for the obesity at different ages responsible could be.

In Coupling analyzes in the mouse, in humans, in the pig, in the Beef, the rat and other model animals were genome-wide QTL (quantitative trait loci; quantitative feature loci) that identifies a Influence on the body mass and have the fat approach. So far, only a few genes have been found that subject to these QTL effects. These were usually major gene effects, i.e. they contributed the majority to the phenotypic variance of a feature (A mutation in the gene PRKAG3 (protein kinase, AMP-activated, gamma 3 non-catatlytic subunit) for example, the muscle glycogen content around 70 and is as the main gene have been identified, Milan et al., Science. 288 (2000) 1248-1251).

A Series of genes with influence on body mass and body composition was identified in monogenic mouse models. The fundamental Meaning of these genes for the regulation of body mass was detected in humans and animals. To the identified genes belong naturally mutant genes (e.g., in the leptin or leptin receptor gene) and genetically modified Genes in transgenic models (e.g., adipocyte-specific gene expression of hydroxysteroid 11-beta dehydrogenase 1) or in knockout models (e.g. Insulin receptor, overview at Butler and Cone; Trends Genet. 17 (2001) 50-54) with changed Growth and fat accumulation. In these drastic models that leads nutated gene usually leads to complete failure of the function of the encoded protein.

Chemically or radiation-induced mutants in the mouse have a special importance in the function of elucidating genes with influence on the characteristic spectrum. Potentially, for example by means of ENU (ethylnitrosourea, ethylnitrosourea), each base can be mutated, as a result of which less drastic changes in the function of genes can be produced (Hrabe de Angelis et al., Nat. Genet. 25 (2000) 444-447; Nolan et al , Nat. Genet. 25 (2000) 440-443). However, mutants only appear in a screen detected if the difference to the baseline is large enough. The identification of such mutations requires repeated backcrossings to show that the observed effect is due to a gene before combining various methods to find the mutated gene.

The Producing artificial mutants is undoubtedly an important one Tool for functional elucidation of Genes and thus for the provision of numerous new, functional candidate genes. Two important questions can However, with these models can not be clarified: first, whether the previously identified and described above mutations also in selected or natural Populations matter for the regulation of body mass play, and secondly, to what extent these genes in the genomic network complex and quantitative features contribute to the phenotype. possibly are some gene variants that have a compensatory task in the Take over regulation can, evolutionary Prefers (Barton and Keightley, Nat Rev. Genet 3 (2001) 11-21). functional Data obtained from the yeast Saccharomyces cerevisiae, demonstrate that interactions between unrelated genes contribute to Example the main cause for Robustness over loss-of-function mutations are, i. Mutations leading to one Loss of function of the affected gene (Wagner, Nat Genet (2000) 355-61).

The Understand the complex regulation of body mass and body composition sets over the QTL mapping beyond identification of the quantitative feature genes (quantitative trait genes, QTG) advance, a very difficult and complex task that has hardly been achieved so far (Nadeau et al., Science 291 (2001) 1251-1255; Threadgil et al., Mamm. Genome 13 (2002) 175-8).

There, As already mentioned, most cases high body weight and obesity, in both humans and animals, are polygenic in nature and are influenced by environmental factors, there is a need identify those genes responsible for the genetic predisposition and expression are responsible.

to solution this task a polygenic animal model (mouse model) was used, that is - at first generally expressed - by distinguishes that in animals in specific tissues (fat, hypothalamus) a set of genes amplified and a set of genes is expressed less expressed, so that of these Genes encoded proteins in different tissues compared to average animals in an increased or decreased proportion be formed. About that There are also certain changes (Polymorphisms) in the sequences of some genes or regulatory Sequences of the same before.

The in the context of the present invention used mouse lines (called as DU6 and DU6i) are characterized by a higher than average body weight and fat accumulation (obesity). They differ of unselected control populations by more than about 2-fold up to about 4 times body weight, by more than about 3 times to about 7 times in the fat mixture and up to about 3 times in muscle mass. This is the case of males / females the selected line DU6i on the 42nd day an average body mass from 60.1 ± 3.9 g / 47.9 ± 4.3 g (52 examined animals), while the body mass average Control animals (DBA / 2) at 15.5 ± 1.7 g / 14.8 ± 1.5 g (13 males / 12 Females), corresponding to an average of 54.0 ± 4.1 g (DU6i) opposite 15.1 ± 1.6 g (DBA / 2) relative to all animals. In addition, lie in the selection lines across from normal animals significant differences in some factors of the nutrient turnover and energy balance such as serum levels of leptin, insulin, IGF-I and IGF binding proteins. By combining data from QTL mapping and differential gene expression analysis genes were identified that are involved in growth and obesity regulation in on high body weight selected mouse lines are involved.

The in the context of the present invention identified differentially expressed genes represent genes, body weight and obesity control at the end of the juvenile developmental phase. The animals were ad libitum fed with a breeding diet that 12.5 MJ / kg metabolic energy and an average content of 22.5% raw protein, 5% crude fat, 4.5% raw fiber, 6.5% crude ash, 13.5% water, 48% N-free Extract, vitamins, trace elements, amino acids and minerals (Altromin Diet 1314, Germany).

Differential gene expression analysis between the two selected high body weight mouse lines (DU6 and DU6i) and the two unselected mouse lines (DUKs and DBA / 2) showed a consensus set of 77 differentially expressed genes in the same way (see Table 1). The genes encode proteins that play a role in metabolism, signal transduction, cell division, and the cytoskeleton play in immune responses.

The following 77 genes were significantly increased (+) or decreased (-) transcribed in the examined obese compared to control animals in epididymal fat tissue: Table 1A: Differentially expressed genes in epididymal fat tissue of DU6 and DU6i lines against DUKs and DBA / 2 at the age of 42 days under standard housing and feeding conditions.

Tabelle 28: Liste der 4 different exprimierten Gene in QTL-Regionen für Wachstum und Fettansatz des polygenen Mausmodells (Hypothalamus):

Table 2A below contains 10 differentially expressed genes from Table 1A above. Table 2B contains 4 differentially expressed genes from Table 1B above. These 10 genes are located in chromosome regions that have been shown to affect body mass and fat accumulation in genetic coupling studies. Such chromosomal regions are also referred to as quantitative trait locus or quantitative trait locus (QTL). Coupling analyzes for QTL mapping were performed in crossover populations between the selected DU6 × DUKs (Brockmann, GA; Haley, CS; Renne, U .; Knott, SA; Schwerin, M. (1998): QTL's effecting body weight and fatness from a Genetics 150: 369-381) and DU6i × DBA / 2 (Brockmann, GA; Kratzsch, J., Haley, CS, Renne, U., Schwerin, M., Karle, S. ( 2000): Single QTL effects, epistasis, and pleiotropy account for two thirds of the phenotypic F ₂ variance of growth and obesity in DU6i x DBA / 2 mice Genome Res 10: 1941-1957, Brockmann, GA, Haley, CS; Wolf, E., Karle, S., Kratzsch, J .; Renne, U .; Schwerin, M., Hoeflich, A. (2001): Genome-wide search for loci controlling serum IGF-binding protein levels of mice J. 15: 978-987). Table 2A: List of the 10 differentially expressed fatty tissue genes of the polygenic mouse model in QTL regions:

Table 28: List of 4 different expressed genes in QTL regions for growth and fat accumulation of the polygenic mouse model (hypothalamus):

In the selected animals were increased transcript levels of Prkca, for Protein kinase C alpha coding gene found. The gene position corresponds to a QTL for increased body weight and fat accumulation in the selected mouse lines. The protein is at G protein and Wnt signaling pathways (G protein and Wnt signaling Pathways) that regulate cell growth and differentiation. PRKCA is an intracellular Signal protein that catalyzes the phosphorylation of substrate proteins and ubiquitous occurs. For the unfolding of its physiological activity alters the protein upon activation its position from the cytosol into cellular membranes. The Cl domain of PRKCA is a diacylglycerol / phorbol ester binding region. The full enzyme activity requires Diacylglycerol, phospholipid and calcium. Diacylglycerol is called Intermediate produced in the biosynthesis of fatty acids and is expected in increased the selected mouse lines available.

The cDNA of H1f2 (H1 histone family, member 2) enters the selected one Animals in an elevated frequency on. Histones are basic core proteins responsible for the nucleosome structure the chromosome fibers in eukaryotes are responsible. The linker histone H1 interacts with linker DNA between nucleosomes and causes the Compaction of chromatin in higher ordered structures.

The Gen Ak2, which is expressed in the selected animals expressively encoded the adenylate kinase 2, which is a mitochondrial enzyme that releases the Phosphorus transfer between ATP and AMP catalyzes. As such it is involved in the regulation of energy metabolism in tissues. So far knockout models for adenylate kinase have not been included a reduced body weight connected.

In the selected animals, the cDNA frequency of the gene Cox8 decreases on. This gene for the cytochrome c oxidase subunit VIII (COX subunit VIII) is another gene that involved in the maintenance of energy homeostasis. cytochrome c is the terminal enzyme of the respiratory chain, which is the electron transfer of cytochrome c causes molecular oxygen. The COX subunit VIII is a nuclear-encoded polypeptide. The function of the core-coded Subunits is not yet known, although it has been suggested that they are involved in the modulation of the catalytic function could be. amendments in the activity from Cox8 can influence the efficiency of energy distribution.

The results obtained according to the invention show that the mRNA levels for Klkbp (kallikrein-binding protein, alias serpin) are increased in the selected animals. KLKBP is a serine (or cysteine) proteinase inhibitor. It binds to kallikrein and inhibits its action. It has recently been demonstrated in rat models that KLKBP is a potential pathogenic factor of diabetic retinopathy and may play a role in animal development and growth. In the dwarf rat, the Klkbp gene is induced by growth hormone (Hatcher et al., FASEB J 13 (1999) 1839-1844). Furthermore, KLKBP has a potential function in vascular biology. Transgenic mice overexpressing kallikrein are chronically hypotensive. Recently, proteome analysis has shown that alterations in the renal kallikrein pathway occur in hypoxia-induced hypertension. Kallikrein is found in the bloodstream and extracellular space. Its affinity towards KLKBP can coordinate and regulate the biological function of kallikrein. Increased transcript levels of Klkbp in adipose tissue, as found in the selected mouse lines of the present invention, can result in increased serum levels of the secreted protein, which can cause reduced kallikrein activity or initiate possible independent actions of the binding protein, as in IGF binding proteins was observed. Thus, increased KLKBP may be a protective factor in the relationship between adiposity and an increased risk of hypertension. This is consistent with the healthy behavior of the selected mice below Stress conditions, while non-selected animals, for example occasionally die unexpectedly.

The Gene Hfe and Igh-Ia, which is the hemochromatosis protein and immunodepline heavy chain Ia are at the Involved immune response. Both genes are in the high body weight upregulated selected mouse lines. The hemochromatosis protein of the mouse regulates duodenal uptake of transferin-bound iron from plasma. In hemochromatosis, a disease that results from a mutation in the Hfe gene causes iron ingested with food increased iron accumulation in liver, Heart and pancreas. In connection with diabetes mellitus 22 was Insulin resistance is observed with excessive accumulation of iron was associated in the liver (Roblin et al., Diabetes Metab 28 (2002) 335-339). So far, there has been a direct effect of the Hfe gene on obesity not established. Hfe knockout mice were on changes of body weight and the composition has not been studied. The hemochromatosis protein is a integral membrane protein with domains for immunoglobulin and class I histocompatibility antigens alpha 1 and alpha 2.

In the inventive, selected Lines is the gene Pctp1 that is responsible for encodes the phosphatidylcholine transfer protein-like factor over control lines significantly downregulated. Phosphatidylcholines are the most general Phospholipid class in eukaryotic cells. This insoluble molecules be between membranes by a highly specific phosphatidylcholine transfer protein (PC-TP) belonging to the superfamily of Steroidogenic Acute Regulatory related protein Transfer (START) domain belongs, (Roderick et al., Nat. Struct. Biol. 9 (2002) 507-511). The same thing Protein was also detected as a serologically defined colon cancer antigen 28, what his possible Role in growth regulation suggests.

The Minichromosome maintenance proteins, a family of 6 conserved polypeptides found in the nucleus of all Eukaryotes are present for the initiation of DNA replication essential. The mRNA transcript levels of Mcmd7 (Minichromosomes maintenance deficient 7, minichromosome maintenance proteins) are in the selected animals compared to non-selected ones Control animals significantly reduced. In neuroblastomas this is MCM7 protein is a direct target of the MYCN transcription factor. Therefore, the Transcript level of Mcmd7 may be a direct measure of cell division activity. From these observations it is concluded that the cell division rate in epididymal Fat tissue in lean control animals is much higher as compared to selected, fat animals. These data are with observations consistent that the Obesity at the age of 6 weeks in selected mice not by hypertrophy but rather by hyperplasia (Timtchenko, D., Kratzsch, J., Sauerwein, H., Wegner, J .; Souffrant, W.-B .; Schwerin, M .; Brockmann, G.A. (1999): Fat Storage capacity is associated with the growth potential with linespecific gene expression and plasma hormone levels. Int. J. Obesity 23: 586-594).

Serine proteases, like Serpina3n, take over important functions, including digestion, blood clotting or the complement system (immune response). Serpins (serine protease inhibitors) inhibit these activities irreversible by imitation of the three - dimensional structure of the normal substrate of the protease [http://users.rcn.com/jkimball.ma.ultranet / BiologyPages / S / Serine Proteases.html].

Also Serpine may play a role during Development [Inglis, J.D. et al. Isolation of two cDNAs encoding novel alpha 1-antichymotrypsin-like proteins in a murine chondrocytic cell live, 1991, Gene 106 (2), 213-220].

Tabelle 4: Zuordnung der Gen-ID Nummern aus Tabelle 3 A und 3B zu den Gen-Namen

In addition to differential expression of the above positional genes (see Table 2A) observed in the adipose tissue compared to unselected control animals, various polymorphisms are also observed in the promoter sequence, which are shown in Table 3A below, with the EMBL sequence Identification numbers are specified. Further, for a gene differentially expressed in the hypothalamus (see Table 2B), various polymorphisms are observed in the promoter sequence shown in the following Table 3B, each indicating the EMBL sequence identification numbers. An assignment to the corresponding gene symbols is contained in Table 4. The sequence comparisons of the genes listed in Tables 3A and 3B are in 1 shown.

Table 4: Assignment of the gene ID numbers from Table 3 A and 3B to the gene names

Tabelle 6: Zuordnung der Gen-ID Nummern aus Tabelle 5 zu den Gen-Namen

In some genes expressed differently in adipose tissue and positioned in QTL regions, additional polymorphisms were found in the 3 'flanking region. The genes with the identified polymorphic variants are listed below in Table 5. The assignment of the gene ID to the gene name is in Table 6. The sequence comparisons of the genes listed in Table 5 are in 2 shown.

Table 6: Assignment of the gene ID numbers from Table 5 to the gene names

The 10 genes that are both different in adipose tissue expressed as well located in such chromosomal regions (see Table 2A) that have an impact on body mass and have shown the fat approach, are genes that are highly likely the genetic causes for the raised Are rich in fat in the selection line. The same goes for the 4 in the hypothalamus different expressed genes, which are also in QTL regions for growth and lipase of the polygenic mouse model (see Table 2B).

The Genes among the 10 (see Table 2A) and 4 (see Table 2B), for the same line-specific polymorphisms in the promoter region and / or in 3 'untranslated Genregion were found, very likely to determine gene variants, directly to the difference in the fat margin between the selected and contribute to non-selected animals.

The other genes from the list of 77 genes (adipose tissue, see Table 1A) or the 23 genes (hypothalamus, see Table 1B) are rather secondary regulated Genes that have increased or decreased as a result of genetically fixed differences reduced gene activity demonstrate. They are with big ones Likelihood of metabolism, the regulation of the energy balance or other control circuits involved.

With in the context of the invention obtained on the basis of the polygenic mouse model Data now allows fundamental conclusions about complex regulation of growth and fat accumulation, that for humans and other animals are relevant. Furthermore, those identified with the model are suitable Genes and polymorphisms for the identification of active substances, the the body mass and influence the lipid content, and thus also of active ingredients, which are suitable for the treatment or prevention of obesity.

wobei es sich bei der Substanz um einen die Körpermasse und den Fettansatz beeinflussenden Wirkstoff handelt, wenn durch Verabreichung der Substanz die Expression eines oder mehrerer der genannten Gene verändert wird. Das vorgenannte Verfahren wird auch als "Screening-Verfahren" bezeichnet.The present invention therefore provides a method for the identification of active substances for influencing the body mass and the lipid charge, in which non-human mammals are administered a substance to be investigated and their influence on the expression of the following genes is determined:

wherein the substance is an active substance which influences the body mass and the lipid charge, if the expression of one or more of said genes is changed by administering the substance. The aforementioned method is also called a "screening method".

The The invention further relates to a process for the preparation of a pharmaceutical Composition for influencing the body mass and the fat deposit, in which an aforementioned method for the identification of active ingredients for influencing the body mass and the fat batch and adding the identified drug to one for administration formulated suitable pharmaceutical composition.

According to a particular embodiment, it is a method in which the active substance for influencing the body mass and the fat mixture is suitable for limiting (reducing) the lipid deposition. In this case, non-human mammals are given a substance to be tested and determined their influence on the expression of the above-mentioned genes, wherein the substance is a body mass and the fat-limiting (reducing) active ingredient when administered by administering the substance Expression of one or more of the genes marked "(-)" in Tables 1A and 1B is increased and / or the expression of one or more of the genes marked "(+)" in Tables 1A and 1B is decreased. The aforementioned method is also called a "screening method" be records.

The The invention thus further relates to a method for producing a pharmaceutical composition for influencing the body mass and the Fettansatzes, in which one of an aforementioned method for Identification of active ingredients for reduction (reduction) the body mass and the fat batch and adding the identified drug to one for administration formulated suitable pharmaceutical composition

By but the invention will also be the identification of active ingredients allows which are capable of increasing the fat intake by being non-human mammals administered a substance to be examined and their influence on the Expression of the aforementioned genes determined, it being in the Substance around one's body mass and increasing the amount of fat Drug is when expression by administration of the substance one or more of the genes marked "(+)" in Tables 1A and 1B elevated and / or the expression of one or more of the in tables 1A and 1B marked "(-)" Gene is degraded. The aforementioned method is also called a "screening method".

The The invention thus further relates to a method for producing a pharmaceutical composition for influencing the body mass and the Fettansatzes, in which one of an aforementioned method for Identification of active ingredients to increase body mass and fat intake performs and adding the identified drug to one for administration formulated suitable pharmaceutical composition.

at the one marked "(-)" Genes are preferably those in Tables 2A and 2B marked with "(-)" Genes, i. Cox8, Pctp1, Mcdm7, Serpina3n and / or the sequence 4930506L07Rik. For those marked with "(+)" Genes are preferably those in Tables 2A and 2B marked with "(+)" Genes, i. Prkca, Hlf2, Akt, Klkbp, Hfe, Igh-la, NA, Ly6a and / or the Sequence 2810037C14Rik. Particularly preferred are those of the aforementioned Genes, for the at the on high body mass and high lipid level selected animals polymorphisms in the promoter region or in the 3'-flanking Area were detected.

at non-human mammals they are preferably rodents, in particular mice. The Invention includes also the use of non-human mammals, especially rodents or mice for use in an aforementioned screening method.

By the present invention, it is thus possible, according to the aforementioned Screening procedures identified drugs for the production of pharmaceutical Compositions for influencing the body mass and the fat deposit, for lowering the body mass and the fat mixture or to increase the body mass and the fat mixture to use.

The The invention further relates to pharmaceutical compositions which contain one or more active ingredients that limit the expression of the above influence designated genes in the aforementioned manner, increase or humiliate.

to Preparation of the pharmaceutical compositions may be the Active ingredients used either individually or in combination and optionally together with pharmaceutically acceptable auxiliary and / or excipients be formulated.

With The present invention relates to active substances which inhibit expression influence the above-mentioned genes in the aforementioned manner, increase or to decrease, for influencing the body mass and the fat deposit, for lowering the body mass and the fat mixture or to increase the body mass and the fat mixture provided.

A concordance of the respective sequence identification numbers for the abovementioned genes listed in the sequence listing is shown in the following table:

The indicated SEQ ID NO. is the same as code 400 in the Sequence Listing number specified by WIPO Standard ST.25.

The Invention will be described below by way of examples, without limited to these to be.

Description of the figures:

1 : Sequence comparisons between DU6, DU6i, DBA / 2, DUKs and ENSEMBL for the genes listed in Tables 3A and 3B.

2 : Sequence comparisons between DU6, DU6i, DBA / 2, DUKs and ENSEMBL for the genes listed in Table 5.

3 A: Chromosome map of the mouse with the QTL positions identified in the DU6i × DBA / 2 crossing and the genes differentially expressed in adipose tissue; on the right side of each chromosome, the QTL regions are shown as bars in different shades of gray. QTLs are abbreviated as follows: BW * for body weight, Lepq * for leptin, Igf1q * for insulin-like growth factor 1, Igfbp * for insulin-like growth factor binding protein, Afw * for abdominal fat weight, Mwq * for muscle weight.
B: Chromosome map of the mouse with the QTL positions identified in the DU6i × DBA / 2 crossing and the genes differentially expressed in the hypothalamus; on the right side of each chromosome, the QTL regions are shown as bars in different shades of gray. QTLs are abbreviated as follows: BW * for body weight, Lepq * for leptin, Igf1q * for insulin-like growth factor 1, Igfbp * for insulin-like growth factor binding protein, Afw * for abdominal fat weight, Mwq * for muscle weight. The loci of C85523 (no symbol) and L20450 (Zfp97) are unknown.

4 : Animal structure for building chromosome substitution lines; i - numbering of the autosomes (i = 1 to 19) and sex chromosome (i = X); a, b designation for partial chromosomes; n - number of offspring per two litters (n = 16); * - animal selected from the offspring with DU6 / DBA (DBA stands for DBA / 2) Genotype on chromosome i and minimal DU6 genome part on all other chromosomes j (j ≠ i).

5 : Differences in body weight on day 42 between CSSs and DBA / 2 mice in different backcross generations (BC). Asterisks mark different levels of significant differences between the CSS and DBA / 2 animals: * P <0.05, ** P> 0.01, *** P <0.001. In BC4 and BCS generations, not all CSS were generated, so there is no data for these lines.

6 : Exemplary profile of IGFBP in serum samples from selected F ₂ progeny; The example shows the large differences in IGFBP concentrations between F ₂ animals. The bands were visualized by phospho-imaging and quantified using the ImageQuaNT software as described in the Materials and Methods section (example section). Serum levels of distinct IGFBPs were measured semi-quantitatively (Box 1: IGFBP-3; Box 2: IGFBP-2; Box 33: IGFBP-4; Box 4: IGF binding affinity between 28 and 30 kDa; S: Standard serum; included in each run for normalizing the different blots).

example 1

Base of all mouse lines of the institute for the biology of farm animals (FBN) is the heterogeneous Breeding line Fzt: DU, at the beginning of the 70s through systematic Crossing of 4 Extracts (NMRI orig., Han: NMRI, CFW, CF1) and 4 Inbred lines (CBA / Bln, AB / Bln, C57BL / Bln, XVII / Bln) and kept in an effective population size of 200 pairs of animals becomes.

• – DU6, Auszuchtlinie, Zucht auf hohes Wachstum
• – DU6i, Inzuchtlinie, abgesplittet aus der DU6, Zucht auf hohes Wachstum
• – DUKs, Kontrolllinie
• – Käuflich erworben wurde die Inzuchtlinie DBA/2.

The following mouse lines of the FBN were used for the investigations for mapping genetic factors for growth and fat deposition:

• - DU6, breeding line, breeding for high growth
• - DU6i, inbred line, split off the DU6, breeding for high growth
• - DUKs, control line
• - The inbred line DBA / 2 was purchased.

The selection scheme is shown below.

When Result of selection via 95 generations increased in the DU6 the body weight from 30 g to 72 g. Also a number of other features of body composition, as well as different serum parameters changed.

For the analysis genetic factors, 2 pedigrees were developed:

Pedigree 1

In the 70th generation, 4 males (DU6) and 4 females (DUKs) were taken from the DU6 and DUKs lines. These animals were crossed 1: 1 to create four segregating F ₂ -design intercross family structures. Controlled and repeated full-sibling mating of F1 generation offspring produced large full-sibling groups (a total of 54 F ₁ and 715 F ₂ offspring). The pedigree with the most F ₂ progeny (341) was used to scan the entire genome.

The animals used for the analysis were characterized phenotypically for Kör weight, abdominal fat, liver, kidneys and spleen.

Pedigree 2

Here, too, intercross family structures were set up using female animals of the inbred line DU6i (extraction from the 4th inbred generation) split off from the DU6 and male animals of the commercially available inbred line DBA / 2. 12 F ₂ subfamilies were formed with a total of 411 offspring (233 males, 178 females) that were phenotypically characterized and genotyped for 93 loci.

The characteristics of the mouse lines mentioned herein are shown in Table 7 below: TABLE 7 Characterization of mouse lines

Example 2

QTL analysis in crossing experiments between the extremely different lines

Cross-breeding experiments between the different-feature lines have identified those chromosome regions in the genome that have a significant influence on body mass, fat deposits, muscle mass and weights of internal organs. The chromosomal sites with influence on the feature development are called QTL (Quantitative Trait Locus). The following crosses were subjected to a genetic linkage analysis for the localization of QTL:
DU6 × DUKs
DU6i × DBA / 2

In all experiments several significant QTL were localized in the genome. In addition to the general characteristics of body mass and fat accumulation, subcomponents were involved in the regulation of the trait complex involved in the linkage analysis and thus receive additional information about locations in the genome that significantly affect these subcomponents. In particular, the newly identified QTLs are factors of nutrient turnover and energy balance such as serum levels of leptin, insulin, IGF-I (insulin-like growth factor) and IGF binding proteins involved in IGF regulation and, in part, independent functions in growth regulation take.

The chromosomal positions and effects are summarized separately for QTL with influence on the body mass and composition and for QTL with influence on the serum concentrations of relevant proteins and hormones in table 8 and table 9. 3 shows the mouse chromosome map with the QTL positions identified in the DU6i × DBA / 2 junction.

The QTL analyzes in the DU6 × DUKs and DU6i × DBA / 2 families are described in more detail below in Examples 3 and 4.

Example 3:

QTL analysis in the family DU6 × DUKs

• (Brockmann, G.A., Haley, C.S., Renne, U .; Knott, S.A .; Schwerin, M. (1998): QTLs effecting body weight and fatness from a mouse line selected for extreme high growth. Genetics 150: 369-381).

Mouse lines:

The Investigation was made with the breeding mouse line DU6, which has been over 70 generations on high body weight was selected as well as at the arbitrarily matched control line DUKs performed. Both lines are from original crosses of four basic populations (NMRI orig., Han: NMRI, CFW, CF1) and four inbred populations (CBA / Bln, AB / Bln, C57BL / Bln, XVII / Bln) of the Research Institute of Agricultural Biology Livestock, Dummerstorf, Germany (Student 1985 Mouse strain Fzt: Du and its use as model in animal breeding research. Arch. Animal Breeding 28: 357-363). The population size in both Lines was 80 pairs per generation. The litter size was by default 9 fixed. Offspring were discontinued on the 21st day. The animals were ad libitum fed with a breeding diet that 12.5 MJ / kg metabolic energy and an average content of 22.5% raw protein, 5% crude fat, 4.5% raw fiber, 6.5% crude ash, 13.5% water, 48% N-free extract, vitamins, trace elements, amino acids and Minerals (Altromin Diet 1314, Germany). The body weight on the 42nd day was in the selected line DU6 (58.3 ± 6.1 g) more than twice as high like that of the arbitrarily paired control DUKs (28.7 ± 2.3g) (BÜNGER, L., U. RENNE, and G. DIETL, 1992, Selection for body weight at 42 days in laboratory mice with and without litter size standardization correlated effects on litter size. Arch. Tierz. 35: 305-319; BÜNGER ET AL., 1990 Results of long term selection for growth traits in laboratory mice. Proceedings of the 4th World Congress on Applied Genetics to Livestock Production, Univ. Edinburgh, Edinburgh, Scotland 13: 321-324). Of the percentage of epididymal fat was in the DU6 line (2.2 ± 0.8%) twice as high as in control (1.1 ± 0.4%) of the same age.

Family tree design:

QTL analysis was performed using pedigrees of F ₂ crossing design. These were created by crossing four males of the high growth selected line DU6 with four females of the control line DUKs. Repeated parental mating and subsequent mating of the F ₁ offspring of the same parents produced large pedigrees with a total of 54 F ₁ progeny and 715 F ₂ progeny. Mating was initially performed at 10 weeks of age and repeated at 6 weeks; the mating was done in all pedigrees at the same time. The structure of the individual pedigrees is summarized in Table 1. The largest pedigree (Identity Number 8) with 341 F ₂ offspring was selected for the genome study. Quantitative measurements made on QTL analysis included body weight, abdominal fat, and the liver, kidney, and spleen weights of all F ₂ individuals. The measured abdominal fat was the gonadal fat in the males and the fat of the perimetrium in females. The ratio of abdominal fat to body weight was defined as the percentage of abdominal fat. The phenotypic data were always taken at the age of 42 days; This is the time for the selection decision in all generations. This age corresponds to the end of the juvenile phase of ontogenesis.

Marker analysis:

At first was the distribution of marker alleles between the lines for 84 loci examined. The markers were chosen to spread over all chromosomes were, with the distance between the markers on average 16.6 cM and the largest interval 42 cM.

The markers were selected for their high variability between suitable inbred lines from the mouse genome database (MGD). Mouse MapPair primers were obtained from Research Genetics (Huntsville, AL, USA). The D11Bhm * primers were a gift from Thomas Boehm (Nehls et al., 1995). YAC / P1 microsatellite markers and several genes across a 3.4 cM interval on mouse chromosomes 11th genome &: 321-331) , D9Fbn1 was developed by Rainer Fürbass in our institute. Although the resolution of the marker mapping of the mouse genome is high, finding an informative marker group for our pedigrees created from crosses of rearing lines was difficult because of parentage between equally present alleles. Over 450 microsatellite markers were tested in pedigree 8 for informative parental alleles before the parents, F ₁ and F ₂ were genotyped for 94 loci covering all chromosomes with an average separation of 12.8 cM. Individuals of pedigrees 3, 4 and 10 were genotyped on chromosome 11 for informative microsatellites previously shown to have an effect on the lipid approach (Das, P., G. Brockmann, L. Meyer, U. Renne, G. Freyer, et al M. Schwerin 1996. The effect of a restricted region of chromosome 11 on body weight in mice under special consideration of the growth hormone gene locus., Arch. Tierz. 39: 185-194).

DNA from mouse tail tissue samples was extracted using the QIAmp Tissue Kit (Qiagen, Germany) by selective binding of DNA to an ion exchange matrix after digestion with proteinase K. The DNA was amplified with Taq polymerase (Appligene, France) using a standard modified PCR protocol (Dietrich et al., Genetics 131: 423-447): 20 ng of template DNA was used in a total reaction volume of 10 μl. After denaturation (96 ° C, 3 minutes) followed by 2 cycles with an annealing temperature of 59 ° C and 57 ° C, the DNA was incubated for 25 cycles of 15 seconds at 94 ° C, 1 minute at 55 ° C and 1 minute 70 ° C amplified. The DNA was automatically distributed for PCR using the Biomek 2000 system (Beckman). Amplifications were performed in 96-well microtiter plates in a 9600 PCR system (Perkin Elmer). The PCR products were dried, redissolved in 3 μl formamide loading buffer and separated on 40 cm long 6% polyacrylamide gels under denaturing conditions. The gels were stained with silver nitrate (Riesner et al 1989 Temperature gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein nucleic acid interactions., Electrophoresis 10: 377-389). After initial fixation in 10% ethanol / 0.5% acetic acid solution for 10 minutes, the gels were immersed in fresh 5.9 mM solution of silver nitrate for 15 minutes and in 375 mM NaOH, 2.3 mM NaBH ₄ , 0.125% formaldehyde for 10 minutes. 37% w / v). After rinsing twice with water, the gels were dried on a Filtrak paper (Germany).

On each gel, the parental marker alleles were loaded on separate lanes and served as standard for genotyping of F ₁ and F ₂ progeny. All results of the genotyping were evaluated twice by independent persons, and runs in which discrepancies occurred were repeated.

Test statistics:

Quantitative differences in the distribution of microsatellite alleles between the lines were assessed using the chi-square test (Rasch et al., 1978, Process Planning Experimental Design and Evaluation, Volume 2, Deutscher Landwirtschaftsverlag Berlin, 884). The heterozygosity indices for the two mouse lines and the genetic distance between the lines were calculated according to Gregorius (Gregorius, A unique genetic distance, Biom. J. 1984, 26: 13-18). A heterogeneity index> 0.5 between the lines in a single locus is consistent with the significance threshold of P <10 ^{-4 of} the chi-square assay.

For linkage analysis were first Pedigree-specific marker mapping with the program CRIMAP (Larder and Green, 1987 Construction of multilocus genetic linkage maps in humans. Proc. Nat. Acad. Sciences (USA) 84: 2363-2367). Because these maps are consistent in content with the published maps were, marker distances became the consensus mapping of the mouse genome (Dietrich et al., 1996 A Comprehensive genetic map of the mouse genome. Nature 380: 149-152) for mapping the QTLs used.

For the analysis of the pedigrees the influences of gender, repeated mating and subfamily (ie F ₂ animals of the same F ₁ parent pair) were first analyzed by analysis of variance (SAS, 1990 SAS User's Guide: Statistics, SAS Inst. Inc., Cary, NC ) and found to be significant and therefore included as fixed effects in the linkage analysis. For the analysis of chromosome 11, each pedigree was assessed separately, followed by combined analyzes of specific pedigrees. In the analysis of several pedigrees, a fixed effect of the pedigree was included in the model.

The data were analyzed by multiple regression as developed for the analysis of interbred crossbreeds (Haley et al 1994 Mapping quantitative trait loci in crosses between outbred lines using least squares., Genetics 136: 1195-207). First, the standard interval mapping model with a single QTL was applied to the coupling group. The estimates obtained were obtained (as described by Zeng (1993 Theoretical Basis for Separation of Multiple Linked Gene Effects in Mapping Quantitative Trait Loci, Proc. Natl. Sci. USA 90: 10972-10976) and Jansen (1993 Interval mapping of multiple quan titative trait loci. Genetics 135: 205-211)) by tailoring background genetic effects to the coupling groups. Background genetic effects were gradually included as co-factors beginning with the highest estimated locus, followed by the second most potent locus until no more QTLs could be detected at the specified level of significance. During this procedure, a background effect was always excluded from the analysis when analyzing its own position. The sex chromosome was divided into a pseudoautosomal region and an X-specific region. In additional analyzes, body weight was included as a co-variant in the analysis of the abdominal fat weight and in the analysis of the percentage of abdominal fat in order to investigate the dependency between the two properties. The combined effect of all significant QTLs on one property (ie variance declared for the F ₂ population) was seen as a reduction of the residual variance in the QTL analysis, in which all QTL positions were co-factorially adjusted compared to Analysis estimated without QTLs. Levels were used for the indicated (α = 0.1), significant (α = 0.05), and highly significant (α = 0.01) coupling, as proposed by Lander and Kruglyak in 1995 (Larder and Kruglyak 1995 Genetic Dissection of complex traits: guidelines for interpreting and reporting linkage results, Nature Genetics 11: 241-247). Chromosome-specific and genome-wide empirical thresholds of the test statistics of the regression analysis were determined using the permutation test proposed by Churchill and Doerge (1994 Empirical threshold values for quantitative trait mapping, Genetics 138: 963-971). The implied level is equivalent to the level at which a false-positive result is expected in a genome study. In a genome of 20 independent chromosome pairs, this is approximately equivalent to using the chromosomal 5% significance level, so that, on average, one of these chromosomes is significant in a study of the entire genome at that level. The chromosome-wide levels vary between the chromosomes depending on their length and the markers they contain. To provide a conservative estimate of the 95% confidence interval for highly significant genetic coupling loci, the maximum value of the test statistic was lowered by 2 LOD and this new value of the test statistic was used to define the chromosome interval in which the QTL was most likely to increase (van Ooijen 1992 Accuracy of mapping quantitative trait loci in autogamous species, Theor. Appl. Genet. 84: 803-811).

As soon as a single QTL was identified on a chromosome the presence of a second QTL by performing a raster search in 2 cM intervals examined. The model of two QTLs was assumed provided a significant improvement over the best possible Model with a QTL at P <0.05 using a variance rate (F) test with 3 degrees of freedom (for the additional additive effect and the dominance effect and the estimated position for the second QTL). Mapping sites are considered genetic Distance from centromere indicated in cM. where a QTL identifies was the interaction of QTL effects with the pedigree, with the within the family tree contained subfamily and with the gender examined and at P <0.05 accepted as significant.

Example 4

QTL analysis in the family DU6i × DBA / 2

• (Brockmann, GA, Kratzsch, J., Haley, CS, Renne, U., Schwerin, M., Karle, S. (2000): "Single QTL effects, epistasis, and pleiotropy account for two thirds of the phenotypic F ₂ variance of growth and obesity in DU6i × DBA / 2 mice ", Genome Res. 10: 1941-1957;
• Brockmann, G.A .; Haley, C.S .; Wolf, E .; Karle, S .; Kratzsch, J .; Renne, U .; Schwerin, M .; Hoeflich, A. (2001): "Genome-wide search for loci controlling serum IGF-binding protein levels of mice ", FASEB J. 15: 978-987).

Mouse lines:

The study was performed with the mouse DU6i line generated by inbred for four generations from the high body weight line DU6. As a contrast line, the commercially available inbred mouse line DBR / 2 (Harlan Netherlands, Horst) was used. The bred line DU6 was selected over 78 generations for high body weight at the age of 42 days (Bünger et al., 1990, loc cit.). Line DU6 is derived from original crosses of four basic populations (NMRI orig., Han: NMRI CFW, CF1) and four inbred populations (CBA / Bln, AB / Bln, C57BL / Bln, XVII / Bln) of the Biology Research Institute farm animals, Dummerstorf, Germany (pupil 1985, loc. cit.). The animals were ad libitum fed with a breeding diet containing 12.5 MJ / kg metabolic energy and an average content of 22.5% crude protein, 5% crude fat, 4.5% crude fiber, 6.5% crude Ash, 13.5% water, 48% N-free extract, vitamins, trace elements, amino acids and minerals (Altromin Diet 1314, Germany). 17 males of the line DU6i and 20 males of the line DBA / 2, which had not fasted, were examined for the characterization of the two mouse lines on the 42nd day.

Family tree design

The QTL analysis was performed using a pedigree of the F ₂ crossing design. These were established by crossing a female of the high-growth inbred line DU6i with a male of the contrast line DBA / 2. Repeated parenting followed by pairing within the subfamilies of 12 pairs of F ₁ offspring created a large pedigree with a total of 411 F ₂ offspring. Mating was initially performed at 10 weeks of age and repeated at 6 weeks.

Phenotypic measurements:

The phenotypic data were taken at the age of 42 days; This is the age of selection in all generations. The quantitative measurements made in the QTL analysis were on the weight of body (BW), abdominal fat (AFW), muscle (MW), liver (LW), kidney (KW) and spleen (SW) and the serum concentration of Leptin, insulin and insulin-like growth factor 1 (IGF-I). F ₂ animals that had not fasted were killed between 9 and 12 o'clock in the morning. In order to obtain the largest possible volume of blood serum for the analysis of the physiological parameters, the animals were decapitated. The measured abdominal fat was the testicular fat in the males and the fat of the perimetrium in the females. The ratio of abdominal fat to body weight was defined as the percentage of abdominal fat (AFP). The weight of the quadriceps, which includes the rectus femulus muscle, the vastus intermedicus muscle, the vastus lateralis muscle, and the vastus medialis muscle, was recorded in a representative manner for muscle development (MW).

leptin became with the "Quantikine Murine "-ELISA (R & D Systems, Wiesbaden, Germany). The determination of insulin in mouse sera was performed by an ultrasensitive rat insulin ELISA (DRG, Marburg, Germany), which a 100% cross-reactivity with mouse insulin showed. IGF-I was analyzed for acid-ethanol extraction (such as previously described) by means of an ELISA in serum (Kratzsch et al. 1993, Measurement of insulin-like growth factor I (IGF-I) in normal adults, patients with liver cirrhosis and acromegaly: experience with a new enzyme immunoassay. Exp. Clin. Endocrinol. 101: 144-149).

Analysis of IGFBP levels in serum:

DU6i and DBA / 2 mice that had not fasted and all F ₂ hybrids were sacrificed by decapitation between 9:00 and 12:00 at the age of 42 days in the morning. The blood was collected and the serum was collected by centrifugation and stored at -20 ° C until analysis. Serum IGFBPs from 208 males and 161 females were analyzed by Western ligand blot analysis (Hossenlopp, P., Seurin, D., Segovia-Quinson, B., Hardouin, S., and Binoux, M. (1986) Analysis analysis of serum insulin-like growth factor binding proteins using western blotting: analyst biochem., 154, 138-143). Serum samples were diluted 1: 5 with sample buffer [50 mM Na ₂ HPO ₄ , pH 7.0; 1% (w / v) sodium dodecyl sulfate (SDS); 50% (w / v) glycerol], boiled for 5 minutes and electrophoresed on an SDS-polyacrylamide gel (5% stacking gel / 12% separating gel) using the Mini Protean, II ^™ system (Biorad, Munich, Germany). The separated proteins were transferred to a nitrocellulose membrane (Millipore, Eschborn, Germany). The blots were blocked with 1% fish gelatin and incubated with [ ¹²⁵ I] IGF-II (10 ⁶ cpm per blot). All incubations and washes were performed at 4 ° C. Binding proteins were visualized on a Phosphorimager Storm (Molecular Dynamics, Krefeld, Germany) and quantified using the ImageQuaNT software (Molecular Dynamics). The signal intensities were corrected for the background and normalized using different standards on different blots ( 6 ).

Serum levels of IGFBPs are reported as relative densitometric values (RDV) over the standard. The sum of all IGFBPs was quantified and defined as total IGF binding capacity. Since IGFBP-2 is regulated differently in mice selected for high body weight and inhibits the body growth of transgenic mice (Hoeflich, A., Wu, M., Mohan, S., Foll, J., Wanke, R., Froehlich, T., Arnold, GJ, Lahm, H., Kolb, HJ, and Wolf, E. (1999) Overexpression of insulin-like growth factor-binding protein-2 in transgenic mice reduces postnatal body weight gain. "Endocrinology 140, 5488- 5496), the amount of IGFBP-2 relative to the total IGF binding capacity in serum (relIGFBP-2) was calculated, whereby the variation in total serum IGFBP levels between different mouse strains was taken into account.

Quantification of IGF-I Serum Concentration:

IGF-1 was determined by an ELISA in serum after acid-ethanol extraction as previously described (Kratzsch, J., Blum, W.F., Schenker, E., Keller, E., Jahreis, G., Haustein, B., Ventz, M., and Rotzsch, W. 1993. Measurement of insulin-like growth factor I (IGF-I) in normal adults, patients with liver cirrhosis and acromegaly: experience with a new enzyme immunoassay. Exp. Clin. Endocrinol. 101: 144-149).

Marker:

The markers have been evaluated for their high variability between appropriate inbred lines from the mouse genome database (MGD, MOUSE GENOME DATABASE, Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Main, World Wide Web (URL: http: // www .informatics.jax.org /) June, 1998). Mouse MapPair primers were obtained from Research Genetics (Huntsville, AL, USA). Over 630 microsatellite markers were tested for informative parental alleles before the parents and F ₂ were genotyped for 93 loci covering all chromosomes with an average separation of 14.1 cM. The marker map is shown in Table 10. Table 10. Couplers for linkage analysis

Example 5

Identification different expressed genes

mouse lines

The Study was conducted with the mouse lines DU6 and its inbred derivative DU6i as lines that are on high body weight were selected. The internal control line of the selection experiment DUKs and the commercially available Inbred strain DBA / 2 (Harlan Netherlands, Horst) were used as contrast lines for the present experiments used. Recently, the same mouse lines for the QTL mapping in crossed populations (Brockmann et al 1998 loc cit, Brockmann et al. 2000 loc. cit., Brockmann et al. 2001 loc. cit.).

The Selection lines DU6, DU6i and the control line DUKs are from Original crosses of four basic populations (NMRI orig., Han: NMRI, CFW, CF1) and 4 inbred populations (CBA / Bln, AB / Bln, C57BL / Bln, XVII / Bln) of the Research Institute of Agricultural Biology Livestock, Dummerstorf, Germany (pupil 1985 loc cit.). The breeding line DU6 was for over 100 Generations of high body weight at an age of 42 days (Bünger et al., 1990 loc cit.). The age of 42 days was the age of selection in all generations. This age corresponds to the end of the juvenile phase of ontogenesis. After 42 days, both the animals have the selection lines and those of the unselected contrast lines the period of fast Growth completed and are post-pubertal.

The Animals were ad libitum fed with a breeding diet containing 12.5 MJ / kg metabolic Energy and an average content of 22.5% crude protein, 5.0% Raw fat, 4.5% raw fiber, 6.5% crude ash, 13.5% water, 48% N-free Extract, vitamins, trace elements, amino acids and minerals (Diet 1314, Altromin, Lage, Germany).

tissue

epididymal Adipose tissue was produced by males, who had not fasted, between 9:00 and 12:00 o'clock in the morning collected. The animals were 42 days old. After the decapitation was immediately collected and weighed the epididymal fat. Same amounts of tissue of 20 animals were within each mouse line in liquid nitrogen Shock frozen and frozen at -80 ° C until preparation stored the RNA.

manufacturing of hybridization probes

For each of the four mouse strains DU6, DU6i, DUKs and DBA / 2, pooled RNA samples were prepared. Prior to RNA isolation, equal amounts of frozen tissue samples from 20 individuals were pooled and homogenized under liquid nitrogen. The association has been used to reduce the individual variability that is high in stock breeding populations. The total RNA was prepared from pooled tissue samples according to the acid-guanidinium-thiocyanate-phenol-chloroform extraction protocol of Chomczynski and Sacchi (Chomczynski and Sacchi 1987 Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.) Analytical Biochem 162: 156-159) modified by CLONTECH (CLONTECH Laboratories GmbH, Heidelberg, Germany). 200 mg of pooled tissue was homogenized at room temperature in a denaturing solution consisting of 2.7 M guanidine thiocyanate, 1.3 M ammonium thiocyanate and 100 mM sodium acetate (Potter S., B. Braun Biotech International GmbH, Melsungen, Germany). For the removal of the fat, the completely homogenized sample was dena incubated on ice for 10 minutes to allow the fat to reach the surface. The fat layer was carefully removed from the reaction vessel and the homogenate was transferred to a fresh reaction vessel. Subsequently, cell debris was removed by centrifugation. The supernatant was transferred to a fresh reaction tube and mixed with twice the volume of saturated phenol (1008 phenol, 15% v / v glycerol, 100 mM sodium acetate (pH 4.0), 18.6% v / v deionized water). After 1 minute vortexing and 5 minutes incubation on ice, 1/5 volume of chloroform was added to the sample, the sample was shaken and placed on ice for an additional 5 minutes. Subsequently, the sample was centrifuged to separate the phases. The upper, aqueous phase was transferred to a fresh reaction vessel. The phenol-chloroform extraction was repeated twice. Finally, the RNA was precipitated by the addition of one volume of isopropanol to the aqueous phase. After centrifugation, the pellet was washed in 80% ethanol, dried and resuspended in deionized water. The integrity and purity of the RNA was controlled prior to cDNA synthesis by formaldehyde-agarose gel electrophoresis and spectrometry. The RNA had A260 / A280 rates of 1.8 or higher.

GeneChip hybridization

The Affymetrix GeneChips Mu11K A and B were used as oligonucleotide microarrays for the Analysis of 13,069 probe groups used that over 11,000 mouse-specific genes and ESTs (expressed sequence tags) (Affymetrix, Santa Clara, CA, USA). For hybridization of the tissue-specific RNAs with the GeneChips became the samples according to the recommendations of the Affymetrix User Guide. The synthesis of the first Stranges was reversed by a T7 (dT) 24 primer and Superscript II Transcriptase (Gibco BRL Life Technologies) using 10 mg total RNA sample was performed. The synthesis of the second strand was according to the Syperscript Choice System (Gibco BRL Life Technologies) using E. coli DNA polymerase I, Ligase performed by E. coli and RNaseH. The smoothing of the fragment ends became performed using T4 polymerase. An in vitro transcription reaction was used to biotin-11-CTP and biotin-16-UTP in the cRNA probe (BioArray HighYield RNA Transcript Labeling Kit, Enzo). The fragmented cDNA was over Night at 45 ° C hybridized to the quality to ensure the probe. "Spikes" were added to the hybridization mix introduced and test 2 arrays (Affymetrix) were a day earlier hybridized. Washing was done using the GeneChip Fluidics station (Affymetrix) according to the protocol carried out by the manufacturer. Dyeing was followed by R-phycoerythrin-streptavidin (Molecular Probes) from an antibody amplification procedure using a biotinylated anti-streptavidin antibody (Vector Laboratories) and goat IgG (Sigma) performed. The scanning was done with a resolution of 3μm, an excitation wavelength of 488nm and an emission wavelength of 570nm using of the GeneArray scanner (Hewlett Packard).

data analysis

The Expression levels were determined using the programs GeneChip Expression Analysis Software System Data Mining Tool (Version 1.2.) "And" Microarray Suite (Version 4.0.1.) "By Affymetrix calculated. At first were the hybridization signals on each GeneChip using all Probe groups scales to the differences in the overall signal intensities between to minimize two arrays, resulting in a more reliable detection of biological relevant changes allowed in the sample. The "decision matrix" of Affymetrix was used to existing, barely existing and non-existent transcript sets to evaluate a gene in a tested RNA. Then followed pairwise comparisons of normalized gene expression signals between GeneChips containing either RNA of a selected mouse line or with RNA of a control mouse line were hybridized. Hybridization of GeneChips with DU6 and DU6i probes as selected mouse lines as well as DUKs and DBA / 2 as unselected mouse lines was used as a kind of replica. The crosswise comparisons DU6 versus DUKs and DU6i versus DUKs on the one hand and DU6 versus DBA / 2 and DU6i versus DUKs on the other were used as replicate comparisons. Genes that are considered positive Hybridization signals detected on at least one of the GeneChips were, were for used the gene expression comparison. Genes that are in the selection line significant compared to the control mouse line as baseline or marginally increased or were degraded, were considered as potentially differentially expressed genes selected. One group of consensus genes was made out of the four pairs Compare between the two selected (DU6 and DU6i) and the two unselected mouse lines (DUKs and DBA / 2) were selected, the all genes included, which were differentially expressed in all four comparisons. Expression differences between the mouse lines are called x-fold increase (positive values) or x-fold reduction (negative values) of gene expression in the selected Mouse line compared with the expression in the unselected Indicated mouse lines.

mapping of genes

All Genes representing the consensus pool of differentially expressed genes in epididymal adipose tissue between large and fat selected and unselected mouse lines were generated using the Sequence information of gene-specific oligonucleotides on the Affymetrix GeneChips, as well as the TIGR, LocusLink, Ensembl and the Human Mouse Homology Database mapped in silico. The mapping position was accepted when a specific single hit in the various databases was found, and the results of the human-mouse homology database consistent with the other results. In cases, at which the mapping position can not be reliably identified was able to use a radiation hybrid mapping Mouse Hamster Radiation Hybrid (RH) panels (leftovers, Huntsville, AL, USA). Gene-specific primers for the RH mapping was for the region of the gene that produced the oligonucleotide sequence the GeneChips included.

Real-time quantification the transcript quantities

The results from the hybridization of the Affymetrix GeneChips with the pooled samples of the selected and non-selected mouse lines, respectively, were used for transcripts of selected differentially expressed positional candidate genes in individual epididymal adipose tissue RNA samples from 5 males at 42 days of age Controlled line. These 5 animals differed from the animals used for the pooled probes for the gene chip hybridization. To verify the hybridization pattern obtained by the Affymetrix GeneChips, real-time fluorometry was performed during the PCR using the FastStart DNA Master SYBR Green I kit in a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). Total RNA of each individual was extracted using the RNeasy kit (QIAGEN, Hilden, Germany) by a method comprising tissue homogenization and selective RNA adsorption on silica gel membranes in the presence of a high salt buffer system. The RNA was quantitated spectrophotometrically at 260 nm and additionally quantified by densitometry of RNA on ethidium bromide stained agarose gels. 1 μg total RNA was used for the RT-PCR in a total volume of 10 μl per reaction. The RT-PCR was performed in parallel for total RNA of adipose tissue from all 20 animals (5 animals per line). CDNA synthesis was performed in the presence of 50 ng oligo-dT (12-18) primers and 10 units of M-MLV RT (H) reverse transcriptase (Promega Corp., WI, USA) according to the manufacturer's recommendations. For the subsequent quantitation of transcript levels, one equivalent of 10 ^-1 to 10 ^-2 μg total RNA was used in the PCR reaction (depending on the gene-specific transcript levels expected from the hybridization signal intensities on the GeneChips). The gene-specific primers for expression analysis were generated from the sequence regions corresponding to the sequence positions of the oligonucleotides representing the gene on the MU11K GeneChips. The primer sequences for gene-specific transcript amplification as well as the PCR conditions are listed in Table 11 (11A: adipose tissue, 11B: hypothalamus). The individual expression data of the genes tested were normalized by the expression level of the RPS29 gene, which was found to be non-differentially regulated in our mouse model. All 20 animals (5 animals per line) were analyzed simultaneously for one gene in a PCR quantitation round. The relative copy number was calculated based on an external standard curve. The x-fold change in transcript levels was calculated as the ratio of the means of normalized copy numbers of all animals of a selected mouse line versus a control mouse line. Positive values indicated higher transcript levels in the selection line compared to control animals; negative values represent genes with lower transcript levels on the selection line compared to the control line. Student's t-test was used to compare normalized transcript levels between different mouse lines.

Example 6

Identification of DNA variants

The DNA sequences of the genes differentially expressed in adipose tissue were determined using Tables 12A (Adipose tissue, promoter area). and 12C (adipose, 3'-untranslated Range) primer determined. The DNA sequences of the hypothalamus are differentially genes were expressed using the methods shown in Tables 12B (Hypothalamus, promoter area) primer determined.

following Protocol was for it observed:

1. Extraction of genomic DNA sequences from the Ensembl database

Around the DNA sequences of the promoters and the 3 'untranslated regions of the selected candidate genes to be able to investigate The genomic sequences were first extracted from the Ensembl database. For the We have the genomic studies of the promoter area Sequences from 1000 bp upstream to 500 bp downstream of the transcription start point every gene of interest exported. For the 3 'untranslated regions of the selected candidate genes were 1000 bp downstream of the last exon from the ensembl database extracted. In the extracted genomic DNA sequences were specific primers for the sequencing of the promoter or the 3'-untranslated Regions derived.

2nd primer design

The specific primer for the sequencing of the promoter or the 3'-untranslated regions, which in the Sequencing were used with the program Primer3 derived.

primer properties

location the primer in the promoter region: Forward primer should be about 1000 bp. upstream of the transcription start point, reverse primer should be in the 1st exon.

location the primer in the 3'-untranslated Region: Forward primer should in the last exon, reverse primer about 1000 bp. downstream of the are the last exons.

In the following, the forward primers and reverse primers are also referred to as F or R primers.
Length of the PCR product: 1200-1300 bp.
Length of the primer: about 20 nt (min 20 nt, max 25 nt)
Melting temperature (T _m ) of the primers: optimally 65 ° C
Difference in annealing temperature between forward and reverse primer: max. 1 ° C

In dependence from the read range in the sequence reaction was one within the first PCR fragment lying additional primer for the following Sequencing to bridge the Gap derived.

3. Preparation of genomic target DNA single mice

The Isolation of the genomic DNA was carried out from the mouse tail with corresponding to the DNeasy Tissue Kit from Qiagen (Hilden, Germany) of the manufacturer's protocol. DNA concentration and quality determination was carried out with the photometer from Pharmacia Biotech (Freiburg, Germany). The measurement was made at 280 nm. For subsequent PCR, the DNA concentration to 20 ng / μl with AE buffer (elution buffer) from the DNeasy tissue Kit from Qiagen set.

4. PCR conditions

PCRs were performed on target specific region-derived primers and isolated mouse tail genomic DNA to amplify the promoter or 3 'untranslated regions of interest, respectively, to provide sufficient material for subsequent sequencing. PCR pipetting scheme for volumes of 10 μl reaction mixture: genomic DNA 1.0 μl (20 ng / μl) 10x reaction buffer 1.0 μl dNTP mix 0.8 μl (10 pmol / μl) MgCl ₂ 0.2 μl (25 mmol / μl) Primer F 0.5 μl (10 pmol / μl) Primer R 0.5 μl (10 pmol / μl) Taq DNA polymerase (Promega, Mannheim, Germany) 0.05 μl (5U / μl) water 5.95 μl PCR program in thermocycler (Biometra, Göttingen, Germany) for amplification: denaturation: 3 min at 94 ° C Followed by 38 cycles denaturation: 15 s at 94 ° C annealing: 1 min at 65 ° C extension: 1 min at 72 ° C Followed by extension: 7 min at 72 ° C 1 min at 15 ° C

5. Characterization of amplification products by agarose gel electrophoresis

Around the quality and specificity they were tested for the amplification products obtained in the PCR applied to a 0.8% agarose gel and separated by electrophoresis and visualized by ethidium bromide. The electrophoresis was at 80 V, 90 mA above 1 hour performed.

6. Cleaning of PCR products

For cleaning DNA amplification fragments immediately after PCR became high Pure PCR product Purification Kit from Roche (Mannheim, Germany) uses. The concentration determination of amplification products carried out semi-quantitatively by means of a concentration series, since a photometric measurement at very low DNA concentrations too big Inaccuracies leads.

7. Sequencing reaction

The sequencing was overlapping on both sides. The target amount of PCR product used was dependent on the length of the fragment and was adjusted according to the recommendation of ABI. During the sequencing reaction, the PCR fragments were again amplified directionally (with F or R primer) and fluorescently labeled. The ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits v2.0 from Applied Biosystems GmbH (Weiterstadt, Germany) was used for this purpose. The sequencing was carried out according to the protocol given by the manufacturer.

Example 7

Generation of chromosome substitution lines (CSS)

According to the experimental plan (see 4 ), a goat of the line DU6i was mated to several females of the control line DBA / 2 for the production of the CSS. Repeated backcrossings to the inbred line DBA / 2 serially transferred one chromosome of the selected line to the genetic background of the recipient line.

For the control the transmission of the target chromosome and for simultaneous control of the genetic background, the entire genome was 148 informative covered by genetic microsatellite markers.

Body weight in the inbred line DU6i was 3.6-fold higher than in DBA / 2 mice. According to the increasing part of the DBA / 2 genome and the concomitant reduction of the DU6i genome, the body weight in the CSS decreased with each backcross to the DBA / 2 strain. Differences in body weight on day 42 between CSSs and DBA / 2 mice in different backcross generations (BC) are in 5 shown at the time of generation of generations BC4 and BC5. Not all CSS were created at the same time in these generations, so no data is shown for some lines. After eight generations of backcrossing, significant effects of the DU6i chromosomes on body weight at the age of 21 days were observed for chromosomes 1, 3, 6, 7 and 14. For the weight gain between the 21st and 42nd day, the chromosomes showed 2, 4, 8, 11, 14, 15, 16, and 17 effects. Chromosomes 2, 5, 11, and 17 were crucial for weight gain between the 42nd and 63rd day. The greatest effect on body weight was observed on chromosome 5, which effect was significant at all ages. The data confirm the results obtained by linkage analyzes in crossover populations.

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (12)

A method of identifying agents for influencing the body mass and fat accumulation, characterized in that non-human mammals administered to a substance under investigation and determining their influence on the expression of the following genes:

wherein the substance is an active substance which influences the body mass and the lipid charge, if the expression of one or more of said genes is changed by administering the substance. Process according to claim 1, in which the active substance for influencing the body mass and the fat formulation is an active substance which is suitable for limiting (reducing) the lipid charge, characterized in that a substance to be examined is administered to non-human mammals and their influence determined on the expression of said genes, wherein the substance is a body mass and the fat mixture limiting (reducing) active ingredient, when by administration of the substance, the expression of one or more of the genes

is increased and / or the expression of one or more of the genes

is lowered. A method according to claim 1, in which the active substance for influencing the body mass and the lipid charge is an active substance which is suitable for increasing the lipid charge, characterized in that a substance to be investigated is administered to non-human mammals and their influence on the expression of the following genes, where the substance is a body mass and fatliquoring enhancing active agent when, by administration of the substance, expression of one or more of the genes

is increased and / or the expression of one or more of the genes

is lowered. Method according to one of the preceding claims, characterized characterized in that the non-human mammal is a rodent. Process for the preparation of a pharmaceutical Composition for influencing the body mass and the fat deposit, characterized in that one carries out a method according to claim 1 and the identified active ingredient is suitable for administration formulated pharmaceutical composition. Process for the preparation of a pharmaceutical Composition for lowering the body mass and the fat mixture, characterized in that one carries out a method according to claim 2 and one the identified active ingredient to a suitable for administration formulated pharmaceutical composition. Process for the preparation of a pharmaceutical Composition to increase the body mass and of the fat mixture, characterized in that a process according to claim 3 performs and adding the identified drug to one for administration formulated suitable pharmaceutical composition. Process according to claims 5 to 7, characterized that the non-human mammal is a Rodent is. Use of a method according to claim 1 identified active ingredient for the preparation of a pharmaceutical Composition for influencing body mass and fat deposits. Use of a method according to claim 2 identified active ingredient for the preparation of a pharmaceutical Composition for lowering body mass and fat. Use of a method according to claim 3 identified active ingredient for the preparation of a pharmaceutical Composition to increase the body mass and the fat approach. Use according to claims 9 to 11, characterized that the non-human mammal used in the method is a rodent.