10. Genomic methods for complex diseases
10.3. Animal models
expression level to an internal control, or housekeeping gene (Housekeeping genes are typically constitutive genes that are required for the maintenance of basic cellular function, and are expressed in all cells of an organism; and their expression level is similar under normal and pathophysiological conditions. Such a gene is e.g. β-actin.) Later specific equipment was developed (real time PCR) for the quantification of the gene expression, and then came the microarrays, with which the expression of all genes could be measured on one chip or array. First, the accuracy and reproducibility of the measurement were not very good and it was very expensive, but later the methods improved considerably and became significantly cheaper, thus it could get into the standard diagnosis methods in some areas.
Nowadays, there has been approaching a novel method, which will probably replace the traditional (but not too old) method. This method is the sequencing, or RNA-seq, which means isolation of RNA sequences, often with different purification techniques to isolate different fractions of RNA followed by high-throughput sequencing (NGS). The advantage of this method relative to the microarray that it provides information on differential expression of genes, including gene alleles and differently spliced transcripts; non-coding RNAs; post-transcriptional mutations or editing; and gene fusions (http://en.wikipedia.org/wiki/RNA-Seq).
10.2.9. Additional microarray-based methods
Several new microarray-based methods have been developed in the recent years. After discovering of the miRNA, products have appeared in the market, with which these could be measured.
Array-comparative genomic hybridization (array CGH) is a technique to detect CNVs. It can be used to create a virtual karyotype, and is capable of detecting new structural variations in tumor tissues, and can be used in prenatal or preimplantation diagnosis, or diagnosing the genomic background of birth abnormalities (http://en.wikipedia.org/wiki/Comparative_genomic_hybridization).
ChIP-on-chip array is a technique that combines chromatin immunoprecipitation ("ChIP") with microarray technology ("chip"). It is used to investigate interactions between proteins and DNA in vivo. The ChIP-seq is the same method using NGS.
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• There are different mouse strains, which differ from each other in disease susceptibilities or other phenotypes. These can be used as animal-models, or through crosses we can study the connection between segregation of genetic markers and phenotypes. These animals can be kept in strictly controlled environments, thus the effects of these can be easier studied.
• At gene level human is not so different from the rest of the animals. The essential genes are usually the same; we differ from the mouse only in 300 genes. But, species like Drosophila melanogaster (fruit fly) are also widely used, and a lot of pathways (like Hippo pathway, which is conserved and plays an important role in organ size control) were first discovered in this animal.
Already two Nobel prizes have been given because of the studies of this species (http://en.wikipedia.org/wiki/Drosophila_melanogaster).
• There are a lot of experiments which in humans may not be carried out from ethical reasons, only in animals.
• There is much easier to get tissues from animals (lung, brain etc.) and measure gene expression, etc. The diagnoses of diseases are much more accurate.
• There are a lot of animal-models for different human diseases.
• There is a possibility to develop genetically modified animals.
Let us see the last two points in more detail. There are two basic types of genetically modified animals used in these studies. One of them is the knock out or KO animals. In KO animals researchers inactivate, or "knock out," an existing gene by replacing it or disrupting it with an artificial piece of DNA.
Among them the mice are the most significant for studying the role of genes which have a known sequence, but whose functions have not yet been determined (http://en.wikipedia.org/wiki/Knockout_mouse). By causing a specific gene to be inactive in the mouse, and observing any differences from normal behavior or physiology, researchers can infer its probable function.
In 2006, 33 research centers in 9 countries founded the International Knockout Mouse Consortium (IKMC), then in July 2011 the International Mouse Phenotyping Consortium aiming to build a huge, shared resource for biomedical research. Mouse embryonic stem cells have been produced, in which researchers have “knocked out”
each of the more than 20,000 specific mouse genes that code for proteins. By growing mice from these cells, researchers can gain insight into the role that the missing genes play in health and disease. The phenotyping effort will aim to probe the anatomy, development, physiology, behavior, and disease traits of 5000 of these mouse lines by the end of 2016 (http://news.sciencemag.org/scienceinsider/2011/09/the-consortium-that-will-launch-.html).
Knocking out genes can also be used for animal-models for different diseases (Table 10.1). In these animals the molecular pathomechanisms or different therapies can be studied.
The other types of the genetically modified animals are called transgenic. Here, the genes are over-expressed, or new genetic information is inserted into the mouse genome. These animals can be used for the same aims as the KO animals.
The over-expressed genes are usually under the regulation of promoters with strong activity. It is also possible that the promoters are only active in certain organ or tissue.
In this way the gene will be over-active only in this organ. E.g. SCGB1A1 is expressed only in the lung, but here it is highly expressed. IL5 gene was introduced after the promoter region of this gene in a mouse strain. The mouse over-expressed the IL5 in its
lung. As IL-5 is an eosinophil colony-stimulating factor, it is a major regulator of eosinophil accumulation in tissues, and can modulate eosinophil behavior at every stage from maturation to survival, IL5 over-expression caused eosinophilia in the lung of this mouse, and asthmatic symptoms developed.
Earlier it was difficult to study essential genes in animals, because lack or over-expression of these genes are often lethal in embryonic development. To avoid these problems conditional transgenic animal models were developed. In these animals the gene will be inactivated or induced in vivo. One of the best-known methods for this is the Cre-Lox recombination. Cre-Lox recombination is a site-specific recombinase technology which allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus.
RNA interference can also be used for transient in vivo gene inactivation. In Caenorhabditis elegans all the genes were studied by inactivating them with RNAi. This method can also be used in more developed animals, even in humans.
10.3.2. Shortcomings of animal models
It is true that the difference between animals and human at gene level is relative small.
But as it turned out (see ENCODE project), about 80% of the genome have certain functions. And at genomic levels the differences are larger. Comparing human and mouse it is also true that some processes are different, or homolog genes can have different functions. Because of these, all the results from animal models have to be confirmed in humans or human systems.
Similarly, the similar diseases can have different pathomechanisms, and there are human diseases for which no good animal models have been developed so far.
E.g. the gene resistin have been discovered in mouse white adipose tissue, which expressed it, and this expression caused insulin resistance, one of the main characteristics of T2DM. Later it turned out that in humans resistin was expressed in macrophages.
10.3.3. Experimental disease models
Because different diseases can be developed by gene manipulations, this can be utilized for developing experimental disease models. It can also be carried out by inducing random mutations by mutagenic agents, then by phenotyping the animals, different strains can be developed by different crosses. Afterwards by genomic screening the genomic background of the diseases can be found out. This method is also capable of studying complex diseases. Such mice are produced e.g. in the Jackson Laboratory (http://jaxmice.jax.org/), from where mice strains with a given phenotype can be ordered. One of the widespread methods for this is ENU mutagenesis. ENU, also known as N-ethyl-N-nitrosourea, is a highly potent mutagen. For a given gene in mice, ENU can induce 1 new mutation in every 700 loci. Mutation is usually induced in male mice, which are then crossed with a wild type female. The G1 progeny can be screened to identify dominant mutations. However, if the mutation is recessive, then G3 individuals homozygous for the mutation can be recovered from the G1 males (see:
http://en.wikipedia.org/wiki/ENU).
There are databases with mouse phenotypes, like Mouse Phenome Database which characterizes strains of laboratory mice to facilitate translational discoveries and to assist in selection of mouse strains for experimental studies (http://phenome.jax.org/db/q?rtn=docs/aboutmpd).
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There are some widespread animal models for polygenic diseases, like Non-obese diabetic (NOD) mouse, spontaneously hypertensive rat (SHR), Dahl salt sensitive rat, New Zealand Obes (NZO) mouse, etc.
Gene Modification Animal Diseases
LDLR KO mouse atherosclerosis
APOE KO mouse atherosclerosis
Ob (LEP in humans:
Leptin)
KO (ob/ob) mouse obesity
LPR (Leptin receptor)
KO (db/db) mouse obesity
TBX21 KO mouse asthma
ANP KO mouse hypertension
SNCA over-expression drosophil
a Parkinson disease Mutant
APP over-expression mouse Alzheimer disease
Table 10.1. Some animal models for human diseases developed by manipulating of a gene