Fifty years after James Watson and Francis Crick discovered the spatial arrangement of DNA, science is standing in the middle of the genome era. Beginning with bacteria and proceeding via the worm Caenorhabditis elegans and the fruitfly Drosophila melanogaster, the genetic code of the most complex organisms has been sequenced, including mouse and man. Yet, the book of the genome remains largely obscure. Even though all characters of the genetic story have been identified, we only understand some words and the meaning of a few sentences, whereas the precise role of many genes remains unknown.
In recent years, the use of genetically engineered animals for functional genetic studies has gained outstanding importance in biomedical research. Particularly, the mouse offers the possibility to overexpress genes in a defined spatial or temporal manner, to inactivate selected genes, or to express mutated genes of the same or different species at a chosen locus. Combinations of these techniques, by sequentially introducing various genetic alterations or by the breeding of several genetically modified mouse lines, offer the possibility to tightly control the expression or the inactivation of genes in a manner that allows the investigator to predetermine not only the type of mutation, but also the developmental time point and the tissue that would be affected.
What can we learn from mutations ?
Despite external morphological differences, the genomes of mouse and man are closely related. Homologous proteins encoded by corresponding genes have mostly identical function and readily interact and cooperate with those of other species. Probably the most straightforward approach that employs animal models in biomedical research is to use mice to replicate a mutation that is known to have a pathologic effect in human patients. It has, for example, long been suspected that a truncating mutation in one allele of the tumour suppressor gene adenomatous polyposis coli (APC) will lead to the formation of a vast number of colon tumours in the affected patient.
In 1992 the group of William Dove published in Science the characterization of a mutant mouse line with the corresponding alteration in the murine APC gene. Affected mice did indeed develop multiple intestinal neoplasias (MIN), tumours similar to those found in human patients with corresponding mutations. The MIN-mouse has since served as a valuable animal model for intestinal tumorigenesis.
This and other tumour models have helped to understand the mechanisms and cellular events that underlie the formation of tumours and their progression to cancer, which is still the primary cause of death in the western population.
Understanding the mechanisms of diseases like Alzheimer's, diabetes, cancer, and various others is the focus of numerous investigations. Whereas some deficiencies are caused by congenital mutations that are easily reproduced in the mouse, others involve somatic alterations that act in a tissue and time-specific manner and can only be copied in their phenotype by using combinations of genetically engineered mouse lines as described above. Even though the process of producing transgenic animals is time consuming and laborious, it has been accepted that the application of specialized animal models in basic research is a prerequisite for the comprehension of the complex pathologies of these disorders and for the subsequent development of specific and refined therapies.
In addition to the generation of genetically modified mice that serve as models for known diseases, transgenic technology can serve as a tool to unravel the molecular and developmental role of genes that have been identified by high throughput sequencing, gene expression projects, or other techniques and have, as yet, an unknown function. This can be performed by the controlled over expression as well as the ablation of the given gene.
In many cases, this will result in morphological defects of the manipulated mouse or mouse embryo. Since for most structures, tissues, and organs in the developing vertebrate organism the basic developmental programmes have already been resolved, these morphological changes can be employed to functionally categorize the given gene. Further genetic and biochemical techniques are then used to find the position of the gene and its corresponding protein in a network of other genes and gene products.
Generation of Animal Models
Basically, there are two different approaches to the generation of animal models. Which approach is used depends on the questions that are to be answered by the analysis of the model. So-called transgenic mice are obtained by the injection of a carefully designed DNA fragment (the transgene) into the nucleus of a fertilized oocyte. A fraction of these injected oocytes will incorporate this DNA fragment at a random position in their genome. Mice that develop from these oocytes will therefore carry the DNA insertion in all cells of their body. In general, the transgenes consist of a sequence that encodes a specific protein and a regulating sequence that controls the expression of the coding sequence in a tissue and time specific manner by utilizing the regulatory machinery that is present in all cells.
In contrast, the generation of targeted mutations, also known as knockout or gene replacement, requires a more complex procedure. DNA constructs, called targeting vectors, have to be designed that carry a central region with the desired mutation. This central region has to be flanked by DNA sequences homologous to both sides of the genomic locus where the mutation is to be introduced. By using embryonic stem (ES) cell technology, and in particular a method called homologous recombination, the targeting vector is used to introduce the desired mutation into the genome of ES cells. The mutated cells are then referred to as targeted or recombinant ES cells. Targeted ES cells are subsequently injected into early mouse embryos, where they participate in the development of the mouse embryo. Thus, the mice develop from the mutated ES cells and the normal early embryos into which they are injected.
Since the early mouse embryos and the ES cells are derived from mouse lines with different coat color (usually black versus white) these chimeric mice can be identified by their mixed coat colour after birth. For the same reason, chimeric mice only carry the mutation introduced into the ES cells in a fraction of their body cells. However, a proportion of their offspring will carry the desired alteration in all cells and are referred to as heterozygous mutants.
The Biomedical Research Campus Berlin-Buch is embedded in the growing biotechnological environment of Germany's capital. The heart and the head of the campus is the Max Delbrück Center for Molecular Medicine (MDC), one of the leading institutions involved in basic research activities in the field of the molecular mechanisms that underlie various disorders. RCC has engaged in a cooperation with the MDC and has brought into existence a Transgenic Core Facility, the TCF Berlin-Buch, that has the aim to serve the MDC with the rapid and cost-effective generation of their custom-tailored animal models. Recently, this service has been extended to clients from other research-based institutions and companies, who are now welcome to request the services of RCC's TCF Berlin-Buch. The cooperation on the campus Berlin-Buch is further intended to combine the experience of both partners in creating high quality animal models, to harness the innovative potential of a vast number of top-class scientists, and to develop new techniques for the generation of genetically modified mice and rats.
-- The author is with RCC Ltd,.www.rcc.ch