Global analysis of gene function in mammals: integration of physical, mutational and expression strategies

John Schimenti
The Jackson Laboratory
Bar Harbor, ME 04609, USA.
207-288-6402
E-mail: jcs@jax.org

The field of Genetics is undergoing tumultuous change after nearly a century of standard approaches to genetic analysis. The Human Genome Project is providing tools and technologies that are changing the ways that we pursue an understanding of gene function, which is the underlying goal in modern and traditional genetics. In this paper, I overview the directions of the genome project as they relate to gene function analysis in mice and humans, and how various modern technologies are coalescing to address this in a powerful way.

The long-term goal of the genome project is to define and understand all the genes specifying the making of a human being. Ultimately, this knowledge will improve the human condition through genetic diagnostics, gene therapy, and improved understanding of biochemical and physiological processes.

The benefits of the genome project are already being realized. High-resolution genetic maps have been generated for humans and other experimental organisms, leading to several notable successes in positional cloning of disease genes. Over the past few years, the completion of genetic maps has been transitioning to physical mapping of genomes and to the characterization of their functional content.

The sequencing of Expressed Sequence Tags (ESTs) is relentlessly moving towards the identification of nearly all transcribed genes in mouse and humans. These ESTs are being placed onto the genetic and physical maps, further facilitating the process of positional cloning. New technologies are being developed to enable the simultaneous analysis of the expression patterns of thousands of genes on a large scale and under different circumstances [Schena, 1995 #56; Chee, 1996 #83; ; DeRisi, 1997 #58]. The physical mapping stage will culminate in the complete sequencing of the human genome and the genomes of other complex model organisms such as mouse, Drosophila and C. elegans.

The most difficult challenge now lies in devising ways to utilize the vast amount of information gathered in the Genome Project to understand how organisms develop and function. The problem of elucidating function for all the 100,000 or so genes in humans/mammals have spawned a new area of research that is being called "functional genomics." Part of the functional genomics field deals strictly with nucleic acids. The philosophy behind it is that much can be learned about the function of genes by simply isolating them, evaluating their transcriptional pattern during development, and predicting molecular function by inference (based on known functions of related genes or protein motifs). Several efforts aimed at generating ESTs (expressed sequence tags) have collectively revealed most of the transcribed genes in humans and model organisms. This information is essential for the application of "gene chips", which enable a rapid determination of what genes are up- or down regulated under different conditions or disease states.

Despite having the entire genome's sequence, and knowledge of what all the genes are and where they are expressed, a true understanding of a gene's function requires more sophisticated genetic analysis. The basic tool of a geneticist is allelism - a difference in genetic composition that manifests itself in a visible, measurable way. Classically, this would involve a mutant. Indeed, this still applies today. In humans and mice, naturally occurring mutations often take the form of diseases. There are a variety of spontaneous mutants in mice that confer all imaginable types of phenotypes, from coat color alterations to neurological effects. However, since spontaneous mutations are not available for all genes, it is necessary to devise methods for creating mutations on a large scale.

The ability to mutate, or "knock out" genes in vitro in mouse embryonic stem cells has revolutionized our ability to understand the function of genes in the context of a whole organism. However, the mouse field has seriously lagged in one major respect: the ability to perform systematic, phenotype-based screens on a scale large enough to statistically covers the entire genome. The experimental obstacles posed by mammalian model systems have made it impractical to efficiently pursue mutagenesis strategies as in invertebrates such as fruit flies. Recent progress, however, has provided the tools to perform in vivo mutagenesis that is efficient enough to enable phenotype screens. Methods have recently been developed to create large chromosomal deletions in ES cells, making it possible to derive mice bearing sets of nested deletions anywhere in the genome. Deletions are useful in modeling human deletion syndromes, and for mutagenesis schemes designed to characterized all gene functions in a given region of a particular chromosome, a strategy called "region specific" mutagenesis.

The general strategy of region-specific saturation mutagenesis is to cross mice that have been mutagenized by a mutagenic chemical (most notably ethylnitrosourea, or ENU) to mice that bear a known chromosomal deletion. The offspring that inherit the deletion from one parent and an altered/mutated gene from the other parent will display a mutant phenotype. This simple breeding scheme allows phenotypic characterization of a large number of mutagenized gametes - sufficient to reach theoretical "saturation," such that a mutation in every gene in the region should have been produced and scored for a novel phenotype. An attractive aspect of a region-specific mutagenesis is that a panel of nested deletions, when available, can be used for fine mapping of a newly induced mutation by complementation analysis.

From a geneticists point of view, mutations are absolutely required to truly understand the function of a gene in the context of a whole organism. Since it is widely recognized that the mouse is the best animal model for human disease and development, there has recently been a tremendous surge in mutagenesis efforts around the world. In conjunction with the completed sequences of the mouse and human genomes, and the chip technologies to characterize gene expression, we are assembling enormously powerful tools to understand the function of genes. These advances are needed, since the number of genes (about 100,000) that specify the making of a human being will take a long time to understand.