Knockout genetics and knockout mice

SIGNIFICANCE: In knockout methodology, a specific gene of an organism is inactivated, or “knocked out,” allowing the consequences of its absence to be observed and its function to be deduced. The technique, first and mostly applied to mice, permits the creation of animal models for inherited diseases and a better understanding of the molecular basis of physiology, immunology, behavior, and development. Knockout genetics is the study of the function and inheritance of genes using this technology.

Knockout Methodology

Before knockout mice, transgenic animals had been generated in which “foreign” DNA was incorporated into their genomes in a largely haphazard fashion; such animals should more properly be referred to as “genetically modified.” In contrast, knockout technology targets a particular gene to be altered. Prior to the creation of transgenic animals, any genetic change resulted from spontaneous and largely random mutations. Individual variability and inherited diseases are the results of this natural phenomenon—as are, on a longer time frame, the evolutionary changes responsible for the variety of living species on the earth. Spontaneously generated animal models of human inherited diseases have been helpful in understanding mutations and developing treatments for them. However, these mutants were essentially gifts of nature, and their discovery was largely serendipitous. In knockout mice, animal models are directly generated, expediting study of the pathology and treatment of inherited diseases.

In a knockout mouse, a single gene is selected to be inactivated in such a way that the nonfunctional gene is reliably passed to its progeny. Developed independently by Mario Capecchi at the University of Utah and Oliver Smithies of the University of North Carolina, the process is formally termed “targeted gene inactivation,” and, although simple in concept, it is operationally complex and technically demanding. It involves several steps in vitro: inactivating and tagging the selected gene, substituting the nonfunctional gene for the functional gene in embryonic stem cells, and inserting the modified embryonic stem cells into an early embryo. The process then requires transfer of that embryo to a surrogate mother, which carries the embryo to term, and selection of offspring that are carrying the inactive gene. It may require several generations to verify that the genetic modification is being dependably transmitted.

Usefulness of Knockout Mice

Knockout mice are important because they permit the function of a specific gene to be established, and, since mice and humans share 99 percent of the same genes, the results can often be applied to people. However, knockout mice are not perfect models, in that some genes are specific to mice or humans, and similar genes can be expressed at different levels in the two species. Nevertheless, knockout mice are vastly superior to spontaneous mutants because the investigator selects the gene to be modified. Mice are predominantly used in this technology because of their short generation interval and small size; the short generation interval accelerates the breeding program necessary to establish pure strains, and the small size reduces the space and food needed to house and sustain them.

Knockout mice are excellent animal models for inherited diseases, the study of which was the initial impetus for their creation. The Lesch-Nyhan syndrome, a neurological disorder, was the focus of much of the early work with the knockout technology. The methodology has permitted the creation of previously unknown animal models for cystic fibrosis, Alzheimer’s disease, and sickle-cell disease, which will stimulate research into new therapies for these diseases. Knockout mice have also been developed to study atherosclerosis, cancer susceptibility, and obesity, as well as immunity, memory, learning, behavior, and developmental biology.

Knockout mice are particularly appropriate for studying the immune system because immune-compromised animals can survive if kept isolated from pathogens. More than fifty genes are responsible for the development and operation of B and T lymphocytes, the two main types of cells that protect the body from infection. Knockout technology permits a systematic examination of the role played by these genes. It has also proven useful in understanding memory, learning, and behavior, as knockout mice with abnormalities in these areas can also survive if human intervention can compensate for their deficiencies. Knockout mice have been created that cannot learn simple laboratory tests, cannot remember symbols or smells, lack nurturing behavior, or exhibit extreme aggression, conditions that have implications for the fields of education, psychology, and psychiatry.

Developmental biology has also benefited from knockout technology. Animals with minor developmental abnormalities can be studied with relative ease, whereas those with highly deleterious mutations may be maintained in the heterozygous state, with homozygotes generated only as needed for study. The generation of conditional knockouts is facilitating study of the genes responsible for controlling the development of various tissues (lung, heart, skeleton, and muscle) during embryonic development. These genes can be explored methodically with knockout technology.

By 1997, more than one thousand different knockout mice had been created worldwide. A primary repository for such animals is the nonprofit Jackson Laboratory in Bar Harbor, Maine, where more than two hundred so-called induced mutant strains are available to investigators. Other strains are available from the scientists who first derived them or commercial entities licensed to generate and sell them.

Double Knockouts, Conditional Knockouts, and Reverse Knockouts

Redundancy is fairly common in gene function: Often, more than one gene has responsibility for the same or similar activity in vivo. Eliminating one redundant gene may have little consequence because another gene can fulfill its function. This has led to the creation of double knockout mice, in which two specific genes are eliminated. Double knockouts are generated by crossing two separate single knockout mice to produce double mutant offspring. Consequences of both mutations can then be examined simultaneously.

Some single knockout mice are deleteriously affected during embryonic development and do not survive to birth. This has led to the generation of conditional knockout mice, in which the gene is functional until a particular stage of life or tissue development triggers its inactivation. The approach is to generate animals with two mutations: The first is the addition of a new gene that causes a marked segment of a gene to be deleted in response to a temporal or tissue signal, and the second is to mark the gene that has been selected to be excised. In these animals, the latter gene remains functional until signaled to be removed.

Knockout methodology involves generation of loss-of-function or null mutations. Its reversal would permit the function of an inoperative gene to be restored. This reversal has been successfully accomplished in mice with the correction of the Lesch-Nyhan defect. Further experimentation may permit it to be applied to humans and other animals. Such targeted restoration of gene function would be the most direct way for gene therapy (the process of introducing a functional gene into an organism’s cells) to cure inherited diseases.

Key terms

• embryonic stem cella cell derived from an early embryo that can replicate indefinitely in vitro and can differentiate into other cells of the developing embryo
• genomethe total complement of genetic material for an organism
• in vitroa biological or biochemical process occurring outside a living organism, as in a test tube
• in vivoa biological or biochemical process occurring within a living organism

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