An Introduction to Gene Targeting

What is a knockout mouse?

A knockout mouse is a laboratory mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA. The loss of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable physical and biochemical characteristics.

Knocking out the activity of a gene provides valuable clues about what that gene normally does. Humans share many genes with mice. Consequently, observing the characteristics of knockout mice gives researchers information that can be used to better understand how a similar gene may cause or contribute to disease in humans.

Examples of research in which knockout mice have been useful include studying and modeling different kinds of cancer, obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease. Knockout mice also offer a biological context in which drugs and other therapies can be developed and tested.

How are knockout mice made?

Researchers begin by harvesting embryonic stem (ES) cells from early-stage mouse embryos four days after fertilization. ES cells are used because they are able to differentiate into nearly any type of adult cell, which means that if a gene is knocked out in an ES cell, the effects can be observed in any tissue in an adult mouse. In addition, ES cells grown in the lab can be used to make knockout mice as long as 10 years after they were harvested.

Gene targeting or homologous recombination is the method researchers use to specifically manipulate a gene in the nucleus of an ES cell. Typically, this is done by introducing an artificial piece of DNA that shares identical, or homologous, sequence to the gene. This homologous sequence flanks the existing gene's DNA sequence both upstream and downstream of the gene's location on the chromosome. The cell's own nuclear machinery automatically recognizes the identical stretches of sequence and swaps out the existing gene or portion of a gene with the artificial piece of DNA. Because the artificial DNA is inactive, the swap eliminates, or "knocks out," the function of the existing gene. This kind the knockout is often referred as constitutive knockout or traditional knockout, as oppose to the conditional knockout described below.

For gene trapping, the vehicle used to ferry the artificial DNA into ES cells often consists of a linear fragment of bacterial DNA. After the artificial DNA is inserted, the genetically altered ES cells are grown in a lab dish for several days and injected into early-stage mouse embryos. The embryos are implanted into the uterus of a female mouse and allowed to develop into mouse pups.

The resulting mouse pups have some tissues in which a gene has been knocked out - those derived from the altered ES cells. However, they also have some normal tissues derived from the non-altered embryos into which the altered ES cells were injected. Consequently, they are not complete knockout mice. It is necessary to crossbreed such mice to produce lines of mice in which both copies of the gene (one on each chromosome) are knocked out in all tissues. Researchers refer to such mice as homozygous knockouts.

What is conditional knockout?

While knockout mice technology represents a valuable research tool, some important limitations exist. About 15 percent of gene knockouts are developmentally lethal, which means that the genetically altered embryos cannot grow into adult mice. The lack of adult mice limits studies to embryonic development and often makes it more difficult to determine a gene's function in relation to human health. In some instances, the gene may serve a different function in adults than in developing embryos.

To overcome these drawbacks, the conditional knockout approach allows researchers to delete the gene of interest in a time- and space-dependent manner. A Cre-loxP or Flip-FRT system is used to excise a critical part of the gene. Often a Cre or Flip transgenic mouse is crossed with the knockout-ready mouse with LoxP or Flip sequences flanking the critical part of the gene. Timing and space-dependence is achieved by the choice of promoter used to drive the Cre gene.

What is knockin?

In contrast to knockout in which a gene or part of a gene is deleted, knockin is the replacement of a gene by mutant version of the same gene using homologous recombination. Knockin is very useful when establishing a disease model of a specific disease-related mutation in human gene.