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
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
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.