How Things Work: Gene Targeting
Earlier this month, biologists Mario Capecchi, Martin Evans, and Oliver Smithies split the Nobel Prize in Physiology or Medicine for their work on gene modification in mice.
The technique for which the three scientists are credited is called “gene targeting.” It is the favored method for creating “knockout mice.”
A knockout mouse is a genetically modified mouse that has had one of its genes purposely deactivated.
Once a gene is deactivated, scientists can make observations and determine the specific function of the gene.
As an analogy to gene deactivation, imagine walking into a room full of power sockets and light switches. Each socket is connected to a switch, and, initially, you do not know which socket corresponds to which switch.
To determine which switch corresponds to which socket, you would have to perform a trial-and-error experiment.
This is precisely what these three scientists have done, only their experiments spanned decades and led to groundbreaking discoveries in genetics.
To understand gene targeting as a whole, one must understand the science underlying each individual’s discovery.
Capecchi studied a natural process called “homologous recombination” in organisms.
Homologous recombination produces an exchange of genetic material between two strands of DNA, leading to genetic variation in a population. It was first discovered in 1958 by Joshua Lederberg.
By 1985, Capecchi discovered that homologous recombination can occur between genes in mammalian cells and artificially introduced DNA. He discovered how to demonstrate that injected DNA can be used to repair damaged DNA through naturally occurring recombination.
Through this technique, Capecchi was able to modify genes. However, his modifications could only target genes with respect to their particular activity.
Smithies, while working independently on a genetic treatment for blood diseases, discovered that genes can be targeted regardless of their activity. That is, Smithies showed that all genes can be modified by homologous recombination.
One should note that the cells used in Capecchi’s and Smithies’ experiments were incapable of breeding gene-targeted mammals. This breeding would require a cell that is part of the germ line.
The germ line of an organism is a line of cells that carries genetic material and is able to pass it on to offspring.
This is where Evans enters the scene. Evans is widely known as the “leader of the stem cell revolution” for recognizing the pluripotent characteristic of embryonic stem (ES) cells. Pluripotent cells can produce all types of specialized tissues.
According to the Nobel Prize website, Evans experimented on mice, and he identified and isolated “the embryonic stem cell of the early embryo, the cell from which all cells of the adult organism are derived.”
Evans was able to show that stem cells could be used to introduce genetic information into the germ line. This was done by injecting one mouse embryo with embryonic stem cells from a foreign germ line, thus creating a “mosaic embryo” that carries stem cells from both germ lines.
A surrogate mother was then impregnated with the mosaic embryo. The resulting offspring were carriers of genes from the foreign germ line.
The stage was now set for gene targeting.
First, embryonic stem cells were harvested from a donor mouse. Then, the gene targeted for inactivation was to be identified and isolated.
Once the gene was identified, scientists constructed a retroviral vector (viral RNA) containing the inactive version of the gene. The embryonic stem cells were then infected with this engineered vector.
Here is where homologous recombination plays a role in gene targeting. The stem cells reproduce naturally, all the while propagating the inactivated gene from the virus throughout the group of stem cells.
The result of this propagation is a healthy population of embryonic stem cells, some infected with modified genes and some that are not.
A method called “Positive-Negative Screening” is then used to separate the two groups. The “normal” cells are discarded and the modified stem cells are injected into a mouse embryo. The engineered embryo is then injected into a surrogate mother.
About 20 days after the injection, “chimeric mice,” which are mice containing genetic material from both the engineered stem cells and the host embryo are born.
Finally, the chimeric mice are bred with normal mice to produce a final liter of mice. This final liter contains both normal mice and “designer mice” that carry and are able to pass down the inactivated gene.
Through gene targeting, ailments such as cancer, diabetes, Parkinson’s disease, and various heart diseases can be modeled in knockout mice and closely studied by researchers.