Gene Targeting

Understanding Gene Targeting and Its Practical Applications in Genetics

Scientists and students interested in gene targeting are at the forefront of one of the most advanced and exciting research areas in human history. The ability to alter specific genes and replace them through homologous recombination has been perfected recently, but it is based on studies dating back to Sternberg and Hamilton’s 1981 research. Gene targeting soon gained worldwide recognition when, in 2007, scientists Mario Capecchi, Martin Evans and Oliver Smithies received the Nobel Prize in Physiology and Medicine using the process to introduce gene modifications in mice with the help of embryonic stem cells.

What Is Gene Targeting?

The gene replacement strategy through the use of homologous recombination is a gene-altering technique that uses genetic recombination to alter a specific endoneous gene. It can be used to delete genes and exons, change individual base pairs through the introduction of point mutations, and introduce specific conditions for the purpose of triggering the modifications it facilitates. It typically requires the generation of a particular vector for each gene that it targets, and it can be utilized on any gene, regardless of its size, function or transcriptional activity.

Permanent vs. Conditional Targeting

One of the main practical advantages of gene targeting is that it doesn’t have to be permanent. In many cases, permanent targeting solutions are used to obtain changes that are expected to stay in place permanently once they are made. However, with the help of conditional targeting techniques, scientists are able to exercise a greater degree of freedom on the modified genes. They can introduce specific conditions that are accurately targeted to trigger the genetic change at a certain time during the organisms’ development, or when a certain tissue-related limitation condition is met.

Using the Targeting Process

There are several gene targeting methods that are currently in use. Moss and laboratory mice are among the most frequently used test subjects, and each require specifically different targeting techniques. For mice, the targeting process involves the injection of altered DNA into mouse embryonic stem cells, in a culture. In the case of moss-based experiments, the DNA is incubated in isolated protoplasts, and then moss filaments are directly screened, typically through the use of antibiotics. These are not the only organisms where genes have successfully been targeted and modified. Sheep, cattle, swine and a variety of fungi were also successfully used as test subjects. Even though these methods still represent remarkable advancements in science, they still have to be perfected, and they are not without their limitations.

Gene Trapping and Targeting

The homology regions of gene targeting cassettes require a specific process that adapts them to a particular gene that is aimed for modification. In contrast with this requirement, gene trapping utilizes a different process whereby scientists apply the random insertion of a cassette. While gene trapping is easier to apply to larger projects, targeting is far more precise and effective when it comes to aiming for specific results. Smaller genes are just as easily targeted as larger ones, and the procedure can also be utilized on genes with low transcriptions that would normally be ignored when it comes to gene trapping.

Practical Gene Mutations and Other Applications

The targeting process has already led to many important successes in the field of genetics and medicine. It is used to study human diseases and the effects that the manipulation of specific genes might have on them. Knockout and knockin mice are primarily used for this purpose. Also, the development of specific genetic mutations in humanized mice have led to the development of accurate human disease models that would have been impossible to create without the use of human test subjects in the past. While gene targeting is still in its infancy when we think of its potential in the next few decades, it lays the foundation for what might be possible in the near and far future with advanced genetics.