Transgenic Techniques

Transgenic techniques have been used for a number of goals: to determine an unknown gene’s function; to analyze the malfunction of a mutated gene; to model human disease; and to provide better agricultural and pharmaceutical products by making transgenic plants and animals. For example, insect-resistant transgenic plants have been engineered. While the benefits of modified plants and animals are far-reaching, there is debate about the ethics of genetically altering plants and animals, and the impact these alterations may have on the environment.

There are several ways to introduce a transgene into the organism. Microinjection is one of these techniques. As its name suggests, microinjection is the process of injecting the transgene into the nucleus of a cell where it is randomly inserted into the host genome. This technique, initiated in 1981, is most commonly used to generate transgenic mice. DNA is injected into the nucleus of a fertilized egg, which is then transferred to a foster mother. If the introduced DNA becomes integrated into the developing embryo’s genome, the offspring will carry the transgene. Two other techniques for random insertion are retroviral and transposable element insertion.

An important application of transgenic technology, introduced in the 1990s, is gene targeting, or the production of “knock-out” organisms. The term “knock out” refers to the ability to disrupt a specific gene, so that it no longer encodes a complete protein. Genes are knocked out by being replaced by a transgene that has been disrupted in vitro, either by the addition of some sequence into the gene itself or by the deletion of part of the gene. Gene replacement occurs when the disrupted transgene is introduced into a cell. Here, it recombines with the recipient’s copy of that gene, inserting itself into the chromosome by homologous recombination.

In mice, transgenes can also be introduced with cultured embryonic stem (ES) cells, using selection techniques to recover the transformed cells. The altered ES cells are then injected into early mouse embryos, and result in a mosaic embryo with normal and transgenic cells. If the altered cells contribute to the germ cells of the mouse, progeny in a subsequent mating will inherit the knocked out gene. These mice can then be mated to produce mice that are homozygous for both copies of the altered gene (both copies of the gene are knocked out). These mice can then be carefully examined to determine what happens when the specific gene is absent. The knockout technique is most commonly applied to mice, insects, and yeast.

Extensions of gene targeting are the “knock-in” approach and conditional mutation. The knock-in approach involves inserting a mutated gene or a similar gene in place of the gene of interest. The newly added gene is expressed at the same time and location as the replaced gene. This method allows scientists to study the effects of mutations in genes as well as discover if certain genes have redundant functions. Conditional mutation is a way of either turning on or turning off the gene of interest, and can be done either in specific tissues or at specific time points.

CRISPR/Cas9 & TALENs

More recently, TALENs (TAL effector nucleases) and CRISPR/Cas9 systems have emerged as powerful new tools for genome modification and transgenics in a variety of organisms and cell types, including mouse, rat, zebrafish, drosophila, C. elegans, and human stem cells and iPSCs (induced pluripotent stem cells).

CRISPR (clustered, regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) and TALENs (transcription activator-like effector nucleases) comprise novel gene editing methods that overcome the challenges associated with previous technologies. Early published research on CRISPR/Cas9, coupled with a growing body of work on TALENs, suggests the potential to pursue therapeutic indications that have previously been intractable to traditional gene therapy, gene knock-down or other genome modification techniques. The CRISPR/Cas9 system, the most recent and exciting approach to emerge, acts by a mechanism in which the Cas9 protein binds to specific RNA molecules. The RNA molecules then guide the Cas9 complex to the exact location in the genome that requires repair. Similarly, TALENs are proteins that can be custom programmed to bind essentially any DNA sequence of interest and to direct gene modification activities to specific targets in the genome. CRISPR/Cas9 and TALENs uniquely enable highly efficient knock-out, knock-down or selective editing of defective genes in the context of their natural promoters, unlocking the ability to treat the root cause of a broad range of diseases.

Source: Biologyreference.com and Editas Medicine