Gene knockout is a method where a gene of interest is deleted in order to observe phenotypic effects of the knockout on the organism. With conditional gene knockout, the deletions can be induced in a specific organ at a specific time in development, rather than being deleted from birth.
Image Credit: Sashkin/Shutterstock.com
Knockout studies yield a great deal of information about gene function. For example, if a total knockout is lethal to the organism early in life, any other effects are impossible to study. However, with a conditional knockout, the gene may be activated during embryonic development, but the deleterious effects can be observed in the adult organism.
The Cre-loxP recombination system is the most commonly used system for conditional gene knockout. The Cre recombinase enzyme enables recombination of DNA by allowing the exchange of information between two strands, resulting in deletion or inversion between the two target loci.
The system can be activated with the addition of agents like tetracycline, an activator of Cre recombinase transcription, and tamoxifen, which transports Cre recombinase into the nucleus. Cre recombinase is not present in mammalian cells, so the knockout activity can’t be activated incidentally.
Conditional self-knockout in retinal degeneration
One example of a developmental gene that remains active in the adult organism is Otx2. In early life, it is a regulator of forebrain and head development, but in adult mice, Otx2 is strongly expressed in the retina.
In humans, Otx2 has been found to play a role in retinal disease and ocular malformations. In a conditional knockout study of Otx2 in mice, scientists saw progressive alterations of retinal pigment epithelium (RPE) and photoreceptor cells. The end result was complete loss of photoreceptors.
The study authors conclude that Otx2 plays a direct role in maintaining those cells, and specifically the process of melanogenesis. The study also proved that embryonal and adult Otx2 protein functions are significantly different.
The findings had a significant impact on the study of human disease, as it was proven that many degenerative diseases of the retina are not developmental pathologies, but rather occur in adults due to other processes. In addition, mutations in Otx2 could represent a new target for the treatment and prevention of late onset retinal disease.
Gene editing
Although Cre-loxP is still the preferred technology for creating conditional knockouts, CRISPR-Cas9 gene editing has also been used for this purpose.
An example of the application of CRISPR-Cas9 for creating knockouts is a 2014 study in Caenorhabditis elegans somatic cell lineages. With the goal of developing a CRISPR-Cas9 knockout system, the researchers targeted an embryonic gene, Coronin, which in humans is associated with neurobehavioral dysfunction.
Coronin regulates actin organization and cell morphology during the process of postembryonic neuroblast migration and neurogenesis in C. elegans. The results of the study showed that Coronin works with another gene, Cofilin, to modulate F-actin organization in migrating neuroblasts.
The advantages of using the CRISPR-Cas9 approach were that it enables rapid production of conditional knockouts, requiring only 1 week in total and that the technology can also produce multiple knockouts at one time.
Some limitations were that the molecular lesions induced by CRISPR-Cas9 are heterogenous, whereas Cre-loxP produces lesions that are precisely defined. In addition, the efficiency of the CRISPR-Cas9 method was quite variable.
Cre-Lox Recombination
Credit: Abnova/Youtube.com
Further Reading
Genetic engineering study explored new strategies to combat heat stress in potato crops