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"I’ve been working with iTL over the past 5 years in the production of 3 different genetically altered mice. Not only did iTL help in the design of the mice, […]” - Raghu Mirmira, MD, PhD University of Chicago.
The gene knockout (KO) method is a popular tool for genetic manipulation that researchers often associate with mouse models designed to study specific diseases and develop new treatment methods. Gene knockouts are often targeted towards specific genes and mutations that are meant to achieve a certain genetic change that can later be passed down through generations of gene knockout models. As we will see, there are numerous techniques and approaches designed to maximize the efficiency of the gene knockout method and facilitate a more straightforward path toward studying gene function in great detail.
In gene knockouts, an organism’s genetic makeup is altered by rendering one or more genes inactive (as the name suggests, “knocked out”). The term “knockout” can refer to the specific gene or to the organism itself. Because of the similarity between human and mouse DNA, by investigating the aftereffects of the loss of a specific gene in mice, scientists are able to extrapolate how the loss or damage of that gene in humans can affect the normal functioning of the human body. Researchers consider the knockout technique to be the opposite of the gene knockin method, which involves the addition of a gene. There are also double, triple and even quadruple knockouts, when multiple genes are eliminated from the body’s natural make up.
The conditional gene knockout method is one of the more special KO methods used by scientists today. It involves gene deletion through a specialized system that can remove the gene based on tissue and/or time-specific criteria. This is an extremely useful innovation that scientists continue to use in studying genes and how they function in normal physiology as well as in disease. To achieve conditional gene knockout, IoxP sequences are introduced around the targeted gene to trigger a viral enzyme known as Cre recombinase, which is able to recombine the sequences and delete the gene found between them.
The gene knockout method has numerous potential applications, from studying the effects of genetically induced diseases, to understanding the impact that the deactivation of a certain gene or group of genes can have on the body and on various areas of the brain that have to do with memory and learning. In these cases, the use of specific knockout methods has led to the more accurate study of nuclear signaling and synaptic activities that has laid the foundation for a new field in neuroscience known as molecular and cellular cognition.
Scientists tasked with perfecting the knockout method are aiming towards increasingly simplified techniques and processes that show quicker results. One such process was discovered along with the use of Paenibacillus polymyxa, a special bacteria with the ability to support nitrogen fixation and produce a number of important active biological components. The new knockout system is designed to help researchers understand the bacterium with greater ease, through a system that’s based on marker exchange mutagenesis directly targeting its main competent cells in order to mediate the deletion and substitution of α-and β-amylase genes. This simple system is an example of the advancements scientists have achieved since the initial inception of the gene knockout method.
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