Setting up a conditional knockout mouse protocol can be a challenging prospect, but it’s also very useful for modern genetic research. Knockout mice have helped scientists and genetic researchers find out more about various genes and understanding the mammalian genome at its core to a greater extent than ever before. Today, protocols such as the Cre-lox recombination system are commonplace, and researchers are also coming up with newer, more complex conditional knockout protocols like the Red-pathway-mediated recombination.
Before covering conditional knockout mouse protocol methods, it’s necessary to highlight the crucial role played by knockout mice. Knockout mouse models are genetically altered mice that are meant to exemplify the way in which turning off a certain gene can affect the body as a whole. Since they were first developed, knockout mice and conditional knockout mouse models have helped scientists find improved treatments and cures to a number of diseases. Moreover, they have even assisted in tackling problematic and often fatal illnesses such as heart disease, diabetes, different forms of cancer, Alzheimer’s disease and many more. With newly developed conditional knockout mouse protocols, it is now possible for researchers to study the genomic changes that happen when a gene is impaired much more closely than in the past.
Conditional knockout mouse protocol techniques and procedures for altering the mouse genome are primarily based on conditional knockouts. This concept was first introduced quite recently in the world of genetics, and is based on the goal of eliminating a specific gene that targets a particular tissue within the body. For example, conditional knockouts can be developed to impair a particular function that only affects the liver or to alter genes that affect the proper functioning of the heart. These methods are extremely valuable due to the possibilities they present, allowing scientists to closely monitor how the impairment of a certain gene can affect the functioning of an organ or the integrity of a specific type of tissue within the body.
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The Cre-lox recombination system is the most commonly utilized system for generating knockouts in any types of animals. Used primarily for knockout mice, the reason why the Cre-lox system is used is because of the ease with which the Cre enzyme is able to recognize the two lox sites within the mammalian DNA and trigger the recombination process between them. This process then leads to the deletion or inversion of specific genes within the lox, depending how they are oriented. This system is most often used with the help of chemicals like tetracycline, which can be introduced to trigger a conditional gene knockout at a specific time.
A more advanced conditional knockout mouse protocol is the Red-pathway-mediated recombination, which was designed to capture various genomic fragments into a Gateway-compatible vector. This technique is used together with other recombineering techniques and simple in vitro gateway recombinations to study the effects of precisely targeted gene disruptions at shorter notice. These types of conditional knockout mouse protocol methods can be used without the need for advanced lab equipment, and are considered more precise than past techniques.
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