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How A Knockout Mouse Is Made

How A Knockout Mouse Is Made by INGENIOUS

How a Knockout Mouse is Made – Types of Methods and Specific Procedures

Understanding how a knockout mouse is made is not necessarily an easy task. In recent years, with advancements in technology and the development of methods that can be easier to understand, the science has become available for transgenic facilities to streamline the knockout mouse process and make it more straightforward than ever before. Creating knockout mice can involve different techniques for producing the desired gene knockout outcome, each with specific advantages depending on the goals of the knockout mouse study.

Knockout Mice: The Beginning of the Process

Before starting on the main steps of creating a knockout mouse, researchers need to generate some critical starting materials. One key starting material is embryonic stem cells (ES cells) harvested from mouse embryos as early as four days after fertilization. These cells will subsequently be used to propagate the knockout gene throughout the genetic makeup of the adult mouse. The advantage of using these types of cells is also that, with their help, the knockout mice can still be created up to 10 years after the cells were initially harvested. Once the ES cells are procured, scientists use one of two primary methods for obtaining a knockout mouse model.

Gene Targeting – A Method of Creating a Knockout Mouse

How a knockout mouse is made depends firstly on the method used for mutating the mouse gene of study. The homologous recombination process is a robust method researchers can use, allowing for specific gene targeting. The method involves the introduction of an artificial gene sequence that is directly based on the target gene sequence, thus the term homologous recombination. The homologous sequences are positioned to flank the existing gene on both sides (“upstream” and “downstream”). In the ES cell, the cellular process of homologous recombination takes over, identifying the matching parts of the sequence and supporting replacement of the original piece of DNA with the engineered gene knockout sequence.

 

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How Does the Gene Trapping Knockout Process Work?

Another strategy of how a knockout mouse is made is known as gene trapping. This method also makes use of ES cells, and it involves researchers using a more random process for introducing an artificial gene and thus knocking out a native gene that is disrupted by that insertion event. An artificial reporter gene can be randomly inserted into the cell’s DNA, and this gene is designed to impair the cell’s natural RNA splicing process when it has been inserted into a native gene locus. The reporter gene’s activity informs researchers about the expression and function of the mouse gene that has been interrupted by the gene trap. Subsequently, these gene trap modified ES cells can be injected into early-stage mouse embryos which can then be used to produce the gene trap knockout mice.

How Knockout Mice Differ from Knockin Mice

Knockout and knockin mice might seem similar, but their purposes are generally different. Knockout mice are designed to have a deleted or mutated gene sequence which renders the gene non-functional (or “null”), whether as a specifically engineered deletion (conventional knockout) or as a reporter expressing construct (gene trap knockout). In contrast, knockin mice are often designed for studying specific mutations of a target gene of study, such a point mutation that results in expression of a desired mutant protein from the modified gene. Knowing how a knockout mouse is made thus opens the possibility of how certain knockin mouse methods work as well.

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Using the Knockout Mice Method to Find Relevant Gene Information – Essential Steps and Useful Information

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