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, however, with advancements in technology and the development of several methods that are typically easier to understand than what had been used in the past, the science has become available to a number of transgenic facilities that have streamlined the process to make it more straightforward than ever before. Creating knockout mice can involve a number of different techniques that are all equally effective, albeit each has its own specific advantages when it comes to using them for particular purposes.
Knockout Mice: The Beginning of the Process
Before starting on the main steps of how a knockout mouse is made, researchers need to follow a few strict procedures. They will first harvest embryonic stem cells (ES cells) from mouse embryos as early as four days after fertilization. These cells will 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, knockout mice can still be created up to 10 years after the cells were initially harvested. Once the cells are procured, the 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 obtaining it. The homologous recombination process is the first method researchers use, and it has to do with specific gene targeting. The method involves the introduction of an artificial gene sequence made to mimic the gene completely. The homologous sequence is positioned to flank the existing gene on both sides (upstream and downstream). Next, the ES cell’s nuclear system takes over, identifying the identical parts of the sequence and automatically replacing the original piece of DNA with the new sequence.
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 complex and random process for introducing an artificial gene. An artificial reporter gene is added randomly to the cell’s DNA, and this gene is designed to impair the cell’s natural RNA “splicing” process. The reporter gene’s activity tells researchers of how the mouse tissue’s existing gene behaves and allows them to subsequently introduce the modified ES cells into early-stage mouse embryos that are then implanted for fertilization.
How Knockout Mice Differ from Knockin Mice
Knockout and knock-in mice might seem similar, but their purposes are entirely different. As shown, knockout mice are designed to replace a gene sequence entirely and render it unusable or for erasing a gene in a random area of the genome in the case of transgenic mice. In contrast, knockin mice are designed for studying the specific mutations of a targeted gene in a specific location within the cell. Knowing how a knockout mouse is made opens the possibility of exploring the details of how certain knock-in methods work as well.