An Introduction to Five Gene Knockout Methods

Posted by beauty33 on February 26th, 2021

A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout. Here, I will introduce five methods.

  1. Conventional Knockout

Conventional gene knockout is through gene targeting, replacing several important exons or functional regions of the gene to be knocked out with Neo Cassette. Such mice do not express the gene product in all tissues and cells in their body. Such knockout mice are generally used to study the effect of a certain gene on the physiology and pathology of mice, and this gene is not embryonic lethal.

  1. Conditional Knockout mice

Conditional knockout mice use gene targeting to place two loxP sites on either side of one or several important exons of the target gene. Before this mouse was crossed with Cre enzyme-expressing mouse, its target gene expression was completely normal. After crossing with mice expressing Cre enzyme specifically, the gene can be knocked out in specific tissues or cells, and the gene is expressed normally in other tissues or cells.

The scope of application of conditional knockout mice is: (1) the gene is embryonic lethal; (2) used to study the physiological and pathological functions of the gene in specific tissues or cells.

  1. Knockin mice (Knockin)

Knock-in mice are through gene targeting, knocking the target gene sequence into the corresponding gene locus of the mouse, and using mouse expression control elements to guide the expression of the target gene.

Such knock-in mice are generally used for drug screening and signal pathway research.

The detailed process of obtaining the chimera and purifying the strain:

  1. Gene knockout mice produced by ZFN technology

ZFN can recognize and bind the designated gene sequence sites and cut them off efficiently and accurately. Cells then use the natural DNA repair process to insert, delete, and modify DNA so that researchers can edit the genome as they please. This was unimaginable in the past. Traditional gene knockout technology relies on homologous recombination that occurs naturally in the cell, and its efficiency is only one part per million, while the gene knockout efficiency of ZFN can reach 10%. Using these technologies for targeted knockout and knock-in of mouse genes can shorten the time from one year to several months.

Designing a specific ZFN in this technology is the most critical link. At present, researchers use computational biology methods to design a highly specific ZFN, but the off target of the ZFN is to cut the place that should not be cut. The problem remains a challenge. For this reason, genetic modification of mice using ZFN technology cannot completely replace traditional techniques.

  1. TALEN technology to make gene knockout mice

TALEN technology is a brand-new molecular biology tool. Scientists discovered that the amino acid sequence of the nucleic acid binding domain of the TAL protein from a plant bacteria has a constant correspondence with the nucleic acid sequence of its target site. Using the sequence module of TAL, it can be assembled into a modular protein that specifically binds to any DNA sequence, so as to achieve the purpose of targeted operation of endogenous genes. It overcomes the inability of the ZFN method to identify any target gene sequence, and the recognition sequence is often affected by upstream and downstream Sequence effects and other issues, while having the same or better flexibility of ZFN, make gene manipulation easier and more convenient. However, due to the problem of off-target, genetic modification of mice using TALEN technology still cannot replace traditional technology.


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