Traditional conditional knockout mice

Mice and rats are the stars of life science laboratories, and successful mouse and rat models are good helpers of life science. Many people may want to design or understand conditional gene knockout mice themselves. Here is a summary for your reference.

The design of conditional gene knockout mice uses the principle of Cre/LoxP or Flipe/Frt. They are all site-specific recombinase systems. Take the Cre/LoxP system as an example. For example, a loxP sequence is placed at both ends of the target DNA sequence to be knocked out. Flox (flanked by loxP) mice were obtained. Flox mice were mated with mice that specifically expressed Cre, and the target gene knockout mice in specific cells were obtained, that is, conditional gene knockout mice. In addition, a gene can be regulated in time and space if it is combined with other induction systems (such as CreERT2) to control CREE expression.

Cre/loxP system is derived from phage and can mediate site-specific DNA recombination. The system consists of two parts: one is a 34bp long DNA sequence (LoxP sequence), which contains two reverse repeats of 13 bp and an 8 bp core sequence. The direction of LoxP is determined by the eight bases in the middle. This LoxP sequence is the site recognized by Cre recombinase, which is a protein composed of 343 amino acids encoded by phage. Cre can mediate the recombination of two LoxP sites. Therefore, the DNA sequence between the two LoxP sequences is deleted. If the cDNA of Cre recombinase is placed under a tissue or cell-specific promoter through genetic engineering, a Cre mouse with tissue/cell-specific expression can be obtained, which is also called Cre tool mouse. Conditional gene knockout mice can be obtained after mating with Flox mice.

The so-called Flox mouse refers to placing the LoxP sequence on both sides of the exon of a gene. The sequence is flanked by LoxP, also known as Flox mouse. This kind of Flox mice is generally obtained by designing and constructing targeted vectors, recombining embryonic stem cells, microinjecting blastocysts and passaging chimeric mice. The mouse is paired with the Cre tool mouse. Because of the expression of Cre, it mediates the recombination of two LoxP site sequences, thus knocking out the sequence between the two LoxP sites. Because the expression of Cre in mice with different Cre tools has tissue/cell specificity, the target gene can be specifically knocked out in different tissues and cells, such as epithelial cells, thymocytes, T cells, B cells, myocardial cells, intestines, lungs and so on.