Conditional Knockout Strategy

Posted by Andrea Brook on February 16th, 2016

Conditional gene knockout is to gain conditional gene expression in specific period, specific tissues and organs, which can be used for study of pathogenic gene function or constructing transgenic animal models to express a gene in specific conditions, specific tissues and organs. The basic principle and implementation method are very similar to those of conditional gene knock-in. While constructing the loxP transgenic animals, there contains a translation termination code on the gene sequence between target gene and promoter, resulting in failure to normal translation of target gene. There is a loxP site on both sides of this segmentation sequence. In the case of the presence of the Cre recombinant enzyme, the deletion sequence of the loxP site can be deleted, so that the target gene can be opened.

Conditional knockout is mainly realized by Cre / 10xP or Ftp / FRT recombination system. These two systems are site specific recombinant enzyme systems, which have been developed as powerful tools for genetic manipulation in vivo and in vitro. The application of these two systems can cause the expression or deletion of the target gene to occur at a certain stage or a particular tissue organ in animal development.

Such gene segmentation containing target gene, gene sequence and loxP sites is usually controlled by the promoter continuously expressing genes(such as p - actin, reduced glyceraldehyde phosphate dehydrogenase), which makes such a functional unit able to continue to obtain the transcription in the organism in different periods, various tissues and organs. However, due to the existence of the segmentation sequence, the target gene can not be translated into a functional protein in these tissues.

Segmentation sequence generally choose the coding sequence of the lacZ or green fluorescent protein (EGFP). The products of those two genes can be used as markers in loxP transgenic animals body to label the presence of target gene.

For Conditional gene knock in, the arrangement of target genes, segmentation sequence, loxP locus and promoter
After building such a loxP transgenic mouse, just let it hybridize with specific CRE transgenic mice, conditional gene knock-in can be achieved. The concrete implementation method is the same with that of conditional gene knockout.

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Andrea Brook

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Andrea Brook
Joined: December 17th, 2015
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