Contributor: Guo Xiaochong Department of Animal Science, China Medical University
Write to the readers who read the literature:
I am here to write some experimental animal knowledge that I might encounter when I look at the literature. I hope to help everyone. In order to facilitate understanding, the language is popular and may not be rigorous enough. If you want to know more about experimental animal expertise, you are welcome to take experimental zoology.
The first episode is about knockout mice.
In Knockout Mice, a complex method is used to make a gene in a mouse not express, so that the mouse exhibits a state of deletion of the gene, which can be used to study the function of the gene.
However, if a gene is particularly important, and the gene deletion may have embryonic lethality, then we will not be able to obtain this knockout mouse, so people have invented conditional gene knockout technology. This technique allows a gene to be silenced at a specific time, within a particular cell or tissue. The method is to first insert a DNA sequence called LoxP on both sides of the target gene (that is, the gene to be knocked out) (LoxP sequence is a 34 bp DNA sequence, 13 bases at both ends are palindrome sequences, middle The 8 bases determine the direction of LoxP. The specific sequence is as follows: 5' - ATAACTTCGTATA - ATGTATGC - TATACGAAGTTAT - 3'
3' - TATTGAAGCATAT - TACATACG - ATATGCTTCAATA - 5'). Then we need to use a transgenic mouse with the Cre enzyme.
The transgenic mice and Cre enzymes are explained below.
Transgenic mice (Transgenic Mice), using molecular biology methods mice had no mice expressing a gene, the gene is artificially inserted may be human, rat, or other species. The first transgenic mouse on the planet expressed the growth hormone gene in the rat, and the mouse grew to the size of the rat.
The Cre enzyme is an enzyme found in bacteriophages that specifically recognizes LoxP sequences and cleaves DNA between two LoxP sequences aligned in the same direction. As mentioned above, we put the LoxP sequence on both sides of the gene to be knocked out. If we put the Cre enzyme in the nucleus, the target gene is cleaved by the Cre enzyme to achieve the target gene knockout. Thus, transgenic mice capable of expressing the Cre enzyme were produced. By tying the Cre transgenic mouse to the mouse in which the LoxP sequence was inserted, a mouse carrying both the Cre and LoxP sequences was obtained. When Cre encounters the LoxP sequence in the nucleus, the target gene is knocked out.
To control which cell or when to knock out the gene of interest, just control the Cre enzyme. When a Cre-transgenic mouse is produced and a specific promoter is placed in front of the Cre enzyme, the Cre enzyme is expressed in the cell or tissue expressed by the promoter, so that only the target gene in the tissue or the cell is knocked out.
The method of controlling gene knockout in time is more subtle. The Cre enzyme gene has been modified, and the modified Cre gene can be transcribed into mRNA or translated into a protein on a ribosome. However, this protein does not have the ability to cross the nuclear membrane, and thus cannot enter the nucleus to function as a DNA-cleaving sequence. When people administer the drug tamoxifen (TM, an estrogen analog) exogenously, the modified Cre enzyme gains the ability to cross the nuclear membrane. Cre then enters the nucleus, meets the genome, and cleaves the sequence between LoxP. So people realized when to administer, when to knock out the gene (of course, give Cre a little time to cross the nuclear membrane). It should be noted that Cre is an enzyme, and like all enzymes, its effect will not be 100%. Perhaps Cre is expressed in 100 cells, and genes in about 70 cells are knocked out.
more content:
Knockin mice, genes knocked into mice. When transgenic mice are made, the foreign gene is randomly inserted into the genome, and it is not necessary to insert the specific position. The gene knock-in technology can accurately insert the foreign gene into the specified position. The principle and method are the same as Knock out, but we don't talk about the principle here. Initially, people used Knockin technology to prepare Cre mice, and Cre was inserted behind the ROSA26 gene, because ROSA26 is a gene expressed in all cells. Cre is placed behind this gene to obtain mice that express the Cre enzyme systemically. Later, Knockin technology was applied in more ways, such as placing the green fluorescent protein (GFP) gene at the end of the target gene to fuse the target gene with GFP. Then, the cells expressing the green fluorescence were observed by fluorescence microscopy. , cleverly found the expression position of the gene.
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