疾病动物模型,基因敲除小鼠模型 z-bio 2021-03-08 310

　　Rats and mice can be said to be stars in life science laboratories. Successful rat and mouse models can be said to be "excellent assistants in life sciences." Many people may want to design or understand conditional knockout mice. .

　　"Conditional knockout mice are designed using Cre/LoxP or Flipe/Frt principles. They are all site-specific recombinase systems. Here we take the Cre/LoxP system as an example. For example, place the loxP sequence at both ends of the target DNA sequence to be knocked out to obtain flox (flankedbyloxP) mice. Flox mice are bred with Cre expressing mice to obtain mice that knock out target genes in specific cells, that is, conditional knockout mice. In addition, when combined with other induction systems that control Cre expression (such as CreERT2), genes can be controlled simultaneously in time and space.

　　The  Cre/loxP system is derived from bacteriophages and can mediate site-specific DNA recombination. The system consists of two components. One is a 34 bp long DNA sequence (LoxP sequence), which contains two 13 bp inverted repeats and an 8 bp core sequence. The direction of LoxP is determined by these eight bases in the center. The LoxP sequence is the site recognized by Cre recombinase. One component is Cre recombinase. A protein composed of 343 amino acids encoded by bacteriophages. Cre mediates the recombination of the two LoxP sites, which may result in the deletion of the DNA sequence between the two LoxP. By genetically modifying Cre recombinase cDNA under tissue or cell-specific promoters, Cre mice (also known as Cre tool mice) that specifically express Cre tissue/cells can be obtained. After mating with Flox mice, you can get conditional knockout mice. The so-called Flox mouse is a LoxP sequence flanking a specific exon of a specific gene. LoxP (also known as Flox mice) Franked the sequence. Such Flox mice are usually obtained by designing and constructing targeting vectors, recombination of embryonic stem cells, blastocyst microinjection and passage of chimeric mice. This mouse is mated with the Cre tool mouse, and Cre expression mediates the recombination of the two LoxP site sequences, thereby knocking out the sequence between the two LoxP site sequences. Different Cre tools can achieve the goal of specifically knocking out target genes in different tissues and cells due to the tissue/cell specificity of mouse Cre expression. Epithelial cells, thymocytes, T cells, B cells, cardiomyocytes, intestines, lungs, etc.

　　How to design the conditional knockout mouse The design mentioned here is mainly the Flox mouse design. The so-called conditional knockout means that there is no abnormal gene expression in all cells except one cell. Under normal circumstances, do not place the LoxP sequence before the first exon. The first exon is usually the promoter. Placing the LoxP sequence can destroy or change the promoter activity. Conditional knockout is usually the knockout of the exon that first caused the frameshift mutation. In this case, it is best not to knock out the exon at the initiation codon ATG. Otherwise, the gene may use the ATG of the ORF to encode a protein that lacks part of the N-terminal sequence, and this part may have all or part of the function of the wild protein. If you choose an exon to knock out (there is a LoxP on each side of the exon), the base of the exon cannot be 3N. Otherwise, the mRNA spliced by the pre-RNA of the new gene will not be able to produce frameshift mutations. Compared with the wild protein, the new protein it produces lacks intermediate sequences. If the number of bases in the exon is 3N +1 or 3N + 2, a frameshift mutation will occur after knocking out the exon, which can achieve the purpose of gene knockout. When screening the exons to be coagulated, the most upstream exon is usually selected as the appropriate exon. Please keep in mind that common DNA analysis software cannot determine the boundary between introns and exons and should be carefully checked. More than 95% of the boundaries follow the gt/ag boundary principle. You can also see the exons and introns of the gene on Ensembl. Most of the results on this website are correct. However, it needs to be carefully checked. After all, the development of knockout mice is a relatively long process and requires special attention. There are not many things to consider in the early days. These principles are only considerations for general topics. In special circumstances, special treatment is required. For example, if you want to knock out a specific domain of a gene, or the exon of the gene is very large, even if the exon has 3N bases, you can knock out at this time.