小鼠模型 z-bo 2020-07-29 704

       In the animal laboratory, living mice and rats can be described as stars, and successful mouse models can be called "good helpers for life sciences." Many people may want to design or understand conditional knockout mice.

　　Conditional knockout mouse design utilizes Cre/LoxP or Flipe/Frt principle. These are all site-specific recombinase systems. Take the Cre/LoxP system as an example here. For example, place the loxP sequence at each end of the target DNA sequence to knock out and obtain flox (flankedbyloxP) mice. Breed Flox mice to mice expressing Cre specifically, or conditionally knock out mice, which can knock out target genes in specific cells. In addition, when combined with other induction systems that control Cre expression (such as CreERT2), it can also regulate genes 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. The first 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. It is a protein composed of 343 amino acids encoded by bacteriophages. Cre can mediate the recombination of two LoxP sites and cause the deletion of the DNA sequence between the two LoxP sites. If the Cre recombinase cDNA is genetically engineered under a tissue or cell-specific promoter, then Cre mice expressing Cre tissue/cells can be used, also known as Cre tool mice. After mating with Flox mice, you can get conditional knockout mice. The so-called Flox mouse refers to the LoxP sequence flanking a specific exon of a specific gene. This sequence is flanked by LoxP, also known as Flox mice. Such Flox mice are usually obtained through targeted vector design and construction, embryonic stem cell recombination, blastocyst microinjection, and passaging of chimeric mice. This mouse is bred together with the Cre tool mouse, and Cre expression mediates the recombination of the two LoxP site sequences, resulting in the sequence knockout between the two LoxP sites. The Cre/mouse expression in mice using different Cre tools is tissue/cell specific, thereby achieving the purpose of specifically knocking out target genes in different tissues and cells. Epithelial cells, thymocytes, T cells, B cells, cardiomyocytes, intestines, lungs, etc.

　　How to design conditional culling mice? The design described here is mainly the design of the Flox mouse. The so-called conditional knockout means that there is no abnormal gene expression in cells other than certain cells. 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 to knock out the exon that originally caused the frameshift mutation. In this case, it is best not to knock out the exon at the start codon ATG. If this is not the case, the gene may use ATG in the ORF to encode a protein lacking part of the N-terminal sequence. This sequence may have all or part of the wild protein function.. When selecting the exon to be knocked out (the exon has both sides One LoxP), the number of exons cannot be 3N. Otherwise, the new gene pre-RNA cannot produce spliced mRNA frameshift mutations. Compared with wild protein, a new protein lacking intermediate sequence is produced. When the number of exons is 3N + 1 or 3N + 2, a frameshift mutation occurs after knocking out the exon, and the purpose of gene knockout can be achieved. When screening the exons to be knocked out (Floxed), usually the most upstream exons are screened for exons suitable for knockout. Please note that common DNA analysis software cannot determine the boundary between introns and exons, so it should be checked carefully. More than 95% of the boundaries follow the gt/ag boundary principle. You can also use Ensembl to check the exons and introns of the gene. Most of the results on this website are correct. However, you also need to double check. After all, the development of knockout mice is a relatively long process and requires special attention. Not much to consider in the early stages. These principles are only considerations for general topics. Special circumstances require special treatment. 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 delete it.