Technical serialization: Application of Cre-loxP site-specific recombination system in the study of conditioned target gene function in mice

Author: Yu Xiaofeng, Chief Scientist of Saiye Biology/Vice President of China

Highlights of the article

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Generally speaking, the strategy of knocking Cre directly into the translation start site of a specific endogenous gene often destroys the expression of the gene at the same time
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Studies have found that, due to the knock-in of Cre into important related genes such as growth and development or disease pathways, the corresponding Cre mice have potential non-specific phenotypes that affect gene function
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For example, in the Foxg1-Cre knock-in mouse model, heterozygous Cre mice have developmental disorders of the mouse forebrain
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Therefore, this strategy is only suitable for related genes that are heterozygously deleted and have no phenotype
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The use of IRES and 2A knock-in strategies avoids the lack of heterozygosity phenotype that may be caused by Cre specific site knock-in
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Regarding the comparison of the two linkage knock-in strategies of IRES and 2A peptide , it is generally recognized that the traditional IRES knock-in strategy will hardly affect the function of endogenous genes.
The expression components, or the removal of the remaining related FRT sequences and other remaining partial sequences, may cause structural changes in the 3'UTR region of the endogenous gene, thereby interfering with the expression level of Cre, making the actual expression level relatively lower than that of the endogenous gene Itself
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The new 2A peptide linkage and knock-in method, the expression of Cre is almost similar to the expression of its endogenous gene, but after cutting the 2A peptide, the remaining about 17-21 amino acid sequence can be attached to the endogenous gene c-terminal protein, which has a potential impact Possibility of endogenous protein function
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Studies have shown that different 2A peptide sequences (P2A, T2A, E2A, F2A) have different self-cleavage efficiencies
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Nevertheless, the application of knock-in 3'end strategy has continuously won its appeal
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BAC transgenic Cre mouse model : After knocking the Cre gene into the specific gene regulatory element sequence of the BAC clone, construct a BAC cloning vector, and then use this BAC vector with pronuclear injection of fertilized eggs to construct a BAC transgenic Cre mouse model
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Although the transgenic technology is also a random integration insertion, it has the advantages of reducing the positional effect caused by the random insertion of plasmid DNA and avoiding the loss of heterozygosity that may be caused by the knock-in of the 5'end of the Cre gene
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It has the advantages of phenotype .
In addition, compared to the expression plasmid transgene with short promoter, BAC vector contains more gene expression-related regulatory element sequences, so it can better reflect the expression characteristics of endogenous genes
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However, the first established mice of BAC transgene will still have different Cre expression activities due to the different insertion sites of the BAC cloning vector
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Therefore, this method cannot solve all the shortcomings of traditional transgenic technology , such as the local influence of the chromatin structure of the insertion site, and the potential risk of gene insertion mutations that cause unreal expression of Cre
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Moreover, while constructing BAC transgenic Cre mice, it may also increase the copy number of the corresponding genes, which interferes with the objective analysis of related gene function research
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Nevertheless, compared to the traditional Cre expression vector with a smaller promoter, BAC transgenic technology still has its obvious advantages
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Safe site knock-in Cre mouse model : The latest Cre mouse model construction strategy
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In order to avoid the influence of many unfavorable factors that may be caused by random gene insertion (such as expression position influence and insertion mutation, etc.
), Cre is targeted to the so-called "safe" sites, such as Rosa26, Hprt and Hipp 11 sites
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Among them, the Rosa26 locus is the most commonly used
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The Rosa26 locus is characterized by the fact that it contains a wide range of promoters for gene transcription to form mRNA, but no translated protein
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Therefore, this gene has no specific function
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Borrowing the characteristics and advantages of the gene locus itself, it is helpful to construct the whole body expressing Cre gene single copy site-directed knock-in mouse model
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Hprt and Hipp11 sites are more suitable for constructing tissue-specific Cre mouse models
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Why build an inducible and regulated CreERT mouse model and its basic characteristics

The specificity and characteristics of the Cre mouse model introduced above are determined by the characteristics of the gene promoter that guides Cre expression or the construction strategy
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Because of the expression of many so-called specific genes, it runs through the embryonic development and maturation process of the entire cell/tissue
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The so-called cell/tissue-specific Cre mice constructed from such gene-specific promoters may have Cre activity starting from the embryonic stage of the mouse
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If the target gene to be studied is a very important gene for mouse growth and development, the early implementation of the Cre-loxP recombination system may lead to the knockout of the target gene during embryonic development, resulting in germline knockout mice.
It is difficult to achieve the purpose of specifically knocking out target genes in cell tissues
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Therefore, how to control the time of Cre expression so that it can be inducible and regulated, that is, through the use of inducers, the time of Cre expression can be controlled, combined with cell/tissue specific Cre characteristics, to achieve spatiotemporal conditional target gene knockout/expression The purpose of the research
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The so-called inducible regulation CreERT system is constructed by fusing the Cre gene with the estrogen receptor (ER) ligand binding domain mutant (ERT)
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The ERT only binds to synthetic ER ligands, such as 4-hydroxytamoxifen (the active metabolite of the drug Tamoxifen)
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In the absence of the inducer, the CreERT fusion protein binds to HSP90 in the cytoplasm and exists in the cytoplasm in an inactive state, and cannot play the role of the Cre-loxP recombination system
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When the drug inducer Tamoxifen (TAM) is provided to CreERT/floxed mice, the Cre protein is separated from the HSP90 protein, enters the nucleus, recognizes the loxP site in the target gene, and exerts the cell/tissue specificity and time specificity of the Cre-loxP site The recombination and cutting effect
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CreERT mice have only one mutation site (G521R) in the ligand binding domain of the estrogen receptor.
The currently commonly used CreERT2 mice introduce 3 mutation sites in the corresponding ligand binding domain (C400V/M453A/L544A).
), greatly improving its sensitivity and specificity to the inducer TAM
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The ability to induce Cre-loxP recombination cleavage at a specific time and location undoubtedly greatly improves the flexibility of experimental research
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At the same time, it also increases the challenge for researchers to actually apply the inducible Cre-loxP-specific recombination system.
For example, for the possible leakage of Cre expression and possible toxic effects of the system, researchers need to apply it in practice.
Zhongdou is cautious
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Challenges and importance of identification of Cre insertion sites in Cre transgenic mouse models

Among the commonly used Cre mouse models, the mouse models constructed using traditional transgenic strategies still account for the largest proportion
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However, for transgenic mouse models, the research on the identification of random gene insertion sites accounts for very little
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Analytical data show that less than 5.
2% (416/8012) transgenic mouse models, studies have reported random gene insertion sites, of which only about 2.
3% (36/1631) transgenic Cre mouse strains, clearly analyzed The insertion sites suggest the challenge of analyzing and identifying random insertion sites of transgenes
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Why do we need to identify and analyze random insertion sites of transgenes? First, it is conducive to the design of specific genotype primers for transgenic mice and the identification of homozygous Cre mice; second, it is conducive to the identification and analysis of related genotypes and the design of specific PCR primers in the experimental research of conditional target genes ; Third, understand the undesirable phenotypes caused by potential insertion mutations, such as the direct or indirect endogenous gene coding sequence caused by random insertion, or the damage and change of nearby related regulatory sequences, and the complex caused by reversal/duplication.
structural change and so on
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Targeted locus amplification (TLA) technology can effectively analyze and identify random transgene insertion sites and the characteristics of inserted genes
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Some researchers have applied this technology to analyze and study more than 40 commonly used transgenic Cre mouse models, and found that there are 30 Cre mouse models, due to random insertion of genes, resulting in structural changes in the corresponding genome, including Twenty-four kinds of mice showed deletion of gene structure, and 6 kinds of mice showed structural duplication
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The endogenous genes of 50% of transgenic Cre mice are damaged, and most of them are the deletion of large DNA fragments and/or the structural changes of genes related to the insertion site
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The change in gene structure caused by random insertion can induce undesired related phenotypes, which affects the accuracy of the experimental results of Cre-loxP recombination specific target genes
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Links to the following text:

Technical Serial No.
2: Application of Cre-loxP site-specific recombination system in the study of conditioned target gene function in mice

Technical Serial No.
3: Application of Cre-loxP site-specific recombination system in the study of conditioned target gene function in mice