Technical Serial No. 3: 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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In order to avoid or reduce the obvious non-specific off-target effects that some Cre mice may cause, in the application of Cre-loxP recombination system for experimental research of target gene editing, it is necessary to design a reasonable PCR identification primer combination for the purpose of analyzing and identifying target genes.
Different situations of recombination targeting, such as heterozygous knockout, homozygous knockout, non-knockout, and wild control of the gene
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Under normal circumstances, when using the mouse tail DNA identification strategy, when performing the so-called conventional specific genotyping, it should not be detected that the target gene has been knocked out, unless the study is to specifically knock out the target of skin epithelial cells.
Gene
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This is also the key to determine whether Cre mice have germ cell knockout effects
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With such a simple identification method, Cre mouse strains without non-specific cleavage can be screened out very quickly to ensure the validity and reliability of experimental research
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Moreover, target cells and control cell populations can also be effectively separated and detected to clarify the efficiency of specific gene knockout
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Another simple method to determine whether Cre mice have germline activity is to mate bi-sexed Cre mice with reporter mice of the corresponding sex, and obtain positive offspring mice (F1), and then with wild mice Mating, analyze the reporter gene expression of its offspring (F2), if the F2 mouse reporter gene expression is more obvious than its F1, it suggests that the Cre mouse may have germline activity
.

In the practical application of the Cre-loxP recombination system, if the goal is to improve the efficiency of Cre recombination and cleavage, you can first establish a single allele of the target gene, knock out the mouse in the germ cell, and then conditionally target it with floxed.
The strategy of mouse reproduction
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But we must be aware of the limitations of this strategy, that is, it is not suitable for the phenotype of heterozygous knockout of the target gene
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In addition, it is not suitable for experimental research that requires knocking out more than two genes
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4.
The combined application of Cre-loxP recombination system and other recombination systems increases the specificity and effectiveness of cell lineage tracing research

The study of cell lineage tracing is based on the activation of Cre in specific target cells and the expression of the corresponding reporter gene
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The most common reporter gene is a fluorescent protein, whose expression specificity and spatiotemporal characteristics completely depend on the characteristics of specific Cre activating recombination
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Therefore, the combination of Cre/CreERT2 mice and reporter mice is often used in cell lineage tracing, behavior, and knockout cell distribution in mosaic tissues
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In order to improve the accuracy and effectiveness of cell lineage tracing, it is important to accurately and effectively label and trace specific cell populations and certain types of special cells in their descendants
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If the reporter gene is non-specifically expressed in other cell populations, or even if the correct target cell population marker is expressed, but does not appear in the expected time window of the research, it may lead to erroneous experimental research results
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The so - called Cre-loxP and Dre-rox dual group system is to use the Cre and Dre dual group enzymes to identify their specific loxP and rox sites, for example, to construct the target genes (such as genes X and Y) related dual group enzyme mice, Namely X-Dre and Y-CrexERT2 mouse models
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The Y-CrexERT2 mouse was constructed by adding a rox site sequence to both ends of the ERT2 sequence in the CreERT2 vector to construct two Cre mouse strains mediated by gene X and Y specific promoters
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Using this dual-group system, combined with reporter gene mouse models (such as R26-LSL-tdTomato), to achieve a specific cell population lineage tracing process, to meet the limited range of positive expression of X-Dre and Y-CrexERT2 at the same time, making more accurate and effective research It is possible to trace specific cell lineages of specific organs or tissues
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The Chinese Academy of Sciences Zhou Bin's research group applied this dual group system to successfully achieve the high efficiency and specificity of target genes in coronary vascular endothelial cells (Tie2-Dre and Wt1-CrexERT2) or cerebral vascular endothelial cells (Tie-2 and Mfsd2a-CrexERT2) Sexual gene knockout or overexpression studies
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The establishment of a new inducible DD-cre mouse model will become a supplement to the Cre-loxP inducible recombination system in the future

Insufficiency in the application of TAM-induced Cre-ERT2 mice, such as the need for multiple administrations, a relatively slow induction and inhibition process; and TAM may bind to the endogenous estrogen receptor in mice, resulting in related side effects, etc.
In order to further improve the system and find a more effective induction method, it has practical significance
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The recent successful development and application of a new inducible DD-Cre mouse model also provides additional options for the application of the Cre-loxP inducible recombination system
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The basic principle of DD technology is based on the fusion of human FKBP12 or bacterial dihydrofolate reductase (ecDHFR) mutation destabilizing dormain-DD (Destabilizing dormain-DD) with any protein of interest (such as Cre)
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The synthesized new DD-related proteins labeled with FKBP12 or ecDHFR can lead to protease degradation
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However, such protein degradation and destruction pathways can be blocked by the antibiotic Trimethoprim (TMP)
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The DD-Cre mouse model is a DD-Cre mouse with a destabilized Cre recombinase system by fusing the DD domain with the Cre gene, and its recombination activity is induced and regulated by the antibiotic TMP, that is, DD-Cre activates the recombination effect as TMP Dependent, so as to realize the site-specific and inducible target gene editing of Cre-loxP recombination system in vivo
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TMP inducers are cheap, non-toxic, fast-induced, can pass through the placenta and blood-brain barrier, and have no endogenous targets in mammals, making TMP an ideal inducer for stabilizing DD-labeled proteins
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Therefore, the application of TMP in vivo to induce protein stabilization is conducive to the rapid delivery of the inducer in experimental animals and its efficient diffusion effect in the nervous system and other surrounding tissues
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Some studies have used R26-CAG-tdTomato or R26 Ai9-tdTomato to report mice, using different concentrations of TMP (8～170 ug/g, intraperitoneal injection once a day or seven consecutive times) 24～48 hours after induction, the recombinant cutting effect is up to Peak, and presents a dose-dependent effect
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Studies have shown that, compared with the CreERT2 system, TMP has a faster inducing effect in the body, making the DD-Cre system an attractive and ideal tool that is widely used in the field of neurogenetic research
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Of course, like other methods, DD-Cre technology may also exhibit a certain degree of Cre background activity
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And the activity will also be affected by certain factors, such as the degradation rate of DD fusion protein, the recruitment of stable binding ligands to fusion with appropriate subcellular domains, and the intervention of DD expression on the function of the fusion protein
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Finally, in the application of the Cre-loxP site-specific recombination system, the recommended guidelines and factors to be considered for the Cre mouse construction strategy are summarized as follows:

(1).
Use site-specific knock-in gene targeting technology to construct a new Cre mouse strain;
(2).
If the site-directed knock-in gene locus is not a heterozygous lethal gene, it is recommended to knock-in Cre into the region of the endogenous related gene coding sequence;
(3) ).
If possible, construct the reporter gene into the newly established floxed target gene targeting mice;
(4).
Cre mice are used as a separate control for target gene targeting studies to exclude non-specific off-target recombination caused by Cre mice.
Function;
(5).
If the target gene is not a heterozygous lethal gene, you can first establish a heterozygous germ cell gene knockout strategy to improve the efficiency of Cre targeting;
(6).
Design a reasonable PCR specific identification primer combination to facilitate genotype identification Analyze different situations such as target gene knockout, wild and specific target gene targeting (flexed); (7).
Use hemizygous or heterozygous Cre mice to reduce possible toxicity and off-target effects;
(8).
If floxed mice are used as additional experimental controls, it will be more perfect;
(9).
The importance of determining the actual dosage of the inducer TAM for pre-experiment;
(10).
Understanding the previous actual application of specific Cre mice, including its non-specific off-target , Reproductive recombination activity, gender and genetic background;
(11).
If two or more target gene recombination and/or target gene editing strategies need to be selected, it is recommended to use mutation type loxP sequences, such as loxN, lox2271 Or lox511; and other similar Dre-rox site-specific recombination systems, etc.
;
(12).
Consider choosing other induction regulation systems, such as the new DD-Cre regulation system induced by the antibiotic trimethoprim (TMP)
.

(13).
DNA length, chromosomal location, epigenetic modification, accessibility of germ cell transcription, etc.
between the target gene 2 x loxP of conditional Floxed mice can also be factors influencing the efficiency of Cre
.

(End of full text)

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

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