Immunity is of particular interest. Potential disadvantages and solutions for the Cre-Loxp system.

As the most widely used conditional knockout system, CRE loxP is of great significance in studying the phenotype and function of specific genes in specific cell types.for the immune system, CRE loxP system is widely used in various scientific problems, to solve cell type specific knockout, mutation, rearrangement and other issues, as well as to conduct lineage tracking research.however, scientists have found that there may be various technical problems in the use of CRE loxP system, which may result in some false negative or false positive results, or seriously interfere with the endogenous fate determination mechanism of cells.in a letter of immunity in 2018, Professor Burkhard Becher from the University of Zurich and Li Fan Lu from the University of California, San Diego, etc. discussed the possible problems of CRE loxP system in detail, and proposed possible solutions.the following three aspects are the key points in the discussion process: 1. CRE is integrated into the genome, and its insertion position has certain limitations and may affect the expression of endogenous genes. At the same time, not all the endogenous gene expressions can truly reflect the expression of cre.many CRES, such as CD19 CRE, replace the original gene position in the genome; some CRES, such as Foxp3 CRE, are the 3'UTR positions inserted with endogenous genes.these two points may cause CRE insertion events to significantly affect the expression of endogenous genes. For example, the difference in the amount of heterozygotes and homozygotes of some genes leads to the difference of downstream signal intensity, or affects the gene modification of many genes at the 3'UTR end after expression.in addition, some CRES are integrated into bacterial artificial genome (BAC), which theoretically include most CIS gene regulatory sequences.but now genomics has a deeper understanding of the remote regulatory elements, and the scheme can not perfectly replace the real regulation of the "simulated gene sequence CRE" by the endogenous gene sequence, so it can not be used as a reasonable scheme for conditional knockout.the questions raised by the authors here can not be completely avoided, but the integration of a fluorescent protein behind CRE can truly indicate whether CRE is correctly expressed in a specific cell lineage, thus avoiding misreading of false positive results. The expression of CRE can not reflect the cutting efficiency of loxP.most of the time, due to the complexity of genome regulation and the different chromatin status of each gene in different cells, CRE has different loxP cleavage efficiency for different gene sites.researchers often insert "loxP stop loxP reporter (lsl-r)" at Rosa26 to indicate CRE efficiency.however, for such reporter genes, the genome state is often open, which makes the efficiency of CRE cleavage higher than that of loxP.the authors also put forward some solutions, focusing on how many CRE loxP target gene sites or protein decreases after CRE treatment compared with the control group, which is best measured at the single cell level.at the same time, we can also detect the direct downstream of target gene and indirectly estimate the efficiency of cre. In conditional knockout, the CRE gene must be highly efficient and genealogically specific.many CRE promoter genes are screened according to the conditional high expression genes.for a very lineage specific promoter gene like CD19, CRE is highly efficient and does not "leak" into other cell lineages. however, many relatively weak conditional promoter genes often have the problem of "leaking" CRE signal into other related cell lineages, especially for some cells with short life cycle (such as neutrophils) The CRE loxP cleavage often leads to "leakage" of CRE loxP cleavage, which results in the remodeling of some cell subsets, and then affects the correct interpretation of the function of the target gene in the next step. the authors suggest that it is better not to use this gene as a cre promoter for incomplete lineage markers (e.g. only high expression). at the same time, PCR should be used to confirm that there is no serious CRE missing signal in the adjacent or similar cell lineage. this article was followed up by extensive discussion. On October 15, 2019, the author of the article, Professor John whery of the University of Pennsylvania, and Professor Boris reizie from New York University published letters in the journal immunity for further details. 1. Professor John wherry's group provided an important supplement to the possible problems of CRE loxP system, that is, inducing CRE loxP system in the period of rapid cell expansion may have significant cytotoxicity. using tamoxifen to induce cre-ert2 into nucleus is a common method in immunology research. however, the effect of this system on cell differentiation and proliferation is rarely reported. In the whery group, in the LCMV infection model, non induced conditional knockout CRE (e8i CRE) did not significantly affect the survival of T cells in the effector cell phase, while the tamoxifen induced group showed a significant decrease in the number of T cells (Fig. 1a-1c). at the same time, the researchers also observed that cre-ert2 of homozygotes had higher cytotoxicity than cre-ert2 of heterozygotes (Fig. 1b-1c). In the whery group, further studies found that tamoxifen treatment of cre-ert2 cells before infection did not affect the number of T cells after infection. therefore, for the induced expression of CRE, researchers should pay attention to the time of tamoxifen treatment and the effect of treatment stage on cell number. Fig. 1. The effect of CRE on the number of T cells under induction conditions. 2. Professor reizie questioned and discussed some points of Professor Lu's discussion paper in 2018. first of all, Professor reizie thinks that the CRE fusion reporter gene proposed by Professor Lu and the detection of target gene quantity at the single cell level still have limitations or difficulties in implementation. importantly, Professor reizie disagrees with the specificity of the CRE reporter fusion reporting system. He uses the CRE system of DC cells as an example. professor reizie thinks that CRE + lsl-r system can still be used as a better reporting system than CRE reporter, and the different signals of lsl-r cleavage just represent that CRE has different endogenous activity in different cell lineages, which is more accurate than merely indicating the expression level of CRE itself. 3. first of all, he said that there was no detailed published data to confirm the main correlation between the reported gene expression and target gene cleavage in lsl-r system. in fact, in some CRE mediated lsl-r systems, there were not only false negative results, but also some false positive results due to the sensitivity and specificity of cre. therefore, it is of great significance to indicate the expression and activity of CRE itself, and to eliminate CRE cleavage of non target cell lineage. finally, Professor Lu concluded that there is no perfect model and perfect solution, but we need to clearly understand the problems of each genetic model before we can better explain our experimental results. original link: 1