The advancement of Cre-loxP mouse modelsIn the late 1980s, Nobel laureates Mario Capecchi at the University of Utah, Oliver Smithies at the University Wisconsin in Madison, and Sir Martin Evans at the University of Cambridge, pioneered approaches for gene targeting in mouse embryo-derived stem (ES) cells and homologous recombination as a system for manipulating genes in the mouse genome.5-7 This work showed the expediency of making specific mutations in ES cells and acquiring genetically customized knockout mice. He closely followed the work on ES cells and used it to develop one of the earliest knockout models of the immune system by rendering mice deficient of B cells.8 However, the technology had its limitations. Rajewsky initially wanted to target the polβ gene in B cells, Marths T cell transgenic line was an attractive alternative.By crossing Marths T cell specific cre transgene with Rajewskys conditional polβ allele, Marth and Rajewsky established mice that lacked DNA polymerase β in T cells.11 “When we did this work, this kind of technology opened the method to do lots of things beyond conditional targeting,” said Rajewsky.”While you can be pretty sure that the cells are mutant, you also require to understand where your cells are to comprehend how they behave. Removal of a DNA Polymerase β Gene Segment in T Cells Using Cell Type-Specific Gene Targeting.
PDFAn inducible Cre-loxP systemThe success of Rajewskys and Marths conditional gene targeting resulted in an explosion of various Cre transgenic lines with expression profiles in various tissues. Scientist developed additional levels of control over gene expression as a natural extension of this starting technology. Mainly, scientists established temporal control to prevent previous challenges that emerged from worldwide gene removal or early developmental Cre recombinase activity. The incorporation of drug- or interferon-responsive promoters permitted researchers to control expression of Cre recombinase. Scientists utilized tetracycline, type I interferon, or tamoxifen to cause promoter activation12-14Andrew McMahon, a developmental biologist from the University of Southern California, and his postdoctoral scientist Paul Danielian, who is now a biomedical editor at General Dynamics Information Technology, fine-tuned Cre recombinase control in vivo with mice utilizing tamoxifen. “With a founding technology like Cre-loxP, a lot of resources got constructed around that,” remembered McMahon.Danielian formerly studied steroid hormone guideline, and his ideas guided their subsequent experiments. The duo fused the ligand binding domain of the estrogen receptor to Cre to create a conditional type of it understanding that it would be sequestered in the cytoplasm in the absence of a ligand. Including a ligand activated this fusion protein and translocated it to the nucleus to trigger Cre activity and cause recombination. This was the very first time anyone had actually conditionally removed gene activity in the establishing fetus of the mammalian system.15 Since then, tamoxifen-inducible Cre-loxP has been one of the most extensively used inducible systems. “Its just an illustration of how powerful genetics is. The more you can remarkably control the procedure that youre working with, the more insight youre going to get into the question that youre asking,” said McMahon.Cre-loxP and CRISPRThe Cre-loxP system stays the gold standard method for conditional gene regulation in mice, however it can be expensive and time consuming. Over the last couple of years, scientists questioned whether to complement this approach with another effective gene editing strategy. They discovered their chance with the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated nuclease, Cas9, which together make it possible for extremely particular DNA changes at exact areas within the genome.16 The Cre-loxP-CRISPR mix stimulated researchers interest in investigating its prospective applications. CRISPR could introduce particular mutations or genetic variations and the Cre-loxP recombination system might specifically excise or incorporate the hereditary components. For Stefan Hans, a developmental geneticist at the Dresden University of Technology, this two-step method used the very best of both innovations for his research studies on nerve cell regeneration in zebrafish.17 The speed of transgenic zebrafish generation and the ability to specifically visualize the target cells were key benefits.”While you can be pretty sure that the cells are mutant, you also require to understand where your cells are to understand how they act. So, this feature is a crucial aspect because the putative mutant cells are identified, and we can easily take and recognize out these cells for analysis,” stated Hans. The mix of these methods was demonstrated in mammalian cells a few years prior, Hans was the very first to utilize it in zebrafish research in 2021.18 The Cre-loxP system left a lasting impact on conditional gene editing, and contemporary advances have taken gene function and advancement to new heights. “Techniques come and go with new technology, Its similar to night and day. When you have a strategy thats lasted 30 years with no replacement technology, I believe thats kind of remarkable,” said Marth. ReferencesSternberg N. Demonstration and Analysis of P1 Site-specific Recombination Using λ-P1 Hybrid Phages Constructed In Vitro. Cold Spring Harb Symp Quant Biol. 1979; 43:1143 -1146. Sternberg N, Hamilton D. Bacteriophage P1 site-specific recombination: I. Recombination between loxP websites. J Mol Biol. 1981; 150( 4 ):467 -486. Saur B. Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Bio. 1987; 7( 6 ):2087 -2096. Saur B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988; 85( 14 ):5166 -5170. Evans MJ, Kaufman MH. Facility in culture of pluripotent cells from mouse embryos. Nature. 1981; 292:154 -156. Doetschman T, et al. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987; 330:576 -578. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987; 51:503 -512. Kitamura D, et al. A B cell-deficient mouse by targeted interruption of the membrane exon of the immunoglobulin μ chain gene. Nature. 1991; 350:423 -426. Gu H, et al. Independent control of immunoglobulin switch recombination at specific switch regions evidenced through Cre-loxP-mediated gene targeting. Cell. 1993; 73( 6 ):1155 -1164. Orban PC, et al. Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci. 1992; 89( 15 ):6861 -6865. Gu H, et al. Deletion of a DNA Polymerase β Gene Segment in T Cells Using Cell Type-Specific Gene Targeting. Science. 1994; 265( 5168 ):103 -106. St-Onge L, et al. Temporal Control of the Cre Recombinase in Transgenic Mice by a Tetracycline Responsive Promoter. Nucleic Acids Research. 1996; 24( 19 ):3875 -3877. Kühn R, et al. Inducible gene targeting in mice. Science.1995; 269( 5229 ):1427 -1429. Metzger D, et al. Conditional site-specific recombination in mammalian cells utilizing a ligand-dependent chimeric Cre recombinase. Proc Natl Acad Sci U S A, 1995; 92( 15 ):6991 -6995. Danielian PS, et al. Adjustment of gene activity in mouse embryos in utero by a tamoxifen-inducible type of Cre recombinase. Cell. 1998; 8( 24 ):1323 -1326. Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial resistance. Science. 2012; 337( 6096 ):816 -821. Hans S, et al. Cre-Controlled CRISPR mutagenesis provides simple and fast conditional gene inactivation in zebrafish. Nat Commun. 2021; 12:1125.
