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Experimental perturbations to investigate cardiovascular physiology
Published in Neil Herring, David J. Paterson, Levick's Introduction to Cardiovascular Physiology, 2018
Neil Herring, David J. Paterson
Some of the issues with gene knockout models can be addressed with tissue- and time-specific knockouts. Tissue- specific knockout is achieved by incorporating locus of X-over P1 (loxP) sites either side of the gene of interest (floxed gene) using homologous recombination as described earlier. LoxP sites direct an enzyme called Cre recombinase that cuts and recombines sequences of DNA at these sites, thereby removing the sequence of DNA between them. The efficiency of the recombination tends to be lower the longer the length of DNA between the two loxP sites. The activity of Cre recom- binase can then be controlled by crossing the mouse with the floxed gene with another mouse expressing Cre recom- binase with a site- or cell-specific promoter as demonstrated in Figure 20.4. Examples of site/tissue-specific promoters with which Cre recombinase can be coupled include: synapsin (neuronal); PRSx8 (catecholaminergic neurons); alpha-myosin heav y chain (MHC; cardiac myocytes); smooth muscle MHC (smooth muscle); or tyrosine-protein kinase receptor Tie-2 (endothelial cells).
Untangling Appetite Circuits with Optogenetics and Chemogenetics
Published in Ruth B.S. Harris, Appetite and Food Intake, 2017
A common way to achieve such explicit specificity is through a two-pronged approach using genetically-engineered mice that express Cre-recombinase in defined neurons, often driven off the endogenous gene promoter, in combination with Cre-dependent viral vectors expressing activity modulators to stimulate or silence neuronal activity using particular wavelengths of light or pharmacologically inert, chemical ligands (Figure 5.1) (Luo, Callaway, and Svoboda 2008). These adeno-associated viruses are designed in such a way that the open reading frame of the coding sequence is inverted, in the antisense orientation, flanked by two pairs of heterotypic, antiparallel loxP-type recombination sites. In the presence of Cre-recombinase, the recombination sites first undergo an inversion of the coding sequence, followed by excision of two sites, leading to one of each orthogonal recombination site oppositely oriented and incapable of further recombination (Atasoy et al. 2008, Schnutgen et al. 2003). This Cre-dependent viral approach is commonly referred to as a FLEX switch or double-floxed inverse open reading frame (DIO) (Atasoy et al. 2008, Witten et al. 2010) and is used to target specific cell types with spatial precision via intracranial surgeries in distinct Cre driver mouse lines.
Anticancer Drug Development with Optical Imaging
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Bohumil Bednar, Guo-Jun Zhang, Cyrille Sur
A conditional Rosa26 knock-in reporter mouse has been generated by Safran et al. (27), where firefly luciferase is placed downstream of loxP-stop-loxP cassette and is activated after delivery of Cre recombinase. This conditional Rosa26 luciferase mouse can be used for detecting tumor development, and metastases in tumor-prone GEM mice that are mediated by Cre-recombination. For example, this mouse strain could be crossed with a mouse that harbors a conditional oncogene transgene or a TSG knockout. Then, an additional cross with a Cre mouse strain or administration of Ad-Cre would deliver Cre recombinase to a certain organ/tissue, and it would allow for imaging of tumor formation and tumor cell migration. By local or systemic administration of Ad-Cre, one can generate a mouse with an organ/tissue-specific luciferase expression. For example, the tail vein injection of Ad-Cre generates dominant expression of luciferase in the liver (Fig. 6A, B), and such reporter mice can be potentially used to evaluate autochthonous liver tumors as well as targeted delivery of novel therapeutic vectors such as siRNA.
Recent progress and challenges in drug development to fight hand, foot and mouth disease
Published in Expert Opinion on Drug Discovery, 2020
Ze Qin Lim, Qing Yong Ng, Justin Wei Qing Ng, Vikneswari Mahendran, Sylvie Alonso
Validation-based insertional mutagenesis (VBIM) is a novel function-based, forward genetic screening method that involves the insertion of a reversible promoter. EV-A71 is a lytic virus, and infected cells typically exhibit massive CPE. Mutations that either silence proviral host factors or drive the expression of antiviral factors are expected to protect the infected cells from undergoing CPE. In addition, the mutant phenotype can be reversed through Cre recombinase-mediated removal of the insert, thereby validating the role of the target gene in the phenotype observed [133]. This approach can be applied to a wide range of mammalian cell lines that can generate high titers of VBIM lentivirus. Using this technique, Nup214 was reported to play an important role during EV-A71 replication [134]. In a separate study using VBIM, KREMEN1 was identified as a novel host receptor for CV-A10 and enteroviruses-A [135]. KREMEN1 is a transmembrane molecule that regulates WNT signaling and promotes the uptake of viral-receptor complexes through clathrin-mediated endocytosis [136,137]. KREMEN1 and KREMEN2-deficient mice remained asymptomatic upon infection with CV-A10, with absence of viral replication in the muscle tissues [135].
Advances in the creation of animal models of paroxysmal nocturnal hemoglobinuria
Published in Hematology, 2021
The Cre/loxP system enables the knocking out of a target gene at a specific time or in a specific cell or tissue. Cre recombinase is derived from phage P1, and the sequence length is 38 bp. The LoxP sequence is about 34-bp long and consists of two 13-bp reverse repeats separated by an 8-bp non-palindromic sequence [23]. Cre recombinase can act on two LoxP sites to cause specific homologous recombination and remove DNA sequences between LoxP sites located in the same direction [24]. Since the Cre recombinant enzyme is controlled by the instantaneous expression promoter, which itself is only expressed and activated in specific cell types or tissues, it becomes possible to pinpoint a particular place to delete a DNA sequence and obtain a CKO mouse [25].
Current models of pulmonary fibrosis for future drug discovery efforts
Published in Expert Opinion on Drug Discovery, 2020
Toyoshi Yanagihara, Sy Giin Chong, Megan Vierhout, Jeremy A. Hirota, Kjetil Ask, Martin Kolb
Interpretation of data from genetically modified mice should, however, be made with caution. There is a possibility of genetic compensation for the missing gene in conventional knockout mice which may alter the outcome of interventions [46]. The observed biology in transgenic mice could be due to disruption of another gene from random integration of transgenes rather than the gene of interest. There can be off-target effects of tools used to generate genetically modified mice. Doxycycline and tamoxifen may have indirect effects on lung development [47]. There is potential toxicity of Cre-recombinase to mammalian cells with prolonged exposure [48,49].