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Applications for Drug Development
Published in George C. Kagadis, Nancy L. Ford, Dimitrios N. Karnabatidis, George K. Loudos, Handbook of Small Animal Imaging, 2018
Jessica Kalra, Donald T. Yapp, Murray Webb, Marcel B. Bally
In vivo cell and molecular imaging is quickly becoming a very important part of a drug efficacy program. Molecular imaging makes it possible to evaluate the mechanism of drug action in vivo. OI is perhaps one of the easiest to implement and least expensive modalities in molecular imaging and has been useful as a first-line approach in understanding cellular and molecular pathogenesis and drug efficacy. Despite its lack of clinical translatability, OI is widely used in vivo to track single cells or molecules in the whole body without the use of ionizing radiation. Reporter gene assays have been used to yield fundamental biological information on transcriptional regulation, signal transduction, protein–protein interactions, cell trafficking, and targeted drug action in vivo at the cellular and molecular levels. For example, Viola et al. inoculated mice with breast carcinoma cells transfected with an HIF-1α luciferase reporter construct (Viola et al. 2008). HIF-1α is a key player in the stress response elicited by low levels of oxygen. Animals were treated with cyclophosphamide or paclitaxel, which caused an increase in HIF-1α protein levels as quantified using BLI (Viola et al. 2008).
In Vivo
Published in Lihong V. Wang, Photoacoustic Imaging and Spectroscopy, 2017
Roger J. Zemp*, Li Li*, Lihong V. Wang
Gene expression imaging, one important branch of molecular imaging, relies on reporter genes and their protein products to provide contrast for imaging. A reporter gene is a nonendogenous segment of DNA that is stably or transiently integrated into the genome of cells, tissues, and even organisms using transgenic technology. The protein product of the reporter gene can be imaged with an imaging modality either directly or by acting on some biochemical assays. The expression of a reporter gene is regulated by its promoters and enhancers [4]. A given reporter gene vector can be constructed with the promoters and/or enhancers of a gene of interest. When signaling events upregulate the expression of a gene of interest, the reporter gene will also be up-regulated since they share the same regulatory elements. Alternatively, a ubiquitously strong promoter, such as human cytomegalovirus (CMV) promoter [5] can be used to provide high levels of reporter gene expression. In such situations, the motivation is to tag cells to track growth and response to therapy. Gene expression imaging has also been used to study such important problems as cell proliferation, angiogenesis, activation of oncogenes and loss of tumor suppressor genes affecting cellular apoptotic programming, and ability to invade and metastasize.
Optimizing Reporter Gene Expression for Molecular Magnetic Resonance Imaging
Published in Shoogo Ueno, Bioimaging, 2020
Qin Sun, Frank S. Prato, Donna E. Goldhawk
Simply stated, a reporter gene encodes protein that can be reliably detected by some method of choice. If the reporter gene is constitutively expressed, it generates a constant label that can theoretically be monitored through all phases of the cell, including its proliferation and differentiation. If the reporter gene is selectively expressed, it imparts a signal that will only be detected when specific TF activity is present, including those that trigger a disease process like inflammation, fibrosis, or metastasis. Traditionally, “reporter gene expression” is a term coined to describe this type of selective gene expression. It is a powerful tool for understanding cellular phenotype and identifying what factors contribute to function and malfunction.
Safe and efficient DNA delivery based on tannic acid-ion coordination encapsulation
Published in Soft Materials, 2023
Liang Liu, Chaobing Liu, Zhaojun Yang, Yiran Chen, Shuheng Wu, Xin Chen
Branched polyethyleneimine (M.W., 30 kDa and 70 kDa), chlorpromazine, methyl-β-cyclodextrin (M-β-CD), and amiloride were purchased from Macklin (Shanghai, China). TA (M.W., 1701.23) was purchased from Beilian (Tianjin, China). Fluorescein isothiocyanate (FITC), ethidium bromide (EtBr), and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich, Inc. (Shanghai, China). EGFP plasmid was used as reporter gene and amplified in Escherichia coli and extracted by a plasmid extraction kit (Omega Bio-tek, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Yuan-Ye Biotech Co., Ltd. (Shanghai, China). HeLa (human cervical cancer) cells, HepG2 (human hepatoma) cells, and 293 T (human embryonic kidney) cells were purchased from the National Infrastructure of Cell Line Resource (Beijing, China).
Whole-cell bioreporters for evaluating petroleum hydrocarbon contamination
Published in Critical Reviews in Environmental Science and Technology, 2021
Bo Jiang, Yizhi Song, Zengjun Liu, Wei E. Huang, Guanghe Li, Songqiang Deng, Yi Xing, Dayi Zhang
Selecting reporter gene needs to be carefully considered for the design of WCBs, dependent on the availability of instrumentation, signal sensitivity, need for additional substrates, ease of cloning and reporter interference or stability in the cell (Harms, Wells, & Meer, 2006; van der Meer, 2010; Yu, Chen, & Mulchandani, 2006). Comparison between different reporter genes was discussed by Hakkila et al. who investigated the performance of four reporter genes, e.g., firefly luciferase (lucFF), luxCDABE, gfp and rfp (red fluorescent protein), in a series of WCBs sensing mercury and arsenate (Hakkila, Maksimow, Karp, & Virta, 2002). The results demonstrated that the expression of lucFF and luxCDABE is far more rapid and 100-times sensitive than gfp and rfp, indicating luxCDABE is a better choice for on-line purpose with higher sensitivity. Huang et al. studied the performance of Acinetobacter sp. ADPWH_lux (luxCDABE as reporter gene) and ADPWH_gfp (gfp as reporter gene) in detecting salicylate and proved the higher sensitivity and shorter response time of ADPWH_lux comparing to ADPWH_gfp (Huang et al., 2005). Nevertheless, the longer maturation and turnover time of GFP makes ADPWH_gfp more suitable in visualizing the presence of salicylate in environmental matrices.
Synthesis of magnetic Fe3O4@Al3+ particles and its application in DNA extraction
Published in Particulate Science and Technology, 2023
Chi Chen, Zhong Zheng, Changxia Liu, Wensheng Yang
The self-replicating plasmid (pUG-PT7-EGFP-TT7) from E. coli BL21 (DE3) was used for DNA extraction. pUG-PT7-EGFP-TT7 represented that the plasmid consisted of pUG36 as vector, T7 promoter, terminator and enhanced green fluorescent protein (EGFP) reporter gene. The forward primers (5′-TCACCTTCACCTTCACCGGA–3′) and reverse primers (5′-AAGCGCGCAATTAACCCTCA–3′) were engineered by Sangon Biotech Co., Ltd (Shanghai, China). The PCR amplified sequence was 793 bp (GenBank accession number: BankIt2518282BankIt2518282OL444940). All other reagents of analytical grade were purchased from the major chemical supply companies.