Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

10.3791/59639 ◽  
2020 ◽  
Author(s):  
Hegang Li ◽  
Jinshan Zhao ◽  
Huaiyuan Qin ◽  
Jingjing Xin ◽  
Flor M. Perez-Campo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Luciferase Reporter System

scholarly journals miR-299-3p suppresses cell progression and induces apoptosis by downregulating PAX3 in gastric cancer

2021 ◽  
Vol 16 (1) ◽  
pp. 266-276
Author(s):  
Zhenfen Wang ◽  
Qing Liu ◽  
Ping Huang ◽  
Guohao Cai
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Western Blot Assay ◽  
Normal Tissues ◽  
Cell Progression ◽  
Luciferase Reporter System ◽  
Proliferation And Invasion

Abstract Gastric cancer (GC) is ranked the fourth leading cause of cancer-related death, with an over 75% mortality rate worldwide. In recent years, miR-299-3p has been identified as a biomarker in multiple cancers, such as acute promyelocytic leukemia, thyroid cancer, and lung cancer. However, the regulatory mechanism of miR-299-3p in GC cell progression is still largely unclear. Cell viability and apoptosis tests were performed by CCK8 and flow cytometry assay, respectively. Transwell assay was recruited to examine cell invasion ability. The interaction between miR-299-3p and PAX3 was determined by the luciferase reporter system. PAX3 protein level was evaluated by western blot assay. The expression of miR-299-3p was downregulated in GC tissues and cell lines (MKN-45, AGS, and MGC-803) compared with the normal tissues and cells. Besides, overexpression of miR-299-3p significantly suppressed proliferation and invasion and promoted apoptosis in GC. Next, we clarified that PAX3 expression was regulated by miR-299-3p using a luciferase reporter system, qRT-PCR, and western blot assay. Additionally, downregulation of PAX3 repressed GC cell progression. The rescue experiments indicated that restoration of PAX3 inversed miR-299-3p-mediated inhibition on cell proliferation and invasion. miR-299-3p suppresses cell proliferation and invasion as well as induces apoptosis by regulating PAX3 expression in GC, representing desirable biomarkers for GC diagnosis and therapy.

scholarly journals A Cell-Based High-Throughput Assay for the Screening of Small-Molecule Inhibitors of p53–MDM2 Interaction

2011 ◽  
Vol 16 (4) ◽  
pp. 450-456 ◽  
Author(s):  
Jing Li ◽  
Shuyong Zhang ◽  
Linghuan Gao ◽  
Ying Chen ◽  
Xin Xie
Keyword(s):  
High Throughput ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Direct Inhibition ◽  
A Cell ◽  
Luciferase Reporter System ◽  
Novel Strategy ◽  
High Throughput Assay ◽  
Two Hybrid ◽  
Rule Out

The p53 tumor suppressor is a potent transcription factor that regulates cell growth inhibition and apoptosis. The oncoprotein MDM2 suppresses p53 activity by direct inhibition of its transcriptional activity and enhances the degradation of p53 via the ubiquitin–proteosome pathway. Overexpression of MDM2, found in many human tumors, impairs p53-mediated cell death effectively. Inhibition of the p53–MDM2 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. To search for new inhibitors of the p53–MDM2 interaction, the authors developed a cell-based high-throughput assay system based on mammalian two-hybrid technology. They also used a dual-luciferase reporter system to rule out false- positive hits due to the cytotoxic effect of compounds. Using this assay, they screened a library consisting of 3840 compounds and identified one compound that activates p53 pathway and induces growth arrest in tumor cells.

scholarly journals Cyp11b1 Is Induced in the Murine Gonad by Luteinizing Hormone/Human Chorionic Gonadotropin and Involved in the Production of 11-Ketotestosterone, a Major Fish Androgen: Conservation and Evolution of the Androgen Metabolic Pathway

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1786-1792 ◽  
Author(s):  
Takashi Yazawa ◽  
Miki Uesaka ◽  
Yoshihiko Inaoka ◽  
Tetsuya Mizutani ◽  
Toshio Sekiguchi ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Metabolic Pathway ◽  
Leydig Cells ◽  
Plasma Concentrations ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Mouse Ovary ◽  
Luciferase Reporter System

We have shown previously that Cyp11b1, an 11β-hydroxylase responsible for glucocorticoid biosynthesis in the adrenal gland, was induced by cAMP in androgen-producing Leydig-like cells derived from mesenchymal stem cells. We found that Cyp11b1 was induced in male Leydig cells, or female theca cells, when human chorionic gonadotropin was administered in immature mice. Expression of Cyp11b1 in rodent gonads caused the production of 11-ketotestosterone (11-KT), a major fish androgen, which induces male differentiation or spermatogenesis in fish. As in teleosts, plasma concentrations of 11-KT were elevated in human chorionic gonadotropin-treated mice. In contrast to teleosts, however, plasma concentrations of 11-KT were similar in both sexes, despite levels of testosterone, a precursor substrate, being about 20 times higher in male mice. Because expression of 11β-hydroxysteroid dehydrogenase type 2, was much higher in the mouse ovary than in the testis, conversion of testosterone into 11-KT may occur more efficiently in the ovary. In a luciferase reporter system that was responsive to and activated by androgens, 11-KT efficiently activated mammalian androgen receptor-mediated transactivation. Our results suggest that the androgen metabolic pathway is conserved between teleosts and mammals, despite sexual dominance and reproductive functions of 11-KT being altered during evolution.

The dual luciferase reporter system and RT‐qPCR strategies for screening of MicroRNA‐21 small‐molecule inhibitors

2019 ◽  
Vol 66 (5) ◽  
pp. 755-762 ◽  
Author(s):  
Yuan‐Yuan Hei ◽  
Yuan‐Xu Guo ◽  
Cong‐Shan Jiang ◽  
Si Wang ◽  
She‐Min Lu ◽  
...  
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Luciferase Reporter System
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