Difference between revisions of "Part:BBa K5267047"

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==Reference==
 
==Reference==
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[1] W. Zhang, T. Takahara, T. Achiha, H. Shibata, and M. Maki, “Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization,” Int. J. Mol. Sci., vol. 19, no. 2, p. 605, Feb. 2018, doi: 10.3390/ijms19020605.
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[1] “Melatonin receptor structure and signaling,” J. Pineal Res., vol. 76, no. 3, p. e12952, Apr. 2024, doi: 10.1111/jpi.12952.
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<br>[2] K. A. Strait, P. K. Stricklett, R. M. Kohan, and D. E. Kohan, “Identification of Two Nuclear Factor of Activated T-cells (NFAT)-response Elements in the 5′-Upstream Regulatory Region of the ET-1 Promoter,” J. Biol. Chem., vol. 285, no. 37, pp. 28520–28528, Sep. 2010, doi: 10.1074/jbc.M110.153189.
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<br>[2] S. Tordjman et al., “Melatonin: Pharmacology, Functions and Therapeutic Benefits,” Curr. Neuropharmacol., vol. 15, no. 3, pp. 434–443, Feb. 2017, doi: 10.2174/1570159X14666161228122115.
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<br>[3] A. H. Rad S. M., A. Poudel, G. M. Y. Tan, and A. D. McLellan, “Promoter choice: Who should drive the CAR in T cells?,” PLOS ONE, vol. 15, no. 7, p. e0232915, Jul. 2020, doi: 10.1371/journal.pone.0232915.

Latest revision as of 07:43, 1 October 2024


P_CMV->MTNR1A->bGH_polyA

Expression of MT1 gene

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
    Illegal BamHI site found at 1255
    Illegal BamHI site found at 1435
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Profile

Name: P_CMV->MTNR1A->bGH_polyA
Base Pairs: 1909bp
Origin: Homo sapiens
Properties: Expression of MT1 gene

Usage and Biology

The melatonin receptors (MTs), specifically MT1 (melatonin receptor type 1) and MT2 (melatonin receptor type 2), are classified under the G protein-coupled receptor (GPCR) family A, with melatonin acting as their endogenous agonist. These receptors are pivotal in the regulation of the circadian rhythm within the human body and are intricately linked to a spectrum of vital physiological processes, including reproductive function, neuronal modulation, and immune system regulation. Furthermore, MTs represent a significant therapeutic target for the amelioration of various pathologies, such as insomnia, affective disorders, and oncological conditions.[1].

Despite the high degree of homology between human MT1 and MT2 receptors, there are considerable differences in their tissue distribution, intracellular signaling mechanisms, and physiological roles. The current dearth of selectivity in melatonin-based pharmaceuticals for either MT1 or MT2 receptors impedes the precision therapy of related disorders. Consequently, the detailed structural elucidation of the MTs agonist binding site is of paramount importance for the development of targeted pharmacotherapeutics.[2]

In light of this, we have engineered a pathway that, upon promoter activation, initiates the synthesis of the MT1 melatonin receptor protein. This strategy is instrumental in the establishment of a cellular assay system designed for the screening of melatonin receptor agonists.(Figure 1)


Figure 1. MT1 gene expression pathway.

Special design

To achieve the objective of driving the expression of the MT1 melatonin receptor, we strategically selected the CMV promoter, a robust promoter derived from the human Cytomegalovirus (CMV), known for its high transcriptional activity in eukaryotic cells. The CMV promoter has been demonstrated to be highly efficacious in facilitating the expression of lengthy and complex genes within HEK-293T cells.[3]

Utilizing the CMV promoter, we initiated the transcription of the MT1 gene within the construct of the gene expression vector, thereby enhancing the expression profile of the MT1 gene. This approach is anticipated to provide a foundation for the development of a cell-based screening platform for melatonin receptor agonists.

Function test

The Green Fluorescent Protein-based Ca2+ indicator, GCaMP, is a prominent tool utilized for the quantitative assessment of intracellular Ca2+ concentration fluctuations within living cellular environments. This genetically encoded biosensor is engineered by fusing the green fluorescent protein (GFP) with the calcium-binding protein Calmodulin (CaM) and a peptide derived from the M13 protein.

Upon binding of calcium ions to the Calmodulin moiety, GCaMP undergoes a conformational change that results in the emission of a fluorescent signal. This property enables the real-time monitoring and visualization of dynamic shifts in intracellular Ca2+ levels, thereby providing valuable insights into cellular signaling pathways and physiological responses.

At the cellular level, melatonin can affect the activity of calcium channels through its receptors, leading to changes in intracellular calcium concentration. The reporting system is designed to be responsive to oscillations in intracellular Ca2+ concentrations.

This part of the system we designed is mostly used for experimental control


Method

We conducted assessments to evaluate the system's response to calcium ions over both extended and abbreviated time periods.

HEK-293T cells were co-transfected with the melatonin receptor plasmid pCJ008 (PCMV-MTNR1A) and the melatonin-stimulated reporting plasmid PCMV-GCaMP. Following a 48-hour stimulation with melatonin, the cells were photographed, with a scale bar of 100 µm for visualization under a wide-field microscope. The data presented are representative images from a single independent experiment.

Concurrently, HEK-293T cells were co-transfected with the melatonin receptor plasmid pCJ008 (PCMV-MTNR1A) and the reporting plasmid GCaMP (PCMV-GCaMP), and this was compared with HEK-293T cells transfected solely with the reporting plasmid GCaMP (PCMV-GCaMP). The data represent the mean ± standard deviation of the relative fluorescence intensity measured 48 hours post-melatonin stimulation, expressed in relative fluorescence units (RFU). (n = 8 independent experiments).

Results

Figure 2. 48-hour long-term calcium ion response

Figure 3. Fluorescence intensity measurement of short-term calcium ion response

Reference

[1] “Melatonin receptor structure and signaling,” J. Pineal Res., vol. 76, no. 3, p. e12952, Apr. 2024, doi: 10.1111/jpi.12952.
[2] S. Tordjman et al., “Melatonin: Pharmacology, Functions and Therapeutic Benefits,” Curr. Neuropharmacol., vol. 15, no. 3, pp. 434–443, Feb. 2017, doi: 10.2174/1570159X14666161228122115.
[3] A. H. Rad S. M., A. Poudel, G. M. Y. Tan, and A. D. McLellan, “Promoter choice: Who should drive the CAR in T cells?,” PLOS ONE, vol. 15, no. 7, p. e0232915, Jul. 2020, doi: 10.1371/journal.pone.0232915.