Difference between revisions of "Part:BBa K5267044"

Revision as of 18:33, 30 September 2024

P_5xNFAT->IgK->Nluc->bGH_polyA

Transpose and respond to calcium ion signals

Sequence and Features

Profile

Name: P_5xNFAT->IgK->Nluc->bGH_polyA
Base Pairs: 1018bp
Origin: Homo sapiens
Properties: The transformation of calcium ion concentration is reported by fluorescence

Usage and Biology

At present, some people have successfully designed a series of repetitive pseudo-palindromic NFAT reaction element guided by nano-luciferase reporter gene system[1].

We developed a series of Ca2+induced NanoLuc reporting systems based on Ca2+ dependent activation of dimer NFAT to monitor the effects of increased Ca2+ concentration in downstream pathways following melatonin receptor response on cells. (Figure. 1)

The system is divided into two parts: the signal response element (encoded by Part:BBa K5267007) and the detection report element (encoded by Part:BBa_K2728003).

HEK293 cells were co-transfected with a newly designed NanoLuc reporter expression plasmid to construct a NFAT response element (RE) -directed Nanoluc reporting system. With the NanoLuc reporter gene, we can detect the activation of the NFAT signaling pathway.[2]

Special design

In order to evaluate the biological implications of heightened intracellular calcium ion (Ca2+) levels, we have engineered a suite of Ca2+-responsive NanoLuc-reporting constructs predicated on the Ca2+-dependent engagement of nuclear factors of activated T cells (NFAT) dimers (Figure 1).

Given the subdued transcriptional impact of an isolated response element, it is a common practice to introduce multiple tandem iterations of said element into the genomic locus proximal to the reporter gene, thereby amplifying the efficacy of the signaling cascade initiation.[1]

The aforementioned reporter constructs incorporate a variable number of tandem repeats (1x, 5x, 6x, and 7x) derived from the NFAT response element (NFAT-RE) within the interleukin-4 (IL4) promoter sequence (5'-TACATTGGAAATTTTTAT-3'). This particular sequence is anticipated to facilitate the transcriptional activation of the NanoLuc reporter genes (Figure 2).

Consequently, this system offers a valuable tool for elucidating the dynamic changes within the signaling network following the activation of melatonin receptors.

Function test

To substantiate the functionality of the aforementioned constructs, human embryonic kidney 293 cells (HEK293) were co-transfected with expression vectors harboring the newly engineered NanoLuc-reporter genes. 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. The optimal configuration of the reporting pathway was ascertained by evaluating and comparing the relative luminescence unit (RLU) expression profiles of the NanoLuc reporter genes, thereby discerning the most efficacious design among the various constructs.

Method

We introduced the expression vectors encoding the novel NanoLuc-reporter constructs into HEK293T cells via co-transfection, followed by the application of melatonin to elicit an intracellular calcium ion (Ca2+) response. The experimental paradigm encompassed three replicate experiments alongside a non-transfected control group (BBa_K5267049). Subsequent to a 24-hour exposure to melatonin, the luminescence intensity of the reporter element NanoLuc (measured as relative light units, RLU) was quantified across all experimental cohorts to assess the melatonin activity induced by the treatment.

Results

HEK-293T cells were co-transfected with melatonin receptor plasmid pCJ008(PCMV-MTNR1A) and plasmids containing different promoters with various copy numbers of NFAT elements pNC008(PNFAT_1-IgK-Nluc), pNC004(PNFAT_5-IgK-Nluc), pNC012(PNFAT_6-IgK-Nluc) and pNC010(PNFAT_7-IgK-Nluc) melatonin stimulation. Data are mean±SD of NanoLuc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments).

In the in the P_5xNFAT->IgK->Nluc->bGH_polyA system, the introduction of 1 nM melatonin did not elicit a statistically significant increase in the mean NanoLuc expression levels compared to the system devoid of melatonin treatment.

Reference

[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.
[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.