In an effort to non-invasively assess the impact of elevated intracellular calcium ion (Ca²⁺) concentrations, we have developed a series of Ca²⁺ inducible NanoLuc reporters predicated on the Ca²⁺ dependent activation of dimeric nuclear factor of activated T cells (NFAT), as depicted in Figure 1[2].
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To non-invasively assess the impact of elevated intracellular calcium ion (Ca²⁺) concentrations, we developed a series of Ca²⁺-inducible NanoLuc reporters based on the Ca²⁺-dependent activation of dimeric NFAT, as illustrated in Figure 1[2]. These reporters incorporate a varying number of tandem repeats (1×, 5×, 6×, and 7×) of a pseudo-palindromic NFAT response element (NFAT-RE) derived from the interleukin-4 (IL-4) promoter sequence (5′-TACATTGGAAAATTTTAT-3′) with minimal CMV promoter (parts:BBa_K5267049). This setup is anticipated to drive the transcription of the NanoLuc reporter gene when NFAT is dephosphorylated due to the significantly increased intracellular Ca²⁺ concentrations (Figure 1).
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<br>These reporters incorporate a varying number of tandem repeats (1×, 5×, 6×, and 7×) of a pseudo-palindromic NFAT response element (NFAT-RE) derived from the interleukin-4 (IL4) promoter sequence (5′-TACATTGGAAAATTTTAT-3′), which is anticipated to drive the transcription of the NanoLuc reporter gene. '''(Figure 1)'''
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<br>To elucidate the effects of intracellular Ca²⁺ concentration increments, human embryonic kidney 293 cells (HEK293) were co-transfected with expression plasmids encoding each of the newly designed NanoLuc reporters. This approach enables the indirect monitoring of the cellular response to fluctuations in intracellular Ca²⁺ concentrations.[3]
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<br>Figure 1. Construction of a pseudo-palindromic NFAT-response element (RE)-directed nanoluciferase(Nanoluc) reporter system.
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<br>'''Figure 1. Schematic representation showing the construction of a pseudo-palindromic NFAT-response element (RE)-directed nanoluciferase(Nanoluc) reporter system.'''
==Function test==
==Function test==
Revision as of 11:55, 2 October 2024
Pmin_5*NFAT promoter
Transpose and respond to calcium ion signals
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]
Profile
Name: Pmin_5*NFAT promoter
Base Pairs: 191bp
Origin: Homo sapiens
Properties: Transpose and respond to calcium ion signals
Usage and Biology
Nuclear factor of activated T cells (NFAT) was first identified over two decades ago as a major stimulation-responsive DNA-binding factor and transcriptional regulator in T cells. NFATs are a family of Ca²⁺-dependent transcription factors that play a central role in the morphogenesis, development, and physiological activities of various cell types and organ systems.
NFAT is widely expressed across different animal tissues and cell types, serving as a key regulatory point in multiple intracellular signal transduction pathways. It plays crucial roles in the immune system, nervous system development, axon growth, and various nervous system diseases. In this project, NFAT is utilized to monitor the effects of increases in intracellular Ca²⁺ concentrations indirectly [1].
Special design
To non-invasively assess the impact of elevated intracellular calcium ion (Ca²⁺) concentrations, we developed a series of Ca²⁺-inducible NanoLuc reporters based on the Ca²⁺-dependent activation of dimeric NFAT, as illustrated in Figure 1[2]. These reporters incorporate a varying number of tandem repeats (1×, 5×, 6×, and 7×) of a pseudo-palindromic NFAT response element (NFAT-RE) derived from the interleukin-4 (IL-4) promoter sequence (5′-TACATTGGAAAATTTTAT-3′) with minimal CMV promoter (parts:BBa_K5267049). This setup is anticipated to drive the transcription of the NanoLuc reporter gene when NFAT is dephosphorylated due to the significantly increased intracellular Ca²⁺ concentrations (Figure 1).