Design and application of gene coding calcium probe GCaMP-X

A gene-encoded, calcium ion technology, applied in the field of calcium ion concentration indicators, can solve problems that need to be optimized

Active Publication Date: 2018-05-04
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the current genetically encoded calcium ion concentration indicator GCaMP remains to be optimized

Method used

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  • Design and application of gene coding calcium probe GCaMP-X
  • Design and application of gene coding calcium probe GCaMP-X
  • Design and application of gene coding calcium probe GCaMP-X

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Research on the relationship between the degree of nuclear aggregation of GCaMP3 and the morphology of corresponding neurons

[0057] At present, many studies have found and reported that when GCaMP is long-term or overexpressed in neurons by virus infection or electroporation, the fluorescence distribution of GCaMP tends to accumulate to the nucleus, and the cell morphology, physiological function and GCaMP monitors Ca 2+ performance will change.

[0058] Therefore, the inventors transfected the fluorescent protein YFP and GCaMP3 plasmids into isolated and cultured cortical neurons, and after 2 days of plasmid expression, the degree of nuclear accumulation of GCaMP3 (the nucleocytoplasmic ratio of GCaMP3 fluorescence N / C ratio) and the corresponding neuron Morphology (total neurite length) was quantified. see results figure 1 .

[0059] Such as figure 1 Shown, (a) Neurite tracking diagrams of representative neurons of fluorescent protein YFP control gro...

Embodiment 2

[0061] Example 2 Study on the Mechanism of GCaMP's Damage to Neurons

[0062]Based on the fact that CaM can participate in and regulate the excitation-transcription coupling of neurons, the inventor speculates that the damage effect of GCaMP on neurons is mainly due to the CaM contained in itself, and that GCaMP can change the gating-signal of LTCC and affect CREB-related gene transcription process.

[0063] First, in order to verify that GCaMP will affect the gating characteristics of LTCC, the inventors used a cell line expression system to conduct experiments, combining GCaMP3 with the carbon-terminal complete Ca V 1.3 channels (α 1DL ) or Ca at the short carbon end V 1.3 channels (α 1DS ) co-expressed in HEK293T cells, using the parameter S Ca (such as S 50Ca =1-I 50Ca / I peakCa , where I 50Ca is the calcium current when the voltage is activated for 50ms, I peakCa is the spike calcium current within 300 ms of voltage-sustained activation) to measure the CDI streng...

Embodiment 3

[0073] Example 3 GCaMP-X and GCaMP3-X C Preparation and effect testing

[0074] The results of Examples 1-2 indicate that existing GCaMPs need to be optimized. Based on the competitive relationship between apo-CaM and the DCT domain of LTCC, the optimization of GCaMP3 can be achieved by introducing a fragment (CaM Binding Motif, CBM) that can weaken the ability of apo-CaM to channel pre-binding. Connecting CBM directly to GCaMP3 is beneficial to the combination of the two in terms of spatial position, which can weaken the pre-binding ability of GCaMP3 and LTCC, and avoid its interference on the gating characteristics of LTCC.

[0075] At the same time, the inventors have also used a special positioning sequence, namely the nuclear export signal (Nuclear ExportSignal, NES) sequence (sometimes also called "nuclear export sequence"), which can bind to the nuclear export receptor in the cell, Export the associated protein to the cytoplasm, further ensuring that the optimized GCa...

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Abstract

The invention discloses an optimized and improved gene coding calcium probe GCaMP-X and basic application thereof. The probe comprises a GCaMP protein, a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence refers to fragments capable of weakening pre-binding capacity between calmodulin un-bound with calcium ions and an L type calcium channel (LTCC); the second amino acid sequence is a localization sequence (intranuclear, extranuclear and other subcellular regions); the first amino acid sequence is located at a nitrogen terminal of the GCaMP protein; and the second amino acid sequence is located at a carbon terminal of the GCaMP protein. Compared with each kind of the conventional GCaMP probes, the optimized GCaMP-X probe has effects of eliminating interference effects on nerve cell LTCC and mediated excitation-transcription coupling thereof and objectively and accurately measuring calcium signals of nerve cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene-encoded calcium ion concentration indicator and an application thereof. Background technique [0002] Genetically-Encoded Calcium Indicators (GECIs) are a class of Ca 2+ Kinetic bioprotein indicators. GECIs can be divided into three categories: (1) Bioluminescent indicators based on photoproteins such as jellyfish, which are essentially different from indicators based on fluorescent proteins, requiring a series of chemical reactions in the molecule to generate fluorescence ; (2) Indicators based on single fluorescent proteins, such indicators include response Ca 2+ Components, such as CaM. CaM fused to fluorescent protein, CaM to Ca 2+ The binding condition of the fluorophore can adjust the degree of protonation of the fluorophore, thereby affecting the spectral properties of the fluorophore. Such indicators include Camgaroos, GCaMPs, Pericams, etc.; (3) Indicators based ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12Q1/02
CPCC07K14/4728C07K2319/60G01N33/5041G01N2333/4727G01N2500/10
Inventor 刘晓冬刘楠杨亚雄何元源
Owner TSINGHUA UNIV
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