Luminescent stem cells and uses thereof

A stem cell and cell technology, applied in the field of recombinant stem cells, can solve the problem of low sensitivity and achieve low cost and reliable drug development process

Inactive Publication Date: 2009-02-18
AXXAM股份公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] Newest A version of the system has also been adapted for fluorescence assays, even with lower sensitivity compared to CCD camera-based devices

Method used

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  • Luminescent stem cells and uses thereof
  • Luminescent stem cells and uses thereof
  • Luminescent stem cells and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0333] 1. Establishment of ES cell lines transfected with photoproteins

[0334] 1.1.1mito ES TBV2 clone

[0335] by linearizing mito with BglII restriction enzyme The pcDNA3 vector of the photoprotein gene was electroporated into ES TBV2 p16 cells to obtain mouse ES TBV2mito Cell lines (Materials and methods). Forty-eight hours after transfection, cells were placed under 200 μg / ml G418 selection. After 8 days of selection, 152 drug-resistant colonies were picked. After morphological analysis, only about 114 were propagated on the MEF layer until they reached confluency in 96 wells / plate in triplicate with:

[0336] 1-2 on gelatin-coated 96 white MTP for testing on a CCD camera-based luminometer at 24 and 48 h after inoculation.

[0337] 2-1 on gelatin-coated 96 white MTP for DNA extraction.

[0338] 3-2 on a feeder layer for freezing and -80°C storage.

[0339] 1.1.2 mito ES TBV2 clone selection

[0340] 4 hours before the measurement, in the dark, at 37 °C, in...

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Abstract

Described is a stable recombinant stem cell able to express an apophotoprotein and produce a bioluminescent signal in the presence of a suitable chromophore as substrate in response to intracellular calcium concentration variation; non human transgenic mammal and cell derivatives thereof produced by said cells; methods for identifying agents modulating the differentiation of stem cells towards a specific cell lineage; methods for identifying a ligand able to stimulate a specific cell lineage target; methods for identifying an antagonist to ligand known to stimulate a specific cell lineage target; uses of stable recombinant stem cells for in vitro testing of toxicity and/teratology of a substance.

Description

[0001] The present invention relates to recombinant stem cells stably transfected with a gene encoding apophotoprotein. More specifically, the present invention relates to non-human pluripotent stem cells, to pluripotent embryonic stem cells, to human and non-human pluripotent tumor cells, to pluripotent adult stem cells and their progenitors. Recombinant photoprotein stem cell lines are used for different purposes, such as in high-throughput screening, in the undifferentiated state to identify agents that stimulate or inhibit differentiation to specific cell lineages, and in the differentiated state to target endogenously expressed Target genes were screened. Background of the invention [0002] Stem cells are unspecialized cells capable of renewing themselves for prolonged periods of time through cell division (1). Furthermore, under certain physiological or experimental conditions, they can differentiate into different cell types, such as beating cardiomyocytes or insulin-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12G01N33/68
CPCC07K14/43595
Inventor S·卡伊纳卡C·努奇S·科拉扎S·洛默尔
Owner AXXAM股份公司
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