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
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[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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