Fig. 5.
Autonomous reporting of transcriptional activity and tissue identification using autobioluminescence. a Two vector inducible or repressible autobioluminescence cassette schematics. The first vector uses a modified tetracycline response element (TETO) to control the expression of the viral 2A-segmented, polycistronic lux operon. In the second vector, CBA drives the expression of either a transactivator (tTA) that provides constitutive lux expression until it is repressed in the presence of doxycycline or a reverse transactivator (rtTA) that inhibits lux expression until doxycycline is present. b For tissue-specific reporting, the luciferase component genes are controlled by the cardiac-specific TNNT2 promoter. c iPSCs with the lux genes under the control of the tetracycline responsive promoter and a separately integrated CBA-driven reverse transactivator (rtTA) produce autobioluminescence when exposed to increasing amounts of doxycycline for either 4 or 24 h. d In contrast, iPSCs with the lux genes under the control of the tetracycline responsive promoter and a separately integrated CBA-driven transactivator (tTA) show a reduction in autobioluminescent output in response to doxycycline exposure. Values are representative of N = 3 replicates. Error bars represent the standard error of the means. p/s/cm2/sr; photons/s/cm2/steradian. e Both wild-type iPSCs and iPSC-derived cardiomyocytes produce autobioluminescence when transfected with Stem-luxCDEF/Stem-luxAB, but only cardiomyocytes produce light when the Stem-luxCDEF/TNNT2-luxAB vectors are used. Data is available at https://osf.io/h5qzj/ [15]