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Figure 1. Design of the autoregulative NTA expression cassette and conditional expression of transgenes in cell lines and in single-transgenic pigs. A) The PtTA promoter drives a bicistronic mRNA encoding a human regulator of complement activation (RCA) linked with a tet-off transactivator (tTA) via a poliovirus IRES. Two constructs carrying the human cDNAs encoding either CD59 (CD59-NTA) or DAF (DAF-NTA) were used. Minimal expression of the transactivator results in tTA binding to the tet-operator sequences and initiates an autoregulative loop. In the presence of exogenous tetracycline the transactivator is inactivated and transcription silenced. B) Conditional expression of DAF in cell culture. NIH3T3 cells were stably transfected with DAF-NTA. A cell pool and a single clone (clone 12) were cultivated in presence (green line) or absence (black line) of doxycycline. Cells were harvested and DAF expression was determined by indirect immunofluorescence using flow cytometry. An overlay of the histograms is presented with nontransfected NIH3T3 cells (red line) as a control. C) Muscle fiber-confined expression of DAF and CD59 in transgenic pigs bearing a single NTA-cassette. Longitudinal (upper row) and cross sections (lower row) of transgenic and wild-type pig muscles after specific antibody (Ab) staining are shown, nuclei are counterstained with hematoxylin. Note that only individual fibers displayed intense Ab staining, whereas neighboring fibers are negative. No expression of the transgenes was detected in several other tissues.
