Tight regulation of transgene expression by tetracycline-dependent activator and repressor in brain.

Methods to temporally and spatially regulate gene mutations will provide a powerful strategy to investigate gene function in the brain. To develop these methods, we have established a tightly regulated system for transgene expression in the forebrain using both a tetracycline (Tc)-dependent transcription activator (rtTA) and a repressor (TetR-Kruppel-associated box). In this system, the repressor binds to the Tc-responsive element (TRE) in the absence of doxycycline (Dox), leading to the repression of leaky activation of TRE-mediated transcription caused by weak binding of rtTA to TRE. Upon Dox administration, only the activator binds to TRE and activates transcription. We tested this system in cultured cells by bicistronically expressing both the regulators using an internal ribosome entry site (IRES). In COS-1, HeLa and SHSY5Y cells, leaky transcription activation led by rtTA in the absence of Dox was repressed without decreasing the level of activated transcription in the presence of Dox. Using this system, transgenic mice were produced that express both the regulators using IRES in the forebrain under the control of the alphaCaMKII promoter and were bred with transgenic mice carrying the TRE-dependent reporter transgene. In reverse transcription-polymerase chain reaction and in situ hybridization analyses of the forebrain in adult double transgenic mice, the treatment of Dox induces reporter mRNA expression, which was not detected before the treatment and after the withdraw of Dox following the treatment. These results indicate that this system allows the tight regulation of transgene expression in a Dox-dependent fashion in the forebrain and will be useful in investigating gene function in the brain.

Generation of microglial cell lines by transfection with simian virus 40 large T gene.

Microglial cells, which were isolated from a primary culture of neonatal rat brain, were transfected with temperature-sensitive simian virus 40 (SV40) large T gene by the calcium phosphate precipitation method. Four weeks after transfection, several colonies were generated, and cloned cells were characterized. One of the cloned cells (RBM129) proliferated actively at 37 degrees C and the dividing rate was significantly decreased at 40.5 degrees C. The expression of large T antigen was detected by western blotting in cells incubated at both 37 degrees C and 40.5 degrees C. The cell line showed high activity of non-specific esterase, isolectin B4 binding and phagocytosis. Also the cells were stained by ED 1 monoclonal antibody. These results indicate that these cells were derived from rat brain microglia, and immortalized by large T gene.

Effect of the replacement of pRoR promoter of lambda dv plasmid by lac promoter on the synthesis of DNA.

Derivatives of lambda dv whose pRoR promoter was replaced by lactose operon (lac promoter) were constructed and cloned in pBR322 plasmid. They were named pLOP-1, 2, and 3, and their structures are shown in Fig. 1. These plasmids were introduced into Escherichia coli (E. coli) lac iq and the effect of the inducer of lac promoter on the synthesis of plasmid DNA was examined. The synthesis of pLOP-2 or 3 DNA was strongly stimulated. pLOP-1, however, responded poorly to the inducer. Plasmids pLOP-2 and 3 were not segregated evenly into daughter cells after the induction and most of the progeny cells did not receive the plasmid. The synthesis of plasmid DNA after induction depended on the function of O and P genes and was inhibited by the addition of rifampicin and chloramphenicol. Most of the plasmid DNA synthesized after the induction cosedimented with the folded host chromosomal complex, suggesting an unusual structure of the DNA. When the inducer was removed, normal segregation of the plasmid resumed and the copy number of plasmid DNA decreased to the original level.