Abstract
Abstract: :
Purpose: In order to study ocular responses to transgene expression, it would be advantageous to be able to regulate the timing and duration of transgene expression. Our goal is to establish a bigenic system for reversible regulation of transgene expression in the eye. Methods: Two different systems have been tested. One is based on the yeast Gal4 transcription factor. The other is based upon a recombinant tetracycline transactivator (tTA). Engineered versions of Gal4 and tTA were linked to ocular promoters (Pax–6 and DREAM) and used to generate transgenic mice. The regulatory elements that are bound by these transactivators (UAS and TRE, respectively) were linked to target transgenes including lacZ. These constructs were used to generate transgenic mice. Bigenic mice, generated by mating transactivator mice with responder mice, were assayed for expression of the target transgenes. Results: Five Pax6–Gal4 transgenic families were generated. In situ hybridization identified three families with good transgene expression in the lens and corneal epithelial cells. Six UAS–lacZ families were generated and mated to the Pax6–Gal4 mice. One UAS–lacZ family (OVE1977) showed no X–gal staining in the absence of Gal4. In bigenic mice, X–gal staining was detected in the eye beginning at embryonic day 10 (E10). This UAS–lacZ reporter family has been mated to mice that express a RU486–regulated version of Gal4 to establish the parameters for inducible and reversible lacZ expression. Six DREAM–tTA families have been generated, along with three TRE–lacZ families. Embryos (E11) that carry both of these transgenes show lens–specific staining with X–gal. Additional studies need to be done to establish the time course for induction and reversion of transgene expression in response to administration of RU486 or doxycycline. Conclusions: Both Gal4 and tTA can be used as transactivators for ocular–specific regulation of trangene expression. These transactivators can now be used for timed studies of signal transduction pathways, transcriptional responses, and cell morphology changes in response to induction or elimination of expression of specific transgenes.
Keywords: transgenics/knock–outs • gene/expression • signal transduction