Aging Aging

Genetically Engineered Mice 365in both cell lines and transgenic animals by replacing the CMV promoter normallyused with one that is cell specific. The gene of interest is placed into asecond vector called the “response” transgene at a multiple cloning site locateddownstream of a Tet response element (TRE) consisting of seven tandem Tetoperator sequences fused to a minimal CMV promoter (pminCMV). The componentsof this system are now commercially available from Clontech. TherTta protein will bind and activate the gene of interest only in the presence ofTet or its more lipophilic derivatives such as doxycyclin (dox). Both highlyinducible and tightly controlled levels of transgene expression are observedupon treatment with inducing agent with little basal expression. The Tet induciblesystem has been recently used to generate transgenic mice containingtetracycline-inducible transgenes in a cell-type specific manner including underthe control of neuronal-specific promoters indicating that dox administeredorally crosses the blood–brain barrier (31,32). In addition, oral dox treatmentseems to have no detrimental effects on body tissues or general toxicity for theanimals (33). The Tet system can be used to drive expression of antisense RNAs tocontrol the activity of endogenous gene expression (34). Like with othertransgenic systems, the site of integration of both activator and responsetransgenes may have an effect of their levels of expression.In addition to the Tet system, other systems are also available that appear tobe effective in inducing both cell-specific and inducible transgene expression.The recently described ecydysone system, for example, also boasts high levelsof inducibility, low basal expression, rapid kinetics, a minimum of confoundinginducing agent-elicited effects, no apparently toxicity to mammalian cellsin vitro or in vivo, and the components are also now commercially availablefrom Invitrogen (35). This system requires the expression of two separate componentsof a modified nuclear Drosophila ecdysone receptor (the Drosophilaecdysone receptor itself and a mammalian retinoic X receptor both can beexpressed from cell-specific promoters); these receptors bind to one anotherand activate expression of the transgene of interest from an ecdysone responsivepromoter upon administration of the steroid compound, muristerone.Induction of transgene expression using this system was reported by the authorsto be greater than that obtained with the Tet system and to have lower levels ofbasal expression; however, there has been only one publication to date in whichthe system has been demonstrated to work in vivo (35).Besides the availiability of systems for tissue-specific and inducible expressionof transgenes, systems have also been developed for the inducible inactivationof gene expression only in certain organs or cell types. Such systemsovercome the problems associated with the possible lethality associated withdisrupting genes during the embryonic period so that the effects of gene losscan be analyzed at later stages in the life of the organism. One such system is