An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 258

knockout of the endogenous gene, however, it is irreversible and the efficiency of
tamoxifen inducibility throughout the body is yet to be demonstrated. The useful-
ness of the Tet-inducible system [21],[22] is critically dependent on the tightness
of transcription induction and suppression. When either the Tet-transactivator
or the target gene is randomly integrated into the genome, they are subjects to
positional effects [23]. It is often necessary to screen through multiple transgenic
lines every time a new transgene is introduced. Attempts to target both tTA and
TRE together into an endogenous gene locus to achieve inducible activation and
inactivation were successful in a few cases [24],[25],[26], but this method is not
applicable to most other genes as the TRE is easily subject to activation from
nearby enhancers independent from tTA. Our iKO technique utilizes each gene's
own promoter to direct the expression of transcriptional activators, e.g. rtTA and
tTA. Therefore it is not limited to certain tissues and available tissue-specific pro-
moter driven transgenic lines. It could be applicable to any gene in any tissue.
Dox-regulated expression can be turned on and off rapidly, i.e. within a few days,
and at any time in development or adult. It also allows analysis of the effects of
gene inactivation in the same animal. The TIGRE locus has been selected to con-
fer little or no basal transgene expression throughout the body while maintaining
high inducibility, enabling stringent control of the on/off switching of the target
gene. Our system also has the flexibility to allow for further improvements. For
example, in some cases it may be more desirable to put the genomic copy of the
gene under TRE promoter into the TIGRE locus instead of the cDNA to more
precisely mimic the expression of the endogenous gene. Also, the IRES we have
used to drive rtTA translation may not work uniformly well in all tissues, and
can be replaced by other approaches such as using the viral 2A-like sequences for
bicistronic translation [27] or direct targeting of tTA/rtTA into the ATG start
codon of the endogenous gene.
Unique chromosomal loci for predicted gene expression provide fundamen-
tal tools for genetic studies. The most widely used is the ROSA26 locus [28] in
which ubiquitous gene expression is achieved by endogenous promoter activity
of this locus. The TIGRE loci identified in this study allow a different mode of
gene regulation - they were selected from hundreds of insertion sites for tight
gene regulation by an exogenous promoter. Therefore, the TIGRE loci offer a
platform for easy insertion of any gene in a tightly regulated locus, applicable to
not only the tetracycline system but also other gene expression systems utilizing
exogenous promoters such as constitutively active promoters, tissue specific pro-
moters, or other inducible promoters regulated by reagents such as ecdysone [29],
mifepristone [30] and streptogramin [31]. Recently a similar approach was also
applied to a human fibrosarcoma cell line to pre-screen optimal integration sites
for transgenes [32]. In addition, here we demonstrated that the stringency of the
regulation at TIGRE loci is further enhanced by the incorporation of insulators,