An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 260

gene's level and this could be a problem for genes that are highly sensitive to gene
dosage effect or show haploid deficiency. The kinetics of the system (days) may be
also too slow for some developmental problems where transient expression of de-
velopmental genes are critical, although it should be noted that it could still work
well in carefully thought-out developmental studies (as in [24]). In addition, it
has been reported that rtTA often can not induce sufficient gene expression in the
brain, at least partially due to developmental inactivation of the TRE promoter
in neurons [36]. It appears that the tTA (Tet-off) system is better suited for the
use in brain [14], as substantial β -gal induction was observed in brain with tTA
(Figure 4F).
Our results of the ApoE iKO mice and the quantitative data using lacZ and lu-
ciferase reporters suggest that the iKO system could be a useful tool in addressing
a variety of biological questions. The two components of the iKO system can be
independently modified, and pairing of their different forms can generate numer-
ous combinations. In KO lines, genes with unique expression patterns are tagged
with a transcription transactivator, which can control, in an inducible fashion, the
expression of a variety of genes derived from TIGRE lines, enabling a number of
additional applications in specific types of tissues or cells. Those include: 1) in-
troducing mutant forms of the target gene into the TIGRE locus for better func-
tional probing of different domains, splice variants, post-transcriptional modifica-
tions (e.g. phosphorylation), or modeling human diseases; 2) humanizing target
genes by placing a human ortholog of the mouse gene under TRE control, which
can facilitate drug efficacy studies; 3) placing a cytotoxic gene under TRE con-
trol to allow inducible cell-type specific ablation; 4) introducing a marker gene
such as GFP, protein interacting probe, or transneuronal tracer, into the TIGRE
locus for cell-type specific tagging, functional analysis, isolation of specific cell
population, or mapping neuronal networks; 5) combining with recently devel-
oped RNAi techniques [37],[38],[39] to down-regulate any genes of interest in a
tissue-specific and inducible manner; 6) re-engineering the TIGRE locus to place
a TRE-driven Cre, and combining it with a tissue-specific rtTA or tTA line and
floxed target genes to achieve inducible region-specific gene knockout [40].
Materials and Methods
screening and isolation of es clones with insertions in target
Genes
Construction of ES cell library infected with the retrovirus and screening and
isolation of ES clones with genes of interest inactivated by the viral insertion is
described previously [18]. Specifically for ApoE, insertions were found by nested