An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 244

regulated (TIGRE) genomic locus, which provides for low basal expression
and high inducibility. Such a locus occurs infrequently in the genome and
we have developed a method to easily introduce genes into the TIGRE site of
mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both
KO and TIGRE lines have been engineered for high-throughput, large-scale
and cost-effective production of iKO mice. As a proof of concept, we have cre-
ated iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensi-
tive and quantitative phenotypic analyses. The results demonstrated reversible
switching of ApoE transcription, plasma cholesterol levels, and atherosclero-
sis progression and regression. The iKO system shows stringent regulation and
is a versatile genetic system that can easily incorporate other techniques and
adapt to a wide range of applications.
introduction
In the post-genome era, a major challenge is deciphering the function of thou-
sands of newly identified genes. One of the main approaches for studying gene
function involves inactivation of genes in cells or animals using random (chemi-
cal or insertional) mutagenesis or gene targeting. A common problem with these
methods stems from the fact that the gene of interest is usually mutated through-
out the animal's life. As a result, 1) in many cases the mutation leads to embryonic
or neonatal lethality, precluding the assessment of the gene's function in later life;
2) in viable mutants interpretation of observed phenotypes is often complicated
by the inability to distinguish the direct effects of the gene loss at the time of
observation from the results of developmental abnormalities caused by the gene
loss earlier in life; 3) in still other cases, life-long absence of a gene product causes
compensatory adjustments of activities of other genes precluding the elucidation
of the function of the gene of interest. Conditional knockout and gene expres-
sion technologies, such as the Cre/lox-mediated tissue-specific knockout [1] and
the tetracycline (Tet) regulated transcriptional activation system [2], can regulate
gene expression in a more spatially and temporally controlled fashion. However,
these technologies are often laborious to establish and the results are frequently
variable.
Here we report the development of a system that provides for the inducible
and reversible gene inactivation in the mouse and can also be readily scaled up for
high-throughput applications. The iKO system is a binary approach based on the
Tet-dependent regulatory technology. It involves the combination of two mouse
lines - a KO line that expresses the Tet-transactivator (tTA or rtTA) in place of the
gene of interest, and a TIGRE (for tightly regulated) line that contains the gene of
interest under the control of the Tet-responsive element (TRE) at a predetermined