An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 246

mutagenesis. The second line (TIGRE) contains an additional copy of Gene X
cDNA (or genomic fragments) driven by TRE-promoter inserted in a specific
locus in the genome (the TIGRE locus), which has been pre-selected for low basal
transcriptional activity and high inducibility. When these two lines are crossed,
rtTA protein produced from the KO allele can activate the TRE-Gene X in the
TIGRE locus only in the presence of Dox. (Alternatively, tTA can be used, which
works in the opposite way - Tet-off instead of Tet-on.)
Figure 1. Schematic diagram of the principle of the iKO system. (A) Two components of the iKO system, a
KO line and a TIGRE line. (B) Generation of iKO mouse by crossing the KO and TIGRE lines. The iKO is a
composite mouse in which: 1) both copies of the endogenous Gene X are disrupted by the insertion; 2) rtTA
is driven by the promoter of Gene X; and 3) the cDNA of Gene X is in the TIGRE locus under the control of
TRE. Functional Gene X is only expressed from the TIGRE locus when rtTA is bound to Dox, and therefore is
inducibly and reversibly regulated by Dox.
Figure 1B illustrates the breeding of KO and TIGRE lines to produce the
iKO mouse, which is homozygous for the KO locus and carries one copy of the
TIGRE allele. The status of the iKO mouse is regulated by Dox. In the absence
of Dox, rtTA protein is produced, but is inactive. As a result, TRE-Gene X is
silent, Gene X protein is not produced and the KO phenotype is manifested. In
the presence of Dox, rtTA stimulates the synthesis of Gene X protein from the
TIGRE locus in the same cells in which the endogenous gene would normally be
expressed. Expression complements the missing endogenous Gene X activity and
leads to phenotypically normal animals. Thus, one can switch between wild type
and KO state of animals by simply adding or removing Dox (e.g. with food).
selection of tiGre Loci
To screen for TIGRE loci, we constructed a Moloney murine leukemia virus
(MoMLV)-based retroviral vector, pRTonZ (Figure 2A), in which the TRE-controlled