Finding Function


Random mutagenesis


Gene Targeting

Conditional Gene Targeting [regulate gene expression in a more spatially and temporally
controlled fashion]


Cre/loxP mediated Tissue Specific KO


Tet regulated TransActivator
Why need an Inducible and Reversible Mouse Genetic Rescue System ???

GOI is mutated through out animals life

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[Developmental
Effect]

3) 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. [Compensatory Effect]
Tet On / Off System

rtTA => rTetR + VP16 from HSV

tTA => Tet R + VP16 from HSV
 Principle of the iKO System
iKO system is a binary approach based on the Tet-dependent regulatory technology

Tet-transactivator
(tTA or rtTA)
at
Endogenous GenX Locus

tightly regulated [TIGRE Locus]

Tet Response Element and cDNA of GeneX
Principle of the iKO System
Selection of TIGRE Loci

to ES cells

cre expression vector Neo Positive

Neo Negative
 cre expression vector

Neo Negative

NeoPositive
 Strategy: Site specific Transgenic mice via invio Electroporation

CMV + cre expression vector

in vivo Electroporate into

mouse testis which contains the
target site of integration
Cre recombination occurs and the
transgene integrate into the presise location
in the genome of
spermatogonial cells of male mouse

CMV

CMV CMV promoter

P promoter
Characterization of gene regulation at TIGRE loci
Screening of class I
clones for
high inducibility

Gene
regulation in mice
generated from
tightly regulated
ES clones
 KO Lines Produced from an ES Cell Library
Mutagenized by a Retroviral Vector

Apolipoprotein E

regulate Cholesterol metabolism & Atherossclerosis progression

Phenotype of ApoE deficiency is quantifiable & very sensitive to leaky expression,

allowing for the evaluation of the stringency of the gene regulation by iKO technology
KO Lines Produced from an ES Cell Library
Mutagenized by a Retroviral Vector
ApoE iKO mice

ApoE -/- ; TRE-ApoE

ApoE gene expression as well as blood cholesterol levels are tightly controlled by DOX

DOX

Present Absent

High ApoE Low ApoE

Low /Normal Blood Cholesterol High Blood Cholesterol

Become WT Become KO
Plasma cholesterol levels in the ApoE iKO, KO and WT group mice
in the absence and presence of Dox.
Atherosclerotic lesion progression
Atherosclerotic lesion regression
Readily scaled up for high-throughput applications

minimizing embryonic lethality

minimizing developmental effects

minimizing compensatory effects

regulation of GOI at TIGER locus

insert GOI at TIGER locus by single step

Efficiency and cost effective

Flexibility
 The advantage s of iKO

1) ability to turn genes on or off at will by adding or removing doxycycline

(Dox) at any time during the animal’s life, thus minimizing embryonic
lethality, developmental effects, and compensatory effects;

2) high degree of regulation to any gene inserted at the TIGRE locus, which
has been selected to confer minimal basal expression and high inducibility,
and to insert any gene of interest in a single step by Cre/loxP recombination

3) efficiency; the design allowing streamlined production of both KO and

TIGRE mice makes it possible to generate iKOs for a large number of genes in
a costeffective manner

4) flexibility; KO and TIGRE lines can be engineered independently and

combined in numerous ways, making a wide range of applications possible.
Additional Pictures
&
Information
 ApoE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents. The
APOE gene is mapped to chromosome 19 in a cluster with APOC1 and APOC2. Defects in
apolipoprotein E result in familial dysbetalipoproteinemia, or type III hyperlipoproteinemia
(HLP III), in which increased plasma cholesterol and triglycerides are the consequence of
impaired clearance of chylomicron and VLDL remnants

APOE[2] is essential for the normal catabolism of triglyceride-rich lipoprotein constituents.

APOE was initially recognized for its importance in lipoprotein metabolism and
cardiovascular disease. More recently, it has been studied for its role in several biological
processes not directly related to lipoprotein transport, including Alzheimer's disease (AD),
immunoregulation, and cognition. Neonates with brain injuries and/or defects who also have
abnormalities in the APOE gene may have an increased risk for cerebral palsy, according to
researchers at the Northwestern University Feinberg School of Medicine. Defects in APOE
result in familial dysbetalipoproteinemia, or type III hyperlipoproteinemia (HLP III), in which
increased plasma cholesterol and triglycerides are the consequence of impaired clearance of
chylomicron, VLDL and LDL remnants.
ApoEKo X ApoE TIGRE

ApoE +/- and TRE-ApoE

expression of TRE-ApoE is
strictly dependent on DOX