Oncogene and tumor

suppressor models
 Animal models help in tumor biology study and develop
new approaches to conquer human cancer
Mouse models ideal for cancer research
Small size, easy handling
Short generation time
High tumor incidence, rapid tumor growth
High nos of syngeneic tumor models
Histocompatibility in inbred strains, allows tumor
transplantation
Genetically best characterized
 Ectopic models

Orthotopic models (express genes in correct anatomical

sites)

Orthotopic models, mimic complexicity of tumor

behaviour in patients, invivo microenvironment
surrounding cancer cells
 Mutagenic agents induce tumorigenesis in mouse
strains, but target genes not known (genetically
uncharacterized)

Transgenic mouse models, stable and precise

introduction of specific mutations into mouse genome
possible
 Transgenesis is the process of introducing an
exogenous gene called transgene, into a living organism,
so that the organism will exhibit new property and
transmit that property to its offspring
 Transgenic mouse model of oncogene

A construct is prepared which has a strong promoter and

gene of interest (oncogene)

This construct is microinjected into pronuclei of fertilized

egg
Homologous recombination occurs, normal gene is
replaced with oncogene
Embryos are implanted into foster mothers
Carrier animals are bred to ensure germline transmission
of construct and multiple independent lines are studied
as each will have different integration sites
 E.g knock-in approach to express bcr-abl oncogene

Limitations
Mutated oncogene is inserted in germ cells, heritable
characteristics, whereas bcr-abl translocation in
leukemia is somatic mutation

Genes involved in cancer also play role in development,

so knocked-in gene expressed in embryos exert
dominant negative effect, result in embryonic lethality
 Cre-lox P system

Cre-lox P system
Cre is site specific DNA recombinase derived from P1
bacteriophage that recognizes 34 bp sequence termed
lox P sites

By homologous recombination, lox P sites can be

introduced in mouse genome

Cre can be expressed in another transgenic mice in

specific tissue and/ or at specific time in development
 Cre-lox P system

Cre-lox P system for expressing oncogene

Steps
Prepare a construct where lox P sites are placed between the
strong promoter and target gene, gene expression is
prevented.

Introduce this construct in pronuclei of fertilized egg to

generate transgenic animal

Similarly generate another transgenic animal which expresses

cre in tissue-specific manner (hematopoietic cells)
 Cre-lox P system

Intercross these transgenic lines

In animals that are born, homologous recombination
takes place, cre excises lox P sites moving strong
promoter close to target gene and trans-activation of
gene only in tissues where cre is expressed

Adv
Tissue specific expression
High levels of expression driven by strong promoter
 Cre-lox P system

Cre-lox P system for developing knock-out mutants

Conditional knock-out mutants lack expression of target

gene only in specific cell type or tissue

Steps
Prepare a construct where lox P sites are located in intronic
regions, flanking the coding region of gene to be inactivated

Introduce this construct in pronuclei of fertilized egg to

generate transgenic animal
 Cre-lox P system

Similarly generate another transgenic animal which

expresses cre in tissue-specific manner (hematopoietic
cells)
Intercross these transgenic lines
In animals that are born, homologous recombination
takes place, cre excises target gene flanked by lox P
sites only in tissues where cre is expressed
 Conditional mouse models for oncogenes

Tetracycline-controlled transcriptional activation

Method of inducible expression of genes where
transcription of genes is reversibly turned on or off in
presence of tetracyline or its derivative doxycycline
Two inducible expression systems used for research in
eukaryotes, Tet-off and Tet-on

Transcriptional activator protein (tTA) is a fusion of

[Tet repressor + VP16 (TF of HSV)]
 Conditional mouse models for oncogenes

tTA protein binds to tet response elements (TRE) located

within inducible promoter results in expression of genes
downstream of the promoter

tTA protein activates gene expression in both Tet-off and

Tet-on system,

Tet-off, activates gene expression in absence of Dox

Tet-on system, activates gene expression in presence of
Dox
 Conditional mouse models for oncogenes

Tet-off system
In absence of Dox, tTA protein binds to DNA at tet
operon, transcription of nearby genes

If Dox added, it binds to tTA protein, renders it incapable

of binding to TRE sequence, no transcription of nearby
genes
E.g bcr-abl fusion protein
Withdrawal of tet, expression of bcr-abl fusion protein,
lethal leukemias develop within 3-11 wks
 Conditional mouse models for oncogenes

Re-addition of tet, no expression of bcr-abl fusion

protein, tumor regression (indicating bcr-abl expression
necessary for transformed phenotype and validating bcr-
abl as leukemia drug target