its regarding conventional genetic engineering method used in cancer gene designinig

1. INSERTIONAL MUTAGENESIS
- BY
AMRITA MOHANTY,
PhD research scholar

2. MUTAGENESIS :
 It is a process by which the genetic information of an organism is
changed in a stable manner resulting in a mutation.
 May occur spontaneously in nature or as a result of exposure to
mutagens or achieved by experimental laboratory procedures.

3. HISTORY OF MUTAGENESIS
 DNA may be modified, either naturally or artificially, by a number of
physical, chemical and biological agents, resulting in mutations.
 In 1927, Herman Muller, first demonstrated that mutation with observable
changes in the chromosomes can be caused by irradiating fruit flies with X-
ray.
 Lewis Stadler also showed the mutational effect of X-ray on barley in 1928
and ultraviolet (UV) radiation on maize in 1936.
 In 1978, Michael Smith discovered site-directed mutagenesis by using
oligonucleotides in a primer extension method with DNA polymerase.

4. TYPES OF MUTAGENESIS
 Directed mutagenesis
 Site-directed mutagenesis
 Insertional mutagenesis
 PCR mutagenesis
 Transposon mutagenesis

5. INSERTIONAL MUTAGENESIS
 Insertional mutagenesis is the phenomenon by which an exogeneous
DNA sequence integrates within the genome of a host organism.
 This event can result in the deregulation of genes in the neighborhood
of the insertion site and can potentially cause a perturbation of cellular
phenotype.
 It has been widely exploited for forward genetics screening aimed at
identifying novel cancer gene.

6. REQUISITES FOR INSERTIONAL MUTAGENESIS
I. Proper oncogenic agent that efficiently integrates in a random
or semi-random fashion into the host genome
II. An animal model that is permissive to tumor formation
III. Efficient technologies to perform retrieval of integration sites
IV.Bioinformatic and static tools to map integrations onto the host
genome and identifying targeted sites, defined as CIS (CIS –
Common insertion site) that are locations hosting candidate
cancer genes.

7. MECHANISM OF (INSERTIONAL MUTAGENESIS)
 Integrating vectors can induce cancer by mutating host genes in
a number of different ways and they can enhance transcription or
translational levels of oncogenes, generated chimeric or
truncated transcripts or inactivate TSG expression.
 Basically, there are three types of insertions –
(i) Enhancer insertions
(ii) Promoter insertions
(iii) intragenic insertions

8. ENHANCER INSERTION
 It is a common mechanism of mutagenesis (virus) and results in the
up-regulation of endogenous gene expression.
 As, regulatory elements may be distal to a gene, enhancer
insertions can be located some distance from the proximal promoter
and may affect the activity of elements via chromatin loops, thus
complicating the identification of the affected genes.
 It may be independent from their orientation, but are classically
upstream of a mutated gene in the antisense orientation or
downstream in the sense orientation, especially for retrovirus.

9. PROMOTER INSERTION
 It occurs when the insertional mutagen integrates in the sense
orientation in or close to the proximal promoter region of an
endogenous gene and uncoupling the cellular gene from its
promoter and placing it under the control of elements found
within the insertional mutagen.
 RNA polymerase transcribes an RNA starting from the promoter
of the insertional mutagen that reads through the introns and
exons of the host gene.
 The integrating vector contains splicing signals that induce the
joining of the first vector – coded exon to those of the host gene
and thus, can result in the translation of high levels of chimeric
transcripts.

10. INTRAGENIC INSERTION
 Intragenic insertions can interfere with the splicing of genes into
which they integrate, as many of insertional mutagens contain poly A
signals (engineered/endogenous), that may elicit premature
termination of gene transcription.
 Truncation of the transcripts may have different consequences on the
resulting protein and result in the expression of altered or neomorphic
alleles.
 viral insertion in the 3’UTR region of a gene may remove mRNA-
destabilizing motifs such as AUUUA hairpins or miRNA target
sequences and thus, resulting in increased levels of truncated but wild-

11. CONTINUED….
 By this mechanism, insertional mutagens may induce the
formation of a 3’ truncated mRNA by removing from the transcript
protein coding exons.
 Alternatively, transcription may start from the integrated
promoter, 5’ truncated mRNA is transcribed and hence, resulting
in C-terminal or N-terminal truncated proteins may possess
oncogenic properties and induce tumorigenesis.
 Using the same mechanism, vector insertions can inactivate a
gene: landing within a gene may result either in an mRNA
encoding an inactive or unstable protein or in aberrant splicing
which abrogate the gene function.

12. INTEGRATING AGENTS
 The integrating agents that are efficiently used so far to identify
new cancer genes by insertional mutagenesis are retroviruses and
transposable elements.
RETROVIRUSES
these are large family of enveloped RNA viruses found in all
vertebrates
 the genome is a homodimer of linear, positive sense, ssRNA of 7-
11Kb, surrounded by a cone shaped protein core.
In the retroviral life-cycle, the genetic information goes from RNA to
DNA and exists in two different forms; as genomic DNA when inside
the viral particle and as proviral double-stranded DNA when
integrated in the host genome.

13. CONTINUED….
 Both the ends of the genome contain terminal noncoding sequences, the so-called
long terminal repeats (LTR), composed of 5’ and 3’ unique sequences (U5 and U3
regions) and of two direct repeats (R) where the transcription start site(TSS) and the
polyadenylation signals (polyA) are located.
 Upon interaction between envelope glycoproteins and cellular receptors, fusion of the
virus and host cell plasma membrane occurs, and the viral capsid containing the RNA
genome enters the cell.
 The viral RNA genome is released in the cytoplasm and subsequently retrotranscribed
into a double-stranded proviral DNA.
 DNA is associated with viral proteins, and translocates to the nucleus, where the
integrase mediates integration of the provirus into the host cell genome.
 Oncogenic retroviruses have been classified in two groups, acute- and slow-
transforming retroviruses
acute transforming retroviruses – it induces the formation of polyclonal cancers
by highly expressing virus-encoded oncogenes with short latency periods (2-3 weeks) .

14. Structure of the genome
and replication life cycle
of
retrovirus

15. RETROVIRUSES AS INSERTIONAL
MUTAGENS
 From many years, insertional mutagenesis have used two types of slow
transforming ssRNA retroviruses: Moloney murine leukemia virus
(MoMLV , ɤ retrovirus ) and mouse mammary tumor virus (MMTV, α
retrovirus).
 These viruses cause lymphomas and mammary tumors in mice.
 Single genomic integration is not enough to promote tumorigenesis
and multiple rounds of proviral insertions within the same genome are
required for the development of cancer.
 By cloning proviral insertion sites from tumor cells it is possible to
identify candidate genes that have contributed to the development of

16. MURINE LEUKEMIA VIRUSES IN HEMATOPOIETIC MALIGNANCIES
 Moloney murine leukemia virus (MoMLV) is the prototypical murine
leukemia virus whose integration have been extensively studied for the
discovery of cancer genes in mice.
 The U3 promoter of MMLV recruits the basal transcriptional
machinery as it contains a TATA box and GC-rich sequences.
 The enhancer element within the virus has binding sites for B and T-
cell–specific transcription factors, such as ETS, NF1, RUNX, and MYB.
 MoMLV mainly induces T and B cell leukemia/lymphoma in mice.

17. CONTINUED….
 By analysing MoMLV integrations in murine tumors, several oncogenes have
been identified, including c-Myc, Pim1, Pim2, Pvt 1.
 Many studies have created recombinant MLVs by substituting LTRs from
other viruses and have tested them in mice.
 In, 2012 , Starkey et al., replaced the MoMLV LTRs with those of feline
leukemia virus and named it MoFe2-MuLV, mainly induced T-cell
lymphoma.
 Interestingly, analysis of MoFe2-MuLV integrations showed that this virus
did not target the same site as MoMLV and indeed led to the identification of
new cancer genes; Mf8t, Jundm2, Ahi1, Rras2.

18. CONTINUED….
 The SL3-3 MLV strain typically induces T-cell lymphomas, but a range of
LTR mutations, such as mutation of RUNX-binding sites, extends cancer
latency and skews tumor formation causing myeloid, B-lymphoid, and
erythroid tumors.
 MLVs are specific for different hematopoietic compartments, for
instance the erythroleukemia-inducing Friend virus is more active in
erythroid cells, whereas the Graffi-1.4 virus induces mainly acute myeloid
leukemia (AML).
 Using MMLV in transgenic E μ-Myc mice induced the development of
B-cell lymphoma and allowed the identification of additional cancer

19. CONTINUED….
• Instead of injecting a viral supernatant in newborn mice, identified CIS in tumors
derived from the recombinant inbred strains, BXH2 and AKXD.
• In BXH2 mice, the MLV is not inherited through the germline, rather is horizontally
transmitted via transplacental infection of implanted embryos.
• In the AKXD strains, the MLV is inherited through the germline, but then somatic
activation of additional recombinant viruses occurs.
• Therefore, the virus starts to replicate and integrates into the genome during early
gestation with mutagenesis continuing throughout the life of the animal causing a life-
long viremia.
• Indeed, studying and comparing the CIS retrieved from these mice allowed identifying
lineage-specific cancer genes involved in hematopoietic malignancies that are also
relevant in human tumors.

20. General outline of the work flow for cancer gene discovery using insertional mutagens

21. MMTV IN MAMMARY CANCER
 Mouse mammary tumor virus (MMTV) is a retrovirus with a similar tumorigenic
behaviour as MoMLV but with a specific tropism for mammary cells.
 Since carcinomas represent the most frequent human malignancies, the ability
of MMTV to hit the epithelial compartment is valuable and has resulted in the
identification of many clinically relevant cancer genes.
 The MMTV life cycle usually begins with the ingestion of infected milk by pups
from their viremic mother and after a few days, the virus infects B cells in
lymphoid tissue of the gut, for example, Peyer's patches.
 Thereby, stimulating a T-cell antigen response, a pool of B and TMMTV-
infected cells is generated as a reservoir of virus, from which MMTV spreads
into different tissues ultimately resulting in transformation of mammary
epithelial cells.

22. CONTINUED…..
Since 1982, MMTV has been used for forward genetic studies when
Nusse and Varmus identified Wnt1 as a frequent insertion target.
 Different studies conducted with MMTV-induced tumors led to the
identification of 12 candidate cancer genes, mainly belonging to Wnt
and Fgf pathways.
 But recently, Theodorou et al., performed the first large scale MMTV
insertional mutagenesis screening in the mouse mammary gland and
160 mammary tumors were screened and identified 33 CIS (among
which 21 were previously unknown MMTV targeted genes).

23. TRANSPOSON MEDIATED INSERTIONAL
MUTAGENESIS
Transposons are DNA sequences that can move from one location on
the genome to another.
 There are two general classes of transposons: retrotransposons and
DNA transposons.
RETROTRANSPOSONS
• They move by a “copy and paste” process (replicative transposition),
transposing via an RNA intermediates, which is then converted to DNA by
retrotranscription, which then inserts in new locations in the genome.
• But the low integration efficiency, the integration of incomplete
retrotranscribed elements and the concomitant induction of
chromosomal aberrations are still limiting their applicability to cancer

24. DNA TRANSPOSONS
 DNA transposons are a class of "cut and paste" transposons that rely
on a transposase enzyme, which recognizes specific DNA sequences
and "cuts" the DNA between them.
 The excised DNA is then reintegrated at another site in the genome.
 DNA transposon-mediated insertional mutagenesis screenings have
been developed in the last decade and allow the generation of tumors
in a wider spectrum of tissues than the ones that are accessible using
retroviruses.

25. SLEEPING BEAUTY SYSTEM
• DNA transposons are actively mobile only in plants and invertebrates, the
Sleeping Beauty transposon system was generated by reverse engineering in
1997, based on sequence comparison between multiple salmonid species of
nonfunctional transposase genes of the Tc1/mariner family that had
accumulated inactivating mutations.
• The Sleeping Beauty system is composed of two elements: a transposase,
that is, the enzyme responsible for mobilization, and the transposon, that is,
the actual mobilized sequence of DNA, which is flanked by the binding sites
for the Sleeping Beauty transposase, the so-called inverted repeats/direct
repeats.
• Transposition occurs when the transposase binds two sites in each inverted
repeat/direct repeat, and the closer the inverted repeats/direct repeats, the

26. CONTINUED….
When the transposase excises a transposon, it leaves behind a three-
base footprint.
 The transposon can then reinsert at any location in the genome
where a TA dinucleotide is present (there are more than 300 million
TA sites in the genome) and during integration, the TA is duplicated.
The cargo of the transposon (the segment contained between the two
inverted repeats/direct repeats) can be any sequence of choice, but
transposition efficiency decreases with increased cargo sizes .
 But, by introducing point mutations in the transposase gene, it has
been possible to significantly increase the level of mobilization.

27. Sleeping Beauty structure
and mechanism of transposition

28. CONTINUED….
 Transposition may be controlled by separating the transposase from the
transposon, thus establishing a nonautonomous bipartite transposon
system.
 In such a bipartite system, the transposon can be mobilized only when
the transposase protein is expressed.
 This may be achieved by generating two transgenic mouse strains: the
first is called the "jumpstarter" strain expressing the transposase gene;
the second is the "mutator" strain carrying the nonautonomous
transposon.

29. PIGGYBAC SYSTEM FOR INSERTIONAL MUTAGENESIS
AND CANCER GENE DISCOVERY
 Other transposable elements have been developed and exploited such as Minos (from
Drosophila hydei) and Tol2 (from Oryzias latipes, the Medaka fish, Zebrafish).
 To date, the only transposon that has been efficiently exploited for cancer gene
discovery as an alternative to Sleeping Beauty is the PiggyBac (PB) system.
 PB is a DNA transposon from the cabbage looper moth Trichoplusia ni that has been
developed to be active in mammalian cells, including mice.

30. CONTINUED….
PIGGYBAC SYSTEM SLEEPING BEAUTY
SYSTEM
Can efficiently mobilise large cargos it can mobilise smaller cargos
they integrate a TTAA tetranucleotide
sequence
they integrate TA dinucleotide sequence
Doesn’t leave any footprints Leaves three base pair footprints
Damages the neighbouring genome near
the mobilisation site
Doesn’t disrupt the genome nearby

31. CONTINUED….
In 2010, in the Science, they published PB insertional mutagenesis to identify
cancer genes in mice and deployed a bifunctional transposon containing 5’ and 3’
consensus sequences for both the Sleeping Beauty and PB transposases.
 The design of the integrating cassettes used was similar to T2/Onc transposon
because they contained a promoter, splice donor, splice acceptor and polyA sites to
generate gain-of-function and loss-of-function mutations.
 Three different types of transposons were developed, each containing a different
enhancer/promoter: MSCV (Murine stem cell virus), CAG, or the phosphoglycerate
kinase (PGK) promoter.
 Nineteen different transposon lines were produced from these three different transposons
each containing a different number of copies resident at different chromosomal loci.

32. Transposons for
insertional mutagenesis

33. CONTINUED….
• Fourteen of these lines were crossed with
mice that constitutively express PB
transposase from the Rosa26 locus.
• But, in case of they observed a high rate of
embryonic lethality in mouse strains that
contain a high copy number of
transposons. However, the strains with
intermediate to low copy number,
transposon arrays tumors formed.
• The tumor latency and the tumor type
were found to be profoundly affected by
the type of transposon.

34. CONTINUED….
 Mobilization of the MSCV transposon resulted in more than 90%
hematopoietic tumors.
 Conversely, the CAG transposon mainly induced solid tumors, including
sarcomas and various carcinomas with poor differentiation, and in some
cases metastasis was observed.
 Mice carrying mobilized PGK transposons developed hematopoietic and solid
tumors, with several mice bearing both types of malignancies.
 Remarkably, 42% of the candidate cancer genes identified by PB insertional
mutagenesis were not significantly hit in the previous studies conducted
using retroviruses or Sleeping Beauty transposons, suggesting that PB
targets a unique spectrum of loci.

35. ADVANTAGES OF INSERTIONAL MUTAGENESIS
SYSTEM
• The recent advance in sequencing
technologies has boosted the analysis of
cancer genomes at different levels,
including identification of point
mutations, chromosomal aberrations, and
epimutations
• High-throughput screening with cDNA
libraries or short hairpin RNA (shRNA)
libraries have also been exploited to
identify oncogenes and TSGs
LIMITATIONS
- These approaches require hundreds or
thousands of tumors to be analyzed to
implicate in cell transformation of those
genes that are less frequently mutated
thus making data from functional
approaches
LIMITATIONS
- cDNA libraries may not express a gene
product at an appropriate level to induce
tumorigenesis, whereas shRNAs are
notorious for off target effects and
incomplete silencing.

36. CASE STUDY

38. INTRODUCTION
 Traditional vertebrate model system such as mouse have now been complemented by
other organisms; rat, diploid frog (Xenopus tropicalis) and Zebrafish (Danio rerio).
 The insertional mutagenesis approach has now being deployed as an appropriate
methodology for genome modification in other model vertebrates that lack ES cells,
such as rat and pig.
 Advantages of the zebrafish include its external fertilization, high fecundity, rapid
development, production of optically clear embryos, and relatively short generation
time for a vertebrate.
 In addition to the high degree of genetic conservation reflected in the developmental
gene pathways and regulatory mechanism, contribute to its emergence as a model for
obtaining insights into fundamental human physiology.

39. CONTINUED….
They established forward genetic tools for the zebrafish include chemical
(N-ethyl-N-nitrosourea [ENU]; and insertional (retroviral) mutagens.
 Here, ENU produced random point mutations in the germline and these
single base pair changes resulted in a high frequency of mutant phenotypes.
 In spite of the high efficiency in generation of point mutations, the major
limitation in this approach, was identification of genes whose mutations are
responsible for the particular phenotype.
 An alternative approach is insertional mutagenesis, in which an exogenous
DNA serves as a mutagen and also functions as a molecular tag for
identifying the gene whose disruption causes the phenotype.

40. METHODOLOGY
Retroviral insertional mutagen in Zebrafish.
 Transposon based insertional mutagen in Zebrafish.

41. RETROVIRAL INSERTIONAL MUTAGEN IN
ZEBRAFISH
• The most extensively studied insertional mutagen to date in zebrafish is the
pseudotyped retrovirus, composed of a genome based on the Moloney murine
leukemia virus and the envelope glycoprotein of the vesicular stomatitis virus .
• They have injected this retrovirus into 1,000-cell to 2,000-cell stage zebrafish
embryos results in chimeric embryos in which different cells have integrations
of the viral sequences in different random sites in the genome.
• By passing these insertions through the germline and inbreeding them, they
have identified more than 500 mutations and about 350 loci; the insertional
nature of the mutagen facilitated the rapid molecular characterization of the
genetic loci, with 335 clones.

42. CONTINUED….
 Retroviruses appear to cause mutations in Zebrafish by several major mechanisms,
including exon disruption and gene silencing caused by insertion into an intron.
 Nearly 30% of mutagenic insertions recovered from a large scale mutagenesis
screening were in exons, basically in the 5’ UTR and such insertions lead to a complete
loss of wild-type gene product.
 And rest most of the other 70% of mutagenic insertions were in introns, as because
the MoMLV prefer to get inserted near the 5’ end of genes and generally these
insertions were in the first intron.
 Hence, these insertions usually result in the reduction or complete abrogation of
endogenous RNA Expression.

43. CONTINUED….
 Intronic insertions can also lead to aberrant splicing, resulting in skipped exons
and either frameshift mutations or internally truncated gene products.
 One major challenge to the field has been the inability to develop a similarly
mutagenic, high-titer retrovirus with robust expression.

44. TRANSPOSON BASED INSERTIONAL MUTAGENS IN
ZEBRAFISH
 They have employed a 5’ gene trap vectors in zebrafish and this trap was used to
increase the mutagenicity of retroviral vector.
 This vector utilized a splice-in and splice-out vector method, with intronic
insertion in the correct orientation inducing a frameshift and probably resulted,
either a truncated protein or a loss of gene product due to nonsense-mediated
mRNA decay.
 The 5’ gene trap contained, a splice acceptor and the green fluorescent protein
(GFP) gene was used in a Tol2 based transposon insertional study in Zebrafish.
 Integration of the gene trap in the proper orientation and reading frame, resulted in
GFP expression in temporally and spatially restricted pattern.
 In this study, thirty six trapped lines were homozygosed with no visible phenotypes,
but only about 5% of zebrafish genes had shown embryonic phenotypes when

45. TRANSPOSON INSERTIONAL MUTAGENESIS IN ZEBRAFISH

46. CONTINUED….
 They concluded that this type of gene traps may not reliably mutate
gene.
 Because many insertions in introns (the type of insertion required to
activate this trap) can abrogate or severely reduce gene expression.
 It may be that many such insertions were not detected as trap events
because the GFP reporter cannot be visualized in the absence of
expression of the endogenous gene.

47. CONTINUED….
 A combined 5’-3’ gene trap (‘gene breaking’) vector was developed to trap genes in
zebrafish.
 They have included a 5’ transcriptional terminator cassette to mutate the
gene in concert with 3’ gene trapping as an alternative strategy to select for
intragenic vector integrations.
 Although the employment of a transcriptional terminator in the gene breaking
trap vector allows suppression of splicing around the trapping vector, the
trapped gene expression domain cannot readily be identified using this basic
approach.
 Alternative approaches to add this feature to gene breaking transposons are
underway.

48. CONCLUSION
 Importantly, the recent studies with PB transposons clearly showed that using novel
tools allowed the identification of novel culprits of cell transformation, which were
elusive in the previous screening.
 Moreover, a-retroviral vectors have been recently developed for gene therapy
Applications.
 Insertional mutagenesis plays also a role in human disease and recent studies have
shown that spontaneous retrotransposon integration has a role in the pathogenesis of
different human tumors.
 The continuous development of new insertional mutagenesis tools, together with the
improvement of sequencing technologies for the retrieval of integration sites, will
continuously boost these forward genetics screenings that promise to significantly