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Gene silencing Techniques

S B Mirza 1314
6th (Afternoon)[Bioinformatics]

10
April 27,
GENE SILENCING TECHNIQUES
2010

Contents
Introduction…………………………………………………………………………………
2
Transcriptional gene
silencing……………………………………………………………..3
Post-transcriptional gene
silencing………………………………………………………..4
Therapeutic applications and
challenges…………………………………………………5
Asthma……………………………………………………………………………………….
6
Knock-Out Mouse[Nude mouse]
………………………………………………………….7
History and
Nomenclature………………………………………………………………...8
Significance………………………………………………………………………………….
8
Immunological Responses of Nude
Mice………………………………………………..8
Uses of Nude Mouse………………………………………………………………………
11
References…………………………………………………………………………………..
12

S B Mirza1314 | GCUF
April 27,
GENE SILENCING TECHNIQUES
2010

Introduction:
“Gene silencing is a general term
describing epigenetic processes of gene
regulation. The term gene silencing is
generally used to describe the "switching
off" of a gene by a mechanism other than
genetic modification. That is, a gene
which would be expressed under normal
circumstances is switched off by
machinery in the cell.”
Cellular components of gene silencing:
 Histones
 Chromatin and heterochromatin
 MicroRNA
 siRNA
 dsRNA
 Dicer
 Transposons

There are different types of gene silencing as follows

1. Transcriptional Gene Silencing:


a) Genomic Imprinting
b) Paramutation
c) position effect
d) RNA-Directed DNA Methylation
e) Transposon silencing
f) Transgene silencing
g) Transcriptional gene silencing

2. Post-transcriptional Gene Silencing:


2
a) RNA interference
b) Nonsense mediated decay

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April 27,
GENE SILENCING TECHNIQUES
2010

3. Meiotic gene silencing:

a) Transvection

Transcriptional Gene Silencing:


is the result of histone modifications, creating an
environment of heterochromatin around a gene that makes it inaccessible
to transcriptional machinery (RNA polymerase, transcription factors, etc.).

Genomic Imprinting:

“genomic imprinting is a genetic phenomenon by which certain genes


are expressed in a parent-of-origin-specific manner. It is an inheritance
process independent of the classical Mendelian inheritance.”

Imprinted genes are either expressed only from the allele inherited from
the mother (eg. H19 or CDKN1C), or in other instances from the allele
inherited from the father (eg. IGF-2). Forms of genomic imprinting have
been demonstrated in insects, mammals and flowering plants.

Paramutation:
2
In epigenetics, paramutation is an interaction between two alleles of a
single locus, resulting in a heritable change of one allele that is induced
by the other allele. Paramutation violates Mendel’s first law, which states

S B Mirza1314 | GCUF
April 27,
GENE SILENCING TECHNIQUES
2010

that in the process of the formation of the gametes (egg or sperm) the
allelic pairs separate, one going to each gamete, and that each allele
remains completely uninfluenced by the other. In paramutation an allele
in one generation heritably affects the other allele in future generations,
even if the allele causing the change is itself not transmitted

Position effect:

Position effect is the effect on the expression of a gene when its location
in a chromosome is changed, often by translocation. This has been well
described in Drosophila with respect to eye color and is known as position
effect variegation (PEV).

The phenotype is well characterised by unstable expression of a gene that


results in the red eye coloration. In the mutant flies the eyes typically
have a mottled appearance of white and red sectors. These phenotypes
are often due to a chromosomal translocation such that the color gene is
now close to a region of heterochromatin. The heterochromatin can
spread stochastically and switch off the color gene resulting in the white
eye sectors.

RNA-Directed DNA Methylation:

RNA-directed DNA methylation RdDM is an epigenetic process first


elucidated in plants whereby small double-stranded RNAs (dsRNA's) are
processed to guide methylation to complementary DNA loci. In the model
plant organism Arabidopsis thaliana, these small dsRNA's may be
generated from three sources:

1 viral replication intermediates,

2 products of the endogenous RNA-dependent RNA polymerase, and

3 transcribed inverted repeats.

These dsRNAs are then processed to direct histone 3 lysine 9 (H3K9)


methylation via Ago4 and the SUVH (Suppressor of Variegation Homolog)
histone methyltransferase family. This H3K9 dimethylation is then 2
putatively bound by the cytosine methyltransferase CMT3, which
methylates cytosines in a non-CG context.

S B Mirza1314 | GCUF
April 27,
GENE SILENCING TECHNIQUES
2010

Post-transcriptional gene silencing:


Post-transcriptional gene silencing is the result of mRNA of a particular
gene being destroyed or blocked. The destruction of the mRNA prevents
translation to form an active gene product in most cases, a protein. A
common mechanism of post-transcriptional gene silencing is RNAi.

Both transcriptional and post-transcriptional gene silencing are used to


regulate endogenous genes. Mechanisms of gene silencing also protect
the organism's genome from transposons and viruses. Gene silencing thus
may be part of an ancient immune system protecting from such infectious
DNA elements.Genes may be silenced by DNA methylation during meiosis,
as in the filamentous fungus Neurospora crassa.

RNA interference (RNAi):

RNA interference (RNAi) is a


system within living cells that
helps to control which genes are
active and how active they are.
Two types of small RNA molecules
– microRNA (miRNA) and small
interfering RNA (siRNA) – are
central to RNA interference. RNAs
are the direct products of genes,
and these small RNAs can bind to
specific other RNAs and either
increase or decrease their
activity, for example by
preventing a messenger RNA from producing a protein. RNA interference
has an important role in defending cells against parasitic genes – viruses
and transposons – but also in directing development as well as gene
expression in general.

Nonsense mediated decay:

Nonsense mediated decay (NMD) is a cellular mechanism of mRNA


surveillance that functions to detect nonsense mutations and prevent the
expression of truncated or erroneous proteins. Following transcription,
precursor mRNA undergoes an assemblage of ribonucleoprotein (RNP)
components followed by regulatory pre-mRNA processing. Large average 2
intron size in eukaryotic cells greatly increases the probability that
aberrant mRNA splicing will result in the presence of a nonsense (stop)
codon (UAA, UAG, UGA) somewhere within the open reading frame. NMD

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April 27,
GENE SILENCING TECHNIQUES
2010

is triggered by exon junction complexes (EJCs) (components of the


assembled RNP) that are deposited during pre-mRNA processing.

Meiotic gene silencing:

Transvection:

Transvection is an epigenetic phenomenon that results from an


interaction between an allele on one chromosome and the corresponding
allele on the homologous chromosome. Transvection can lead to either
gene activation or repression. Formally (see quote from Lewis, below), it
can also occur between nonallelic regions of the genome as well as
regions of the genome that are not transcribed.

Strategies:

Gene Silencing Technique Offers New Strategy for Treating, Curing


Disease:

Turning on or off technique:


A new technique aimed at directly controlling the expression of genes by
turning them on or off at the DNA level could lead to drugs for the
treatment or cure of many diseases, say researchers at UT Southwestern
Medical Center.
"Virtually every disease starts at the level of malfunctioning gene
expression, or viral or bacterial gene expression," said Dr. David Corey,
professor of pharmacology and biochemistry. "This is an approach that 2
could theoretically produce a drug for the treatment or cure of almost any
disease."
Some’ll be disscussed here:

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April 27,
GENE SILENCING TECHNIQUES
2010

 Asthma
 Knock-out Mouse[Nude Mouse]

Asthma:

Asthma is a disease affecting the airways that carry air to and from your
lungs. People who suffer from this chronic
condition (long-lasting or recurrent) are
said to be asthmatic.

Now, John Shannon and colleagues, at


Cincinnati Children's Hospital Medical
Center, have demonstrated that LPCAT1
has a crucial role in the generation of
surfactant in vivo in mice and that the activity of this protein must be
maximal for the transition from the womb to air breathing. They therefore
speculate that decreased LPCAT1 expression, as a result of mutations in
the gene responsible for making this protein, might underlie the fatal

respiratory distress syndrome.

Knockout Mouse:

A knockout mouse is a genetically engineered mouse in which one or 2


more genes have been turned off through a gene knockout. Knockout
mice are important animal models for studying the role of genes which
have been sequenced, but have unknown functions. By causing a specific

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April 27,
GENE SILENCING TECHNIQUES
2010

gene to be inactive in the mouse, and observing any differences from


normal behavior or condition, researchers can infer its probable function.

THE NUDE MOUSE

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April 27,
GENE SILENCING TECHNIQUES
2010

ude mouse is a genetic mutant that has a deteriorated or removed


thymus gland, resulting in an inhibited immune system due to a
greatly reduced number of T cells.

The phenotype or main outward appearance of the mouse is a lack of


body hair, which gives it the "nude" nickname. The first nude mutation
2
was observed in 1962. The Nude mouse is valuable to research because it
can receive many different types of tissue and tumor grafts, as it mounts
no rejection response. These xenografts are commonly used in research
to test new methods of imaging and treating tumors. The genetic basis of

S B Mirza1314 | GCUF
April 27,
GENE SILENCING TECHNIQUES
2010

the nude mouse mutation is a disruption of a gene named the FOXN1


gene.

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April 27,
GENE SILENCING TECHNIQUES
2010

History and Nomenclature:


Nude mice have been bred since at least 1937. They were discovered and
first bred by Miroslav Holub (1923-1998), a distinguished Czech
immunologist and a renowned poet.
The Nomenclature for the nude mouse has changed several times
since their discovery. Originally they were described as ‘nu’ and this was
updated to ‘Hfh11nu’ when the mutated gene was identified as a
mutation in the HNF-3/forkhead homolog 11 gene. Then in 2000 the gene
responsible for the mutation was identified as a member of the Fox gene
family and the nomenclature was updated to ‘Foxn1nu’.

Significance:
A mutant mouse said to be nude because it is hairless due to the
presence of two copies of the gene ‘nu’. Because they lack a thymus,
Nude mice cannot generate mature T lymphocytes. Therefore they are
unable to mount most types of immune responses, including:

1. antibody formation that requires CD4+ helper T cells


2. cell-mediated immune responses, which require CD4+ and/or CD8+ T
cells
3. delayed-type hypersensitivity responses (require CD4+ T cells)
4. killing of virus-infected or malignant cells (requires CD8+ cytotoxic T
cells)
5. graft rejection (requires both CD4+ and CD8+ T cells)

Picture: Placental and foetal weight and litter size were studied on the
18th day of pregnancy in the athymic nude mouse. Placental weight and
litter size were unaffected by the absence of a thymus. Foetal weight was
reduced, probably as a result of the health status of such animals.

Immunological Responses of
Nude Mice:
Mice homozygous for the
mutation nude lack a thymus.
They are deficient in lymphocytes
in thymus dependent areas of peripheral lymphoid organs. They lack cells
bearing ‘θ’ which is a cell surface antigen found on thymocytes and 2
thymus dependent cells. In a preliminary communication it have been
reported that nu/nu mice have impaired heterograft rejection and low
immunoglobulin levels.

S B Mirza1314 | GCUF
April 27,
GENE SILENCING TECHNIQUES
2010

In 1969 Miller and Mitchell gave evidences, that thymus-derived cells


and antibody producing cells form two populations, but nu/nu mice shows
a deficiency in both thymus-derived cells and in antibody forming cells,
but how both the populations are effected? This was defined as, it could
occur either because the nude mutation affects the
maturation/differentiation of both cell types, for instance, by preventing
differentiation of a common precursor, or may be because the normal
development of one cell type is dependent on the normal development of
the other.

The low Immunoglobulin level is suggestive to be as due to B-cell


defect. If Nude mice do lack a normal B cell population, the interesting
question arises as to the primary site of the block. nudes lack mature T
cells, but there is no intrinsic defect in T cell precursors, rather it is the
lack of the normal inductive environment provided by the thymus that
causes the block in T cell differentiation. The proposed block in B cell
development imposed by nude could either be intrinsic to B cell
precursors or secondary to a defect in a tissue that normally induces B
cell differentiation. If a defect in inductive tissue exists, it is an entirely
open question as to whether the thymus itself provides the inductive
signals, for instance, by means of a hormone.

Uses of Nude Mouse:


“Not only have Nude mice answered many questions in immunology, but
their ability to maintain human tissues have made them useful tools for
investigating, for example, the properties of human cancers.”
Many of the uses of Nude mice are:
• Self-recognition specificity expressed by T cells from nude mice,
Absence of detectable Ia-restricted T cells in nude mice that do Exhibit
self-K/D-restricted T cell responses
• Functionality of T cells in athymic nude mice if inserted from mice
having thymus
• Lack of mature B cells in nude mice With X-linked immune deficiency
which shows that lack of B-cells in nu/nu mice is due to a recessive
gene associated with nu/nu mice and X-linked immune deficiency
• It is also observed fact that truncated Galectin-3 inhibits tumor growth
and metastasis in Orthotopic Nude mice model of human Breast cancer
• Suppression of Tumor cell growth was observed both in Nude Mice and
in culture by n-3 Polyunsaturated Fatty acids when mediated through
Cyclooxygenase-independent pathways
• The influence of synthetic polymeric agents on the immune
responsiveness of congenitally athymic (nude) mice was investigated
by determining the effects of in vivo treatment with polynucleotides 2
and polymeric haptenated antigens on splenic theta-bearing cells, on
mitogen stimulation and on plaque-forming cell responses to thymic
dependent and thymic independent antigens. Contrary to in vitro data,

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April 27,
GENE SILENCING TECHNIQUES
2010

no evidence was obtained to demonstrate in vivo restoration of these


immune parameters by the use of non-specific immune enhancers
• Nude mice are now a days widely used by combining two different
deficient strains, as Nude mice and xid mice, Nude mice and SCID mice
etc
• The H-2 complex and M locus have been defined for two incipient
inbred strains of mice carrying the nu mutation. The strains differ at
both loci from each other and cells from nu/nu mice of these allogeneic
strains have been used to examine the role of T cells in the one-way
mixed leukocyte reaction (MLR)
• Antibody response to many of tumor causing viruses, for example,
Simian virus tumor antigen when reconstituted with T-cells

These six photographs show 6 different skin xenografts surviving on nude


mice.

A. Human (mammal) skin after


60 days.

B. Cat (mammal) skin at 51


days.

C. Chicken (bird) at 32 days;


the feathers were already
present when the graft was
made.

D. Chameleon (reptile) at 41
days.

E. Fence lizard (reptile) at 28


days.

F. Tree frog (amphibian) at 40 days.

S B Mirza1314 | GCUF
April 27,
GENE SILENCING TECHNIQUES
2010

References:
1) e Nude Mouse in Experimental and Clinical Research (Vol.1). Fogh, J;
Giovanella, B.C. (eds) Academic Press, 1978, ISBN 0-12-261860-2
2)
The Nude Mouse in Experimental and Clinical Research (Vol.2).
Fogh, J; Giovanella, B.C. (eds) Academic Press, 1982, ISBN 0-12-
261862-9

3) Handbook of Mouse Mutations with Skin and Hair Abnormalities:


Animal Models and Biomedical Tools By John P. Sundberg
Published by CRC Press, 1994

4) http://jaxmice.jax.org/faq/bulletin/bulletin06.html

5) http://jem.rupress.org

6) http://www.karger.com

7) (OMIM)http://en.wikipedia.org/wiki/Mendelian_Inheritance_in_Man

8) Benjamin Cohen and David Ng, Artwork by Jane Wang – 2006

9) Proc. Natl. Acad. Sci. USA Vol. 81, pp. 886-888, February 1984
Medical Sciences

10) Proc. Natl. Acad. Sci. USA Vol. 81, pp. 886-888, February 1984
Medical Sciences

11) Clin. exp. Immunol. (1971) 8, 305-317 by H. H. Wortis

12) Wilkinson, Lawrence S.; William Davies and Anthony R. Isles


(November 2007). "Genomic imprinting effects on brain
development and function". Nature Reviews Neuroscience 8 (11):
832–843. doi:10.1038/nrn2235. PMID 17925812.
http://www.nature.com/nrn/journal/v8/n11/abs/nrn2235.html.
Retrieved 2008-07-01.

13) DeChiara, Thomas M.; Elizabeth J. Robertson and Argiris Efstratiadis


(February 1991). "Parental imprinting of the mouse insulin-like
growth factor II gene". Cell 64 (4): 849–59. doi:10.1016/0092-
8674(91)90513-X. PMID 1997210.
http://linkinghub.elsevier.com/retrieve/pii/0092-8674(91)90513-X.
Retrieved 2008-07-01.
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