Download The central dogma of molecular biology describes the flow of

Methods in Gene Silencing
The central dogma of molecular biology describes the flow of genetic
information within a biological system:
1. The dogma is a framework for understanding the transfer of
sequence information between sequential information-carrying
biopolymers, in the most common or general case, in living
organisms. There are 3 major classes of such biopolymers: DNA
and RNA (both nucleic acids), and protein.
2. The general transfers describe the normal flow of biological
information: DNA can be copied to DNA (DNA replication), DNA
information can be copied into mRNA (transcription), and
proteins can be synthesized using the information in mRNA as a
template (translation).
Transcription
Main article: Transcription (genetics)
Transcription is the process by which the information contained in a
section of DNA ( gene) is transferred to a newly assembled piece of
messenger RNA (mRNA). It is facilitated by RNA polymerase and
transcription factors. In eukaryotic cells the primary transcript (premRNA) must be processed further in order to ensure translation. This
normally includes a 5' cap, a poly-A tail and splicing. Alternative
splicing can also occur, which contributes to the diversity of proteins
any single mRNA can produce
Genes are regulated at either the transcriptional or posttranscriptional level. So
1. Transcriptional gene silencing (TGS) : is the result of histone
modifications, creating an environment of heterochromatin(
histone methylation) around a gene that makes it inaccessible
to transcriptional machinery (RNA polymerase, transcription
factors, etc.), Promoters silenced.
2. Post-transcriptional gene silencing ( PTGS): 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.( promotor active,
gene is hypermethylated in coding region,This resently called
RNAi.
Other names of post-transcriptional gene silencing (PTGS) :
1. Gene silencing: Scientific Exploitation: Knockouts and
Knockdowns epigenetic processes of gene regulation, the
ability of exogenous double-stranded RNA (dsRNA) to
suppress the expression of the gene which corresponds to the
dsRNA sequence., which lead mRNA to unable to make a
protein during translation, that occurs without a change in
DNA sequence…
a. Gene silencing: is naturally triggered when a viral pathogen
invades a cell in a plant or animal and enables that plant or
animal to stop the virus from replicating and causing
disease.
b. Although use of this natural mechanism by scientists is still
at the early research stage. Gene silencing technology is
enabling researchers around the world to protect animals
from diseases and develop new crop varieties
2. RNA silencing: refers to a family of gene silencing effects by
which the expression of one or more genes is down regulated
or entirely suppressed by the introduction of an antisense
RNA molecule. The most common and well-studied example
is RNA interference, in which endogenously
expressed microRNA or exogenously derived small interfering
RNA induces the degradation of complementary messenger
RNA. It also plays an important role in defending plants
against viruses. Enzymes detect double stranded RNA (that is
not normally found in cells) and digest it into small pieces that
are not able to cause disease. This can be performed under
normal conditions or in the context of a disease.
3. RNA interference: is a biological process in which RNA
molecules inhibit gene expression, typically by causing the
destruction of specific mRNA molecules. Or Small Interfering
RNAs (siRNAs) are short, double-stranded RNA molecules that
can target mRNAs with complementary sequence for
degradation via a cellular process termed RNA interference
(RNAi)
Short history :
1. 1990 Jorgensen :
Introduction of transgenes homologous to endogenous genes often
resulted in plants with both genes suppressed!
*Called Co-suppression
**Resulted in degradation of the endogenous and the transgene
mRNA
2. 1995 Guo and Kemphues:
injection of either antisense or sense RNAs in the germline of C.
elegans was equally effective at silencing homologous target genes
3. 1998 Mello and Fire:
-extension of above experiments, combination of sense and
antisense RNA (= dsRNA) was 10 times more effective than single
strand RNA
4.The importance of this technology is reflected by the fact that the
2006 Nobel prize for medicine was awarded for the discovery of RNA
interference by Craig Mello and Andrew Fire.
What is RNA interference /PTGS?
1. dsRNA needs to be directed against an exon, not an intron in
order to be effective.
2. homology of the dsRNA and the target gene/mRNA is required
3. targeted mRNA is lost (degraded) after RNAi
4. the effect is non-stoichiometric; small amounts of dsRNA can wipe
out an excess of mRNA (pointing to an enzymatic mechanism)
5. ssRNA does not work as well as dsRNA
General guidelines for target sequence selection include: General
Design Guidelines for siRNA
1. Find 21 nt sequences in the target mRNA that begin with an
AA dinucleotide.
Beginning with the AUG start codon of your transcript, scan for
AA dinucleotide sequences. Record each AA and the 3' adjacent
19 nucleotides as potential siRNA target sites.
2. Select 2-4 target sequences
• Avoid areas with a GC content of >70% or <30%, •Since a 4-6
nucleotide poly(T) tract acts as a termination signal for RNA pol III,
avoid stretches of > 4 T's or A's in the target sequence when
designing sequences to be expressed from an RNA pol III promoter
•Since some regions of mRNA may be either highly structured or
bound by regulatory proteins, we generally select siRNA target sites
at different positions along the length of the gene sequence. We
have not seen any correlation between the position of target sites on
the mRNA and siRNA potency.
•Compare the potential target sites to the appropriate genome
database (human, mouse, rat, etc.) and eliminate from consideration
any target sequences with more than 16-17 contiguous base pairs of
homology to other coding sequences.
3. Design appropriate controls:
A complete siRNA experiment should include a number of controls to
ensure the validity of the data.
Two of these controls are:
•A negative control siRNA with the same nucleotide composition as
your siRNA but which lacks significant sequence homology to the
genome. To design a negative control siRNA, scramble the nucleotide
sequence of the gene-specific siRNA and conduct a search to make
sure it lacks homology to any other gene.
•Additional siRNA sequences targeting the same mRNA. Perhaps the
best way to ensure confidence in RNAi data is to perform
experiments, using a single siRNA at a time, with two or more
different siRNAs targeting the same gene. Prior to these
experiments, each siRNA should be tested to ensure that it reduces
target gene expression by comparable levels.
Vectors: pSilencer 4.1-CMV Expression Vectors
The pSilencer 4.1-CMV vectors employ a powerful CMV promoter to
drive high level expression of cloned hairpin siRNA templates in a
wide
variety of cell types. They also include an antibiotic resistance gene
that
provides a mechanism to select for transfected cells that express the
1. Mammalian selectable markers
The pSilencer 4.1-CMV puro siRNA expression vector contains a
puromycin
resistance gene to enable antibiotic selection in mammalian cells.
Antibiotic selection can be used to enrich for cells that were
successfully
transfected with pSilencer 4.1-CMV puro by killing off cells that
lack
the plasmid. Short term antibiotic selection is very useful for
experiment
systems where low transfection efficiency would otherwise
preclude
detection of a reduction in target gene expression. For long-term
gene
knockdown studies, the puromycin resistance gene makes it
possible to
select cell populations, or clonal cell lines, that stably express the
hairpin
siRNA. Puromycin in culture
medium at 50–4000 ng/mL is commonly used to select cells with
an
integrated plasmid containing the resistance gene. Cells without
the resistance gene are normally killed within 3–5 days.
Modified CMV promoter for
siRNA expression : The pSilencer 4.1-CMV puro System employs a
modified
Cytomegalomavirus (CMV) promoter to drive expression with
RNA pol II, and includes a modified simian virus-40 (SV40)
polyadenylation
signal downstream of the siRNA template to terminate
transcription.:
pSilencer 4.1-CMV plasmids
are supplied ligation-ready
The pSilencer 4.1-CMV siRNA Expression vectors are linearized
with
both BamH 1 and Hind III to facilitate directional cloning. They are
also purified to remove the digested insert so that it cannot religate with
the vector. This greatly increases the percentage of clones bearing
the
hairpin siRNA template insert after ligation, reducing the time and
effort required to screen clones. A basic pSilencer 4.1-CMV puro
vector
map is shown in Figure 1 on
Designing siRNA Hairpins Encoded by siRNA Expression Vectors and
siRNA Expression Cassettes
These designs produce an RNA transcript that is predicted to fold
into a short hairpin siRNA as shown in Figure 1. The selection of
siRNA target sequence, the length of the inverted repeats that
encode the stem of a putative hairpin, the order of the inverted
repeats, the length and composition of the spacer sequence that
encodes the loop of the hairpin, and the presence or absence of
5'-overhangs,
For traditional cloning into pSilencer vectors, two DNA
oligonucleotides that encode the chosen siRNA sequence are
designed for insertion into the vector (Figures 2 and 3). In general,
the DNA oligonucleotides consist of a 19-nucleotide sense siRNA
sequence linked to its reverse complementary antisense siRNA
sequence by a short spacer. Ambion scientists have successfully
used a 9-nucleotide spacer (TTCAAGAGA), although other spacers
can be designed. 5-6 T's are for cloning into the pSilencer 4.1-CMV
vector, nucleotide overhangs containing the Bam H1 and Hind III
restriction sites are added to the 5' and 3' end of the DNA
oligonucleotides, respectively added to the 3' end of the
oligonucleotide.
Optimizing Antibiotic Selection Conditions:
Cell type, culture medium, growth conditions, and cell metabolic
rate
can all affect the optimal puromycin concentration for selection
of pSilencer
4.1-CMV-transfected cells. Identify the lowest level of puromycin
that kills nontransfected cells within approximately 5 days by
testing
puromycin concentrations from 50–4000 ng/mL while keeping all
other culture conditions equal. See step 1. Puromycin titration
(kill
curve) below.
Using the pSilencer 4.1-CMV siRNA Expression
Vector
1.
a.
b.
c.
Cloning Hairpin siRNA Inserts into pSilencer 4.1-CMV
Prepare oligonucleotide
Anneal the hairpin siRNA template oligonucleotides
Ligate annealed siRNA template insert into the pSilencer 4.1CMV vector
d. Transform E. coli with the ligation products
e.
f.
g.
h.
Expected results
Identify clones with the
hairpin siRNA insert
Pick clones, isolate plasmid DNA, and digest the plasmid with
BamHI
i. and HindIII to confirm the presence of the ~55 bp siRNA
insert.
j. Purify pSilencer 4.1-CMV plasmid for transfection
pSilencer 4.1-CMV plasmid preparations must be free of salts,
proteins,
and other contaminants to ensure efficient transfection.
2. Transfecting pSilencer 4.1-CMV into Mammalian Cells:
a. Transfect cells and culture 24 hr without selection
Transfect the purified plasmid into the desired cell line, plate
transfected
cells at the plating density and culture for 24 hr without
selection
It is important to include two non-transfected control cultures.
One is
subjected to puromycin selection to control for the fraction of
cells that
survive selection; it will help determine the effectiveness of the
transfection
and selection. The second control is grown without puromycin
selection as a positive control for cell viability.
b. Add medium containing puromycin
Add culture medium
puromycin identified
containing the concentration
of
3. Selecting Antibiotic-Resistant Transfected Cells:
pSilencer 4.1-CMV siRNA expression vectors can be used in
transient
siRNA expression assays, or to create cell populations or a
clonal cell line
that stably expresses your siRNA. Note that with normal
(nontransformed)
and primary cell lines, it may be difficult to obtain clones that
stably express siRNA. For these types of cells, it is recommend
choosing
the antibiotic selection strategies outlined in sections 1 and 2
below
a. Short term puromycin selection for enrichment of cells that
transiently
express the siRNA: Culture the cells for 1–3 days in the
puromycin-containing medium, Analyze the population for an
expected phenotype and/or the expression of the target gene.
b. Selecting a population of cells that stably express the
siRNA: Creating a population of cells stably expressing the
siRNA involves
treating cells with puromycin for several days to eliminate
cells that were
not transfected. The surviving cell population can then be
maintained
and assessed for reduction of target gene expression.
c. Selecting for clones that stably express the siRNA :
the goal is to create a clonal cell line that expresses
the hairpin siRNA template introduced with pSilencer 4.1CMV. Cloning
stably expressing cell lines is advantageous because strains
that
exhibit the desired amount of gene knockdown can be
identified and
maintained, and clones that are puromycin-resistant but
which do not
express the siRNA can be eliminated.