Source: http://www.patentsencyclopedia.com/app/20100173407
Timestamp: 2016-07-23 23:11:03
Document Index: 780439588

Matched Legal Cases: ['art 1', 'art 2', 'ART3', 'ART(3', 'art5', 'art5', 'art5', 'art5', 'art5', 'art1', 'art2', 'ART(3', 'ART3', 'ART(3', 'ART(3', 'art5', 'art5', 'art5', 'art5', 'art5', 'art5', 'art5', 'art5', 'art5', 'art5', 'art5', 'art5', 'art5']

INHIBITION OF GENE EXPRESSION - Patent application
Patent application title: INHIBITION OF GENE EXPRESSION
Krzysztof Wypijewski (Dundee Tayside, GB)
Christophe Lacomme (Edinburgh Lothian, GB)
Gyorgy Hutvagner (Dundee Tayside, GB)
Csaba Hornyik (Dundee Tayside, GB)
Jane Shaw (Tayside, GB)
Jennifer Stephens (Dundee Tayside, GB)
Patent application number: 20100173407
There is provided a method of inhibiting gene expression by locating a
5'-5 donor splicing site sequence in the 3' UTR of a gene or within
coding sequence containing the stop codon of a gene. Stability of
homologous mRNA in trans is also adversely affected leading to a
reduction in expression. A polynucleotide containing a 5'-donor splicing
site sequence in either coding sequence containing the stop codon or the
3' UTR is also provided and in one embodiment is in the form of a vector.
The 5'-donor splicing site sequence can be present in multiple copies,
for example as a tandem repeat. In one embodiment the 5-donor splicing
site sequence has the sequence 5'-MAGGTRAGTA-3' where M is A or C and R
is A or G.Claims:
1. A polynucleotide comprising a gene having an exon containing a stop
codon and said polynucleotide having a transcription terminator for said
gene, wherein the gene has a sequence derived from a 5' splicing site
located upstream of the transcription terminator and within the exon of
the gene containing the stop codon.
2. The polynucleotide as claimed in claim 1 wherein said gene encodes a
protein or a portion thereof.
4. The polynucleotide as claimed in claim 1 wherein said gene is in cDNA
5. The polynucleotide as claimed in claim 1 wherein the 5' splicing site
sequence is located downstream of the stop codon.
6. The polynucleotide as claimed in claim 1 wherein the 5' splicing site
sequence is located upstream of the stop codon.
7. The polynucleotide as claimed in claim 1 wherein the 5' splicing site
sequence is located within 20 to 1000 nucleotides upstream of the
9. The polynucleotide as claimed in claim 1 wherein two or more copies of
the 5' splicing site sequence are present.
10. The polynucleotide as claimed in claim 9 wherein the two or more
copies of the 5' splicing site sequence are present in tandem.
11. The polynucleotide as claimed in claim 1 wherein the 5' splicing site
sequence is the sequence 5'-MAGGTRAGTA-3' (SEQ ID NO: 1) where M=A or C
and R=A or G, or a variant thereof in which 1, 2, 3 or 4 nucleotides are
substituted or deleted.
12. The polynucleotide as claimed in claim 11 wherein the 5' splicing site
sequence is the sequence 5'-CAGGTAAGTA-3' (SEQ ID NO: 2) or a variant
thereof in which 1, 2, 3 or 4 nucleotides are substituted or deleted.
16. A polynucleotide vector having a 5' splicing site sequence located
upstream of a transcription terminator wherein two or more copies of the
5' splicing site sequence are present in tandem.
17. A polynucleotide vector having a 5' splicing site sequence located
upstream of a transcription terminator, wherein said vector comprises at
least a portion of an open reading frame containing a stop codon or at
least a portion of a 3'UTR, wherein said 5' splicing site sequence is
located in the 3'UTR or open reading frame.
18. The vector as claimed in claim 16 wherein said vector comprises at
least a portion of a 3' UTR of a target gene, wherein said 3' UTR portion
comprises at least one copy of the 5' splicing site sequence.
19. The vector as claimed in claim 16 wherein said vector comprises at
least a portion of an open reading frame of a target gene, wherein said
open reading frame contains a stop codon, wherein said open reading frame
20. The vector as claimed in claim 16 wherein said vector is able to
insert said 5' splicing site sequence within the 3'UTR of a target gene.
21. The vector as claimed in claim 16 wherein said vector is able to
insert said 5' splicing site sequence within an open reading frame of a
target gene, said open reading frame containing a stop codon.
22. The vector as claimed claim 16 wherein the 5' splicing site sequence
is located within 20 to 1000 nucleotides upstream of a transcription
23. The vector as claimed in claim 16 wherein the 5' splicing site
24. The vector as claimed in claim 23 wherein the 5' splicing site
25. The vector as claimed in claim 23 which further includes a
26. The vector as claimed in claim 25 wherein the polyadenylation signal
is AAUAAA.
27. The vector as claimed in 16 wherein said vector includes a
polynucleotide sequence of a target gene in a form suitable for
28. The vector as claimed in claim 27 which includes a full length
sequence of the target gene.
30. The vector as claimed in claim 27 wherein the target gene is a
chimeric gene.
31. The vector as claimed in claim 26 wherein said vector comprises at
35. A method of reducing expression of a target gene wherein said gene
comprises at least a portion of either an open reading frame containing a
stop codon or of a 3'UTR, said method comprising modifying the portion of
open reading frame or 3'UTR by inserting a 5' splicing site sequence
therein and upstream of a transcription terminator of said target gene.
36. The method of claim 34 wherein said 5' splicing site sequence is
inserted into the open reading frame of said gene.
37. A method of reducing expression of a target gene, said method
comprising providing a vector comprising, in functional relationship, a
transcription initiator, a targeting sequence and a transcription
terminator, said vector further comprising a 5' splicing site sequence
upstream of said transcription terminator, and wherein 21 nucleotides of
said targeting sequence has at least a 95% sequence identity to 21
nucleotides of the target gene.
38. The method as claimed in claim 37 wherein 21 nucleotides of the
targeting sequence has 100% sequence identity to 21 nucleotides of the
39. The method as claimed in claim 37 wherein said targeted sequence is in
an open reading frame of the target gene.
40. The method as claimed in claim 37 wherein said targeted sequence in a
3' UTR of the target gene.
41. A host cell containing a polynucleotide of claim 1.
42. A host cell containing a polynucleotide vector of claim 16.
49. The vector as claimed in claim 24 which further includes a
50. The vector as claimed in claim 17 wherein said vector includes a
expression.Description:
[0001]The present invention is concerned with a method to inhibit gene
expression. In particular the method concerns inhibiting gene expression
by the inclusion of a 5'-donor splicing site sequence within the
3'-untranslated region of an expressed gene or with the exon containing
the stop codon of the expressed gene.
[0002]The selective regulation of gene expression is important in the
control of many physiological processes. Control of gene expression could
therefore allow treatment of many diseases and could also be utilised to
manage infections due to pathogenic organisms such as viruses, bacteria,
fungi and protozoa. Additionally, the ability to control gene expression
would also have significant utility in gene discovery, particularly in
determining the function of the product of a particular gene. The
regulation of gene expression in cell model systems or transgenic
organisms would benefit academic research as well as the commercial
[0003]One naturally occurring method of gene regulation utilises sequence
specific DNA binding proteins called transcription factors. Binding of
the transcription factor to its target gene results in either the
activation, enhancement or inhibition of gene expression. Exemplary
transcription factors include NF-kappa B, steroid hormone receptors (such
as progesterone) or zinc finger proteins.
[0004]Artificial manipulation of gene expression is currently performed
using antisense technology, small molecule regulators, and gene
[0005]Anti-sense technology is the most common approach to achieve
gene-specific interference. This technique relies upon the provision of
an oligonucleotide able to specifically hybridize with a portion of a
target nucleic acid, which may be DNA, cDNA or, more usually, RNA. The
hybridisation of the oligonucleotide to the target nucleic acid affects
the ability of the target nucleic acid to replicate or undergo normal
transcription (if DNA) or affects translocation within the host cell,
translation, splicing or catalytic ability (if RNA). Generally, the
expression or function of the target nucleic acid will be inhibited by
the binding of the anti-sense oligonucleotide. The oligonucleotide
selected may be a small interfering RNA (siRNA) or double-stranded RNA
(dsRNA) or may alternatively be a small hairpin RNA (shRNA).
[0006]Where the target nucleotide acid is RNA, the anti-sense, shRNA or
dsRNA approach is termed RNA interference (RNAi), a form of
post-transcriptional gene silencing (PTGS) (Voinnet 2001, Trends Genet
17:449-459). In animal cells, RNAi technology has the disadvantage that
the short oligonucleotide used to generate shRNA or dsRNA must be
specific to the target nucleic acid in order to avoid unintentional
interference with other genes, so that selection of suitable
oligonucleotides can be difficult, and the process of selection rather
laborious. Moreover, long dsRNA and its analogs trigger the interferon
response and the induction of associated proteins kinases pathways,
therefore masking the phenotype associated to RNAi gene knock-down.
[0007]Endogenous mechanisms associated with RNAi are believed to have
evolved to protect host against RNA viral infection. Other examples of
post-transcriptional control of mRNA turnover as part of the host quality
control mechanism includes nonsense-mediated decay (NMD) (Hilleren and
Parker 1999, Ann Rev Genet 33:229-260; Mitchell and Tollervey, 2001, Curr
Opin Cell Biol 13:320-325) and the elimination of uncorrectly matured
mRNA that are recognised as aberrant RNAs.
[0008]Normal gene regulation, particularly in eukaryotes, relies upon
polyadenylation--the covalent linkage of 50 to 250 adenosine residues to
the 3' end of an mRNA molecule. The polyadenosine (poly-A) tail protects
the mRNA from degradation by exonucleases and is also required for
translation efficiency and mRNA export (L1 and Hunt, 1997, Plant Physiol
115:321-325; and Gallie, 1993, Rev Plant Physiol Plant Mol Biol
44:77-105).
[0009]Regulation of the poly-A tail addition involves a choice between
several poly-A sites on a single pre-mRNA molecules, therefore generating
mRNAs with different 3' untranslated regions (3'UTR) sequences impacting
on mRNA stability, localization and translatability. The 5'-donor
splicing site (5'ss) sequence of Bovine papillomavirus (BPV-1) has been
identified as a cis-element mediating inhibition of gene expression
involved in the regulation of expression of late genes L1 and L2 coding
for two capsid proteins (Furth and Baker, 1991, J Virol 65:5806-5812;
Furth et al., 1994, Mol Cell Biol 14:5278-5289). In animals it is
believed that a U1 small nucleolar ribonucleoprotein (U1 snRNP) particle,
normally involved in recognition of the 5' splice site during pre mRNA
splicing, binds upstream of a poly-A site inhibiting its usage. This
inhibitory mechanism is reminiscent of U1A autoregulation (Boelens et
al., 1993, Cell 72:881-892). By this means U1A autoregulates its
production by binding its own pre-mRNA and inhibiting polyadenylation
(Gunderson et al, 1994, Cell 76:531-541).
[0010]So far the mechanism of inhibitory activity of a 5'ss in a 3'-UTR
has been described only in mammalian cells (Furth et al., 1994, Mol Cell
Biol 14:5278-5289). It was reported more recently that binding of a
mutated U1 snRNA at complementary sites located within the terminal exon
of pre-mRNA, directs the mRNA for degradation (Fortes et al., 2003, Proc
Natl Acad Sci USA 100:8264-8269). A similar approach has been described
where a modified U1snRNP harbouring in its 5'-end, a sequence
complementary to the 3'-UTR led to the degradation of the target mRNA
(Liu et al, 2004, Nucl Acid Res 32:1512-1517; Rowe et al., US
2003/0082149).
[0011]We have now found that 5'ss sequence when placed at the 3'-UTR could
inhibit the expression of the modified gene in cis and affect the
stability of mRNA in cis and of homologous mRNA in trans. Further
investigations have shown that the 5'ss sequence can be placed within the
open reading frame and inhibit expression.
[0012]In addition, we have found that inclusion of a 5'ss sequence within
the 3'UTR of a given gene triggers degradation of the modified gene in
cis and the degradation of homologous genes in trans. This mechanism
consists of two distinct steps: alteration of polyadenylation status and
mRNA degradation. Although the detailed mechanism of degradation is not
known, the experiments reported herein clearly show that a 5'ss sequence
located in an inappropriate context can function as an inhibitory element
of polyadenylation and expression. The down-regulation in cis is not
abolished by the p19 silencing suppressor protein suggesting that this
mechanism of gene expression inhibition is distinct from other
PTGS-associated pathways involving the siRNA pathway and the RNA-induced
silencing complex (RISC).
[0013]U1 snRNP is a ribonucleoprotein complex that functions primarily to
direct early steps in spliceosome formation by binding to the pre-mRNA
exon-intron boundary (Brown and Simpson, 1998, Annu Rev Plant Physiol
Plant Mol Biol 49:77-95). Nucleotides 2-11 of the 5' end of U1 snRNA base
pair bind with the 5'ss of the pre mRNA.
[0014]We propose that interference between ribonucleoproteins binding to
these 5'-splice donor sites (such as U1 snRNP) located in the vicinity of
the polyadenylation signals of the RNA transcript will interfere with
polyadenylation and therefore would lead to an incompletely processed,
immature pre-mRNA that would be recognized as aberrant and eventually
[0015]Knock down approaches based on splicing components have been
previously described (Fortes et al, 2003, Proc Natl Acad Sci USA
100:8264-8269). In that approach a modified U1snRNA (part of the U1snRNP
splicing component) which carries a 10-nucleotide modified guide-sequence
making it complementary to targeted mRNA, binds to the target sequence
and subsequently interferes with the polyadenylation process and gene
expression in mammalian cells (Rowe et al, US Patent Publication No.
2005/0043261). Such an approach is time-consuming as it requires
individual U1 snRNA-based vector generation for each gene target with the
risk of poor specificity due to the short guide sequence.
[0016]Splicing is a process of maturation of mRNA transcripts, which is
essential for gene expression. The splicing process is common between
eukaryotes (from plants to mammals) and the splicing signals are
conserved (Brown and Simpson, 1998, Annu Rev Plant Physiol Plant Mol Biol
49:77-95). Moreover, as previously stated BPV-1 uses a similar mechanism
of regulation to inhibit the expression of late genes in animal cells
(Furth and Baker, 1991 J Virol 65:5806-5812; Furth et al., 1994, Mol Cell
Biol 14:5278-5289). Therefore we predict that the 5'ss-mediated
inhibition could operate both in plant and animal kingdom for
high-throughput knock-down of endogenous genes. By preparing suitable
expression vectors, transfection of native or transgenic GFP mammalian
and C. elegans cell cultures can be achieved. Monitoring GFP expression
at the protein level, mRNA level, fluorescence and its effect in trans,
further targeting selected candidate genes such as collagen or
osteocalcin can also be used.
[0017]The present invention therefore provides a modified polynucleotide
comprising a gene having an exon containing a stop codon and said
polynucleotide having a transcription terminator for said gene, wherein
the gene has a sequence derived from a 5' splicing site (hereinafter
"5'ss sequence") located upstream of the transcription terminator and
within the exon containing the stop codon.
[0018]As indicated above and in example 1, it should be understood that
the 5'ss sequence in each aspect of the present invention may not be
involved in any splicing event. In this regard, the 5'ss sequence(s)
could be considered as being "unpaired", that is to say that the or each
5'ss sequence would not pair with a 3'ss sequence in a splicing event to
excise nucleotide sequence lying between a 3'ss sequence and the 5'ss
[0019]In one embodiment, the reference to "transcription terminator"
refers to the location at which transcription is terminated in vivo or in
vitro using cellular extracts.
[0020]In one embodiment the 5'ss sequence is located up to and including
10000 nucleotides upstream of the transcription terminator, for example
up to and including 5000 nucleotides upstream of the transcription
[0021]In one embodiment, the 5'ss sequence is located within 20 to 1000
nucleotides upstream of the transcription terminator.
[0022]Optionally the 5'ss sequence is located up to and including 50, 100,
200, 300, 400, 500, 600, 700, 800 or 900 nucleotides upstream of the
[0023]In one embodiment, the modified polynucleotide comprises a
polyadenylation signal downstream of the 5'ss sequence.
[0024]As indicated above, where the 5'ss sequence is located in coding
sequence, it must be located within the same portion of coding sequence
as the stop codon for the gene. The 5'ss can be located upstream or
downstream of the stop codon. The 5'ss sequence must not be separated
from a polyadenylation signal by an intron. Of course, where the gene of
interest does not contain introns, there will be no such restriction on
the location of the 5'ss sequence.
[0025]As used herein the term "derived from" means that the 5'ss sequence
has at least 6 contiguous nucleotides copied from at least a portion of a
5' splicing site. The 5'ss sequence can have at least 10 contiguous
nucleotides copied from a 5' splicing site.
[0026]In one embodiment, the 5'ss sequence inhibits gene expression.
[0027]In one embodiment, the 5'ss sequence is located downstream of the
stop codon for the gene.
[0028]In one embodiment, more than one copy of the 5'ss sequence may be
inserted. For example two or more copies of the 5'ss sequence can be
inserted as repeats whether in tandem or separated by one or more
nucleotides. The copies of the 5'ss sequence can be inserted as direct
repeats. Optionally, three, four or five copies of the 5'ss sequence can
be present. In one embodiment 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20 copies of the 5'ss sequence can be present. In one
embodiment two 5'ss sequences are present as tandem repeats. Multiple
copies of these tandem repeats can be present, for example 2, 3, 4, 5, 6,
7, 8, 9 or 10 copies of the tandem repeats can be present.
[0029]In one embodiment a spacer of from 0 to 10000 nucleotides between
each 5'ss sequence can be present. Where more than two copies of the 5'ss
sequence is present, each spacer can be independently selected. Where
tandem repeats of two 5'ss sequences as referenced above are present,
these can be spaced apart from other 5'ss sequences (optionally also in
the form of tandem repeats) by a spacer.
[0030]In one embodiment, the spacer can be selected from 0 to 5000
nucleotides, for example from 0 to 1000 nucleotides. In one embodiment
the spacer is from 0 to 100, for example 0 to 50 nucleotides, for example
the spacer can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.
[0031]The polynucleotide described above can be used to promote mRNA
instability, both in cis and in trans. Where the effect is observed in
trans the mRNA affected is transcribed from a gene (the target gene)
having a targeted sequence which is identical or homologous to at least a
portion of the gene of the polynucleotide. In one embodiment the targeted
sequence is at least 21 nucleotides in length and has homology with a 21
nucleotide portion of the polynucleotide. In one embodiment "homology"
can be considered as at least 90% sequence identity, for example 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% sequence identity, with the
portion of the polynucleotide.
[0032]The percent identity of two amino acid sequences or of two nucleic
acid sequences may be determined by aligning the sequences for optimal
comparison purposes (e.g., gaps can be introduced in the first sequence
for best alignment with the sequence) and comparing the amino acid
residues or nucleotides at corresponding positions. The "best alignment"
is an alignment of two sequences which results in the highest percent
identity. The percent identity is determined by the number of identical
amino acid residues or nucleotides in the sequences being compared (i.e.,
% identity=number of identical positions/total number of
positions×100).
[0033]The determination of percent identity between two sequences can be
accomplished using a mathematical algorithm known to those of skill in
the art. An example of a mathematical algorithm for comparing two
sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.
Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)
Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs
of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporated
such an algorithm. BLAST nucleotide searches can be performed with the
NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences
homologous to nucleic acid molecules of the invention. BLAST protein
searches can be performed with the XBLAST program, score=50, wordlength=3
to obtain amino acid sequences homologous to protein molecules of the
invention. To obtain gapped alignments for comparison purposes, Gapped
BLAST can be utilised as described in Altschul et al. (1997) Nucleic
Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform
an iterated search which detects distant relationships between molecules
(Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the
can be used. See http://www.ncbi.nlm.nih.gov.
[0034]Where high degrees of sequence identity are present there will be
relatively few differences in amino acid sequence. Thus for a 21
nucleotide sequence, change of a single nucleotide will result in a 95%
sequence identity, whereas change in 2 nucleotides in the sequence will
result in a 90% sequence identity.
[0035]The present invention further provides a polynucleotide vector
having a 5' splicing site sequence (hereinafter a "5'ss sequence")
located upstream of a transcription terminator.
[0036]The 5'ss sequence can be located in at least a portion of 3' UTR of
a gene or within at least a portion of an open reading frame (ORF) of a
gene. Where the 5'ss sequence is placed in an ORF, it must not be
separated from the stop codon by an intron, that is the ORF contains a
[0037]In one embodiment the polynucleotide vector comprises a
transcription initiator (promoter and/or start site). The transcription
initiator will be in a functional relationship with the terminator and
5'ss sequence, i.e. will be located upstream of the 5'ss sequence. The
transcription initiator will be able to permit transcription of DNA to
RNA, for example in a host cell or in vitro, ie. can bind transcription
factor(s) that recruit RNA polymerase which initiates transcription.
[0038]In one embodiment the polynucleotide vector includes at least one
site to facilitate insertion of a targeting sequence between the
transcription initiator and transcription terminator. In one embodiment
the site is a restriction enzyme site. Suitable examples are well known
in the art. In one embodiment the site is a site-specific recombination
site. Suitable examples are known in the art.
[0039]The targeting sequence can be selected according to the intended use
of the polynucleotide vector.
[0040]In one embodiment the targeting sequence is at least 21 nucleotides
[0041]The targeting sequence can be a portion of a gene encoding a
protein, a portion of 3'UTR of a gene encoding a protein, or a portion of
a gene for an untranslated RNA.
[0042]In one embodiment the polynucleotide vector of the present invention
comprises, in functional relationship, a transcription initiator, a
targeting sequence, and a transcription terminator, wherein said vector
further comprises a 5'ss sequence upstream of the transcription
[0043]In one embodiment the polynucleotide vector of the present invention
is suitable for inhibiting gene expression of a target gene in a cell.
[0044]The target gene will have a targeted sequence having homology with
the targeting sequence of the polynucleotide vector according to the
invention. In one embodiment, the targeted sequence comprises at least 21
(consecutive) nucleotides which have homology with the targeting
sequence. In one embodiment, "homology" can be considered as at least 90%
sequence identity with the targeting sequence, for example 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the targeting
sequence. In one embodiment the targeting sequence is at least 21
(consecutive) nucleotides.
[0045]In one embodiment, the gene targeted for inhibition of expression
(the target gene) does not form part of the polynucleotide vector of the
present invention. The polynucleotide vector is able to inhibit
expression of the target gene in trans. Optionally the target gene is
endogenous to the cell. In one embodiment the target gene is located in
the genome of the cell.
[0046]In one embodiment the cell is a eukaryotic cell. For example, the
cell can be an animal cell (optionally an insect or mammalian cell, such
as a human or non-human cell) or can be a plant cell.
[0047]The vector can be designed to insert the 5'ss sequence within the
3'UTR of a target gene. In such an embodiment, the vector can
conveniently also include restriction enzyme sites either side of the
5'ss sequence to facilitate its insertion at the required location. In an
alternative embodiment the vector can include site specific recombination
sites to facilitate its insertion at the required location.
[0048]The vector can be designed to insert the 5'ss sequence at a location
within an open reading frame (ORF) of a target gene, wherein said
location is not separated from the stop codon by an intron. In such an
embodiment, the vector can conveniently also include restriction enzyme
sites either side of the 5'ss sequence to facilitate its insertion at the
required location. In an alternative embodiment the vector can include
site specific recombination sites to facilitate its insertion at the
required location.
[0049]In an alternative embodiment the present invention provides a
polynucleotide vector able to insert a 5' splicing site sequence such
that expression of a target gene is reduced.
[0050]In an alternative embodiment, the vector is an expression vector and
includes a full length sequence or a partial sequence of the target gene.
In this embodiment, the 5'ss sequence is located within the 3'UTR and
downstream of the stop codon, and upstream of the transcription
terminator of the target gene. Alternatively the 5'ss sequence is located
within the 3'UTR of an unrelated gene, as well as inside sequence derived
from the target gene. The 5'ss sequence will normally be located upstream
of the transcription terminator and downstream of the last exon
containing the stop codon of the target gene (i.e. downstream of the stop
codon). In one embodiment, the 5'ss sequence will be upstream of the stop
codon and the transcription terminator. Optionally, the 5'ss sequences
will be upstream of the transcription terminator and downstream of a
coding region of a gene without a stop codon. Optionally, the 5'ss
sequence will be downstream of a coding or non-coding cDNA portion of a
gene, located upstream to the transcription terminator. Optionally, the
5'ss sequence will be located upstream of an uninterrupted open reading
frame (ie. an open reading frame without an intron or 3' acceptor
splicing site) and a transcription terminator.
[0051]As indicated above, 2, 3, 4, 5 or more copies of the 5'ss sequence
can be present. Optionally the 2 or more copies of the 5'ss sequence are
arranged as direct repeats or in tandem. Optionally spacer(s) can be
present between copies of the 5'ss sequences or between tandem copies of
the 5'ss sequences, if present.
[0052]In one embodiment, the 5'ss sequence (whether in a vector or
otherwise) is the sequence 5'-MAGGTRAGTA-3' (SEQ ID No. 1), where M=A or
C and R=A or G. Variants derived from this sequence in which four or less
nucleotides have been substituted or deleted are also covered.
Optionally, only one, two or three nucleotides have been substituted or
[0053]In one embodiment the 5'ss sequence (whether in a vector or
otherwise) is the sequence 5'-CAGGTAAGTA-3' (SEQ ID No. 2) or a variant
[0054]In one embodiment, the vector of the present invention can include
the sequence 5'-MAGGTRAGTA-3' (such as 5'-CAGGTAAGTA-3') or a variant
thereof as defined above, together with a polyadenylation signal. A
suitable polyadenylation signal is AAUAAA.
[0055]The target gene can be any gene of interest. The target gene can be
a structural gene (ie. transcribed to mRNA which is then translated to a
protein or polypeptide) or can encode non-translated RNA such as tRNA
miRNA or rRNA. The target gene could be a full length or partial cDNA or
encode a partial protein/polypeptide. The target gene could be endogenous
to the host cell or could be heterologous (foreign) to the host cell. The
target gene could also have been manipulated genetically, for example to
alter its expression product, for example the target gene could be a
[0056]In one embodiment the gene includes a targeting sequence of 21
nucleotides able to bind specifically under stringent conditions to a
targeted sequence of at least 21 nucleotides in length. The targeting
sequence can be selected from translated or non-translated nucleotide
[0057]Stable hybridisation of polynucleic acids is a function of hydrogen
base pairing. Hydrogen base pairing is affected by the degree to which
the two polynucleotide strands in the duplex are complementary to each
other and also the conditions under which hybridisation occurs. In
particular salt concentration and temperature affect hybridisation. One
of ordinary skill in the art would be aware that the effective melting
temperature (E Tm) of the polynucleotide duplex is controlled by the
ETm=81.5+16.6(log M[Na+])+0.41(% G+C)-0.72(% formamide)
[0058]Where hybridisation is conducted under stringent conditions, only
sequences having a high degree of complementary base pairs will remain in
duplex form. As used herein the term "stringent conditions" with respect
to hybridisation refers to wash conditions of 0.1×SSC at 60 to
68° C. Optionally, the wash conditions can include a suitable
concentration of SDS, for example 0.1% SDS.
[0059]In one embodiment the vector of the present invention includes a
3'UTR for replacing at least a portion of the 3'UTR of a target gene. The
3'UTR will comprise at least one copy of a 5'ss sequence, such as
5'-MAGGTRAGTA-3' or a variant thereof, together with a polyadenylation
signal, such as AAUAAA. In one embodiment the 3'UTR will comprise at
least one copy of the 5'ss sequence 5'-CAGGTAAGTA-3' or a variant
thereof, together with a polyadenylation signal, such as AAUAAA.
[0060]In one embodiment the vector of the present invention is an
expression vector comprising a gene to be expressed, wherein said gene
has a 3'UTR comprising at least one copy of a 5'ss sequence (such as
5'-MAGGTRAGTA-3' or a variant thereof) together with a polyadenylation
signal, such as AAUAAA.
[0061]The vectors of the present invention can be used to transfect or
transform host cells and the host cells cultured in conventional culture
media according to methods known or described in the art.
[0062]Incorporation of cloned DNA into a suitable vector, transfection or
transformation of host cells and selection of the transfected or
transformed cells are all processes well known to those skilled in the
art and numerous suitable methods are described in the literature (see,
for example, Sambrook et al., Molecular Cloning: A laboratory Manual,
3rd edition, Cold Spring Harbor Laboratory Press, 2001).
[0063]In one embodiment, the expression vector is for expression in plant
[0064]In one embodiment, the expression vector is for expression in animal
[0065]The present invention also includes host cells containing the
modified polynucleotide or vector of the present invention.
[0066]The present invention further provides a method of reducing
expression of a target gene, said method comprising modifying the ORF
upstream of the stop codon or the 3'UTR of the target gene by inserting a
5'ss sequence therein. The 5'ss sequence will normally be located
upstream of the transcription terminator. In one embodiment the 5'ss
sequence is located downstream or upstream of the target gene stop codon
if the sequence is intronless. The 5'ss sequence is located between the
last intron and transcription terminator if the sequence lacks a stop
codon. That is to say the 5'ss sequence must not be separated from a
polyadenylation signal by an intron.
[0067]As indicated above, 2, 3, 4, 5 or more copies of the 5'ss sequence
can be inserted. They can be arranged either as direct repeat or as
multiple copies spaced apart from each other and located within suitable
relative distance to the transcription terminator and poly(A) site to
mediate inhibition of gene expression. By "suitable relative distance" we
mean that each copy of the 5'ss sequence can be independently separated
from the polyadenylation site by a distance of from 1 nucleotide up to
1200 nucleotides.
[0068]In one embodiment the 5'ss sequence can be 5'-MAGGTRAGTA-3' (for
example 5'-CAGGTAAGTA-3') or a variant thereof.
[0069]The present invention will now be further described with reference
to the following, non-limiting, examples and figures in which:
[0070]FIG. 1:
[0071]Schematic representation (not to scale) of the binary constructs and
agromixes (right side) used to agroinfiltrate N. benthamiana leaves and
N. benthamiana leaves observed under UV light at 3 dpi. LB, left border
of the T-DNA, 35S, 35S CaMV promoter, GFP, green fluorescent protein,
NOS-Ter, nopaline synthase transcription terminator, RB, right border of
the T-DNA (NB: the T-DNA elements are represented only for the GFP
construct and are present in all constructs used for agroinfiltration).
The 5'-donor splice site is represented by an arrow in sense (1×S)
or antisense orientation (1×A) or two arrows in sense (2×S)
or antisense orientation (2×A).
[0072]FIG. 2:
[0073]Confocal laser scanning observation of biolistically transfected
onion epidermal cells with constructs A: GFP-5'ssas (1×A), B:
GFP-5'ss (1×S), C: GFP-2×5'ssas and D: GFP-2×5'ss
(2×S). All images were taken at 3 dpi and at the same gain and are
representative from at least three independent biolistic events (1 cm=15
[0074]FIG. 3:
[0075]Analysis of GFP protein and mRNA levels during RNAi and
5'ss-mediated inhibition. A: western blot analysis of GFP level in leaves
agroinoculated with GFP (lane 1), hpGFP+GFP (lane 2),
GFP-2×5'ssas (lane 3) or GFP-2×5'ss (lane 4). Leaf discs
samples were harvested and pooled from at least two different leaves from
three different plants. B: Semi-quantitative RT-PCR to monitor GFP and
ubiquitin mRNA levels in GFP, hpGFP+GFP, GFP-2×5'ss. Both RT-PCR
products corresponding to GFP and ubiquitin mRNAs have been assessed.
Lane 1, molecular weight, lane 2, non-template control, lanes 3-7-11, 22
cycles, lanes 4-8-12, 25 cycles, lanes 5-9-13, 28 cycles, lanes 6-10-14,
31 cycles. Samples were taken from two different leaves per plant from
three independent plants.
[0076]FIG. 4:
[0077]GUS staining of leaves agroinfiltrated with GUS or GUS-2×5'ss
constructs. A schematic representation (not to scale) of GUS and
GUS-2×5'ss is presented. 3 leaf discs from two different leaves per
plant from four independent plants (labelled 1, 2, 3 and 4) were excised
and stained as described in material and methods. Top row: samples from
leaves inoculated with GUS construct, bottom row: samples from leaves
inoculated with GUS-2×5'ss construct.
[0078]FIG. 5:
[0079]5'ss mediates inhibition of gene expression in trans.
[0080]Agroinfiltrated N. benthamiana leaves observed at 3 dpi under UV
illumination. A: agroinfiltrated leaf with agromixes GUS-2×5'ss,
GFP, GUS+GFP, GFP-2×5'ss+GFP, GUS-2×5'ss+GFP. B: Assessment
of GFP fluorescence by spectrofluorimetry for the abovementioned
agromixes, values are expressed as arbitrary units of GFP fluorescence
emission as described in Material and Methods. C: agroinfiltrated leaf
with agromixes PDS-2×5'ss, GFP, PDS+GFP, GFP-2×5'ss+GFP,
PDS-2×5'ss+GFP. D: Assessment of GFP fluorescence by
spectrofluorimetry for the abovementioned agromixes, values are expressed
as Arbitrary Units (AU) of GFP fluorescence emission as described in
[0081]FIG. 6:
[0082]Local and systemic effect of p19 on RNAi and 5'ss-mediated
inhibition. A: N. benthamiana transgenic 35S::mGFP5-ER::NOS 16c lines
agroinfiltrated with hpGFP (top panels) or GFP-2×5'ss (bottom
panels) in absence of p19 (left panels) or co-agroinoculated with p19
(middle and right panels) observed under UV illumination at 8 dpi. The
right panels are a close-up of the boxed area from the middle panel. The
arrow indicates the border of the inoculated patches appearing red for
GFP-2×5'ss. B: Systemic N. benthamiana leaves observed under UV
illumination at 6 dpi, 8 dpi and 13 dpi with or without p19 from 16c
plants inoculated with hpGFP (upper panels) or GFP-2×5'ss (lower
panels). C: Northern blot analysis of GFP mRNA levels (upper panel) of
systemic leaves at 13 dpi from plants agroinoculated with GFP,
GFP-2×5'ss, hpGFP and empty binary vector control. Bottom panel: UV
picture of SYBR Safe staining of ribosomal RNA from the corresponding
samples as a loading control.
[0083]FIG. 7:
[0084]Effect of different transcriptional terminators on 5'ss-mediated
inhibition. A: Nucleotidic composition of the 3'UTR of GFP-2×5'ss
(SEQ ID No. 3) and GFP-MCS-2×5'ss-OCS (SEQ ID No. 4). For
GFP-2×5'ss construct, the GFP stop codon TAA is upstream the
inserted tandem 5'ss (in bold) including the SacI and AscI restriction
sites. The 2×5'ss element is boxed. The entire NOS terminator
sequence is presented downstream the 2×5'ss element with the
canonical polyadenylation signal AAUAAA underlined in bold. For
GFP-MCS-2×5'ss-OCS, the boxed area encompass the multiple cloning
site (MCS) and the tandem repeat 2×5'ss. The entire OCS terminator
sequence is presented downstream with putative polyadenylation signal
AAUGAA underlined in bold. B: UV picture at 3 dpi of a N. benthamiana
agroinfiltrated leaf with agromixes GFP-2×5'ss+GFP (1), GFP+GFP
(2), GFP-MCS-2×5'ss-NOS+GFP (3), GFP-MCS-2×5'ss-OCS+GFP (4)
and GFP-OCS+GFP (5).
[0085]FIG. 8:
[0086]Assessment of mRNA polyadenylation level in GFP-2×5'ss and
[0087]A: Semi-quantitative RT-PCR to monitor GFP mRNA levels in GFP and
GFP-2×5'ss. Using first strand cDNA generated using an oligo dT
primer (upper panels) or random hexamers (lower panels). Lane 1,
molecular weight, lane 2, non-template control, lanes 3-7, 22 cycles,
lanes 4-8, 25 cycles, lanes 5-9, 28 cycles, lanes 6-10, 31 cycles.
[0088]B: The ubiquitin mRNA levels were assessed on the same samples used
for monitoring GFP mRNA levels. Ubiquitin primers (material and methods)
were used to amplify the ubiquitin cDNA PCR product. Lane 1, molecular
weight, lane 2, non-template control, lanes 3-6, cDNA primed with
oligodT, lanes 7-10, cDNA primed with random hexamers. Lanes 3-4 and 7-8,
22 cycles, lanes 5-6 and 9-10, 25 cycles. Lanes 3, 5, 7, 9,
GFP-2×5'ss, lanes 4, 6, 8, 10, GFP.
[0089]FIG. 9:
[0090]Assessment of the level of transcriptional readthrough.
[0091]A: Schematic representation (not to scale) of the GFP-2×5'ss
construct with the position of 2×5'ss elements (black arrows), the
NOS terminator (boxed in grey) and the position of a putative intron
(dotted rectangle). The positions of the oligonucleotide primers are
indicated (small arrows). The nucleotidic sequence of the 3'-UTR region
(SEQ ID No. 3) is presented with the 2×5'ss element (boxed), the
canonical polyadenylation site AAUAAA (bold underlined), an additional
putative polyadenylation site AAUAAU (bold underlined), and a putative
3'-acceptor splicing site (AG, underlined in bold). B: Semiquantitative
RT-PCR of GFP and GFP-2×5'ss of readthrough products from cDNA
synthesized using oligo dT or REV1 primers and PCR amplified using
FWD/REV1 primers combination (upper panel), or cDNA population
synthesized using oligo dT and REV2 primers and PCR amplified using
FWD/REV2 primer combination after 25 cycles of PCR amplification. C:
Semiquantitative RT-PCR of GFP cDNA population amplified from samples
GFP, GFP-2×5'ss and GFP-2×5'ss-Fp19 using FWD and oligo dT
primer (Material and methods) at 25 and 28 cycles (upper and lower panel
respectively). Ubiquitin mRNA levels were assessed similarly in these
samples and showed equal amplification of PCR products for each samples
(data not shown). NTC: non-template control.
[0092]FIG. 10:
[0093]Real-time RT-PCR determination of normalised relative amounts of pds
mRNA levels (±SE) in PDS-2×5'ss, control empty binary vector or
uninoculated N. benthamiana leaves at 3 dpi and 7 dpi. Value represents
the mean from three independent plants per construct sampling two
different leaves per plant (n=6). A schematic representation of N.
benthamiana PDS cDNA with the position of primers used for Real-time
RT-PCR analysis and the cDNA portion cloned into the binary vector is
presented. LB: left border of the T-DNA, 35S: 35S CaMV promoter, white
rectangle "S": PDS cDNA fragment, tandem thick black arrows: 2×5'ss
sequences, black rectangle: NOS-Ter, nopaline synthase transcription
terminator, RB, right border of the T-DNA.
[0094]FIG. 11:
[0095]Comparison of level of gene expression down-regulation of plasmids
carrying one or two impaired 5'ss located in a 3'-UTR. GFP--control;
GFPart 1--1 copy 5'ss sequence; and GFPart 2--2 copies 5'ss sequence.
[0096]FIG. 12:
[0097]Relative fluorescence (arbitrary units) for GFP-ART
(pGFP-2×5'ss), GFP (pBINmgfp5-ER), Spacer50
(pGFP-5'ss-spacer50-5'ss) and Spacer1000 (pGFP-5'ss-spacer1000-5'ss). No
significant difference to downregulation observed in cis or in trans.
[0098]FIG. 13:
[0099]Enzymatic activity of β-glucuronidase as relative fluorescence
units per minute per mg of total protein. GUSART (pGUS-2×5'ss),
GUSART3 (pGUS-6×5'ss) and GUS (pBI121) (control).
[0100]FIG. 14:
[0101]Enzymatic activity of β-glucuronidase as relative fluorescence.
GUSART (pGUS-2×5'ss), GUSART(3) (pGUS-6×5'ss) and GUS
(pBI121) and GFP-ART (pGFP-2×5'ss).
[0102]FIG. 15:
[0103]Agroinfiltrated Nicotiniana tabaccum leaves observed at 3 dpi under
UV illumination. A: agroinfiltrated leaf with agromix GFP
(pGFP-2×5'ss) in Nicotiniana tabaccum var Xanthii, B:
agroinfiltrated leaf with agromix GFP (pGFP-2×5'ss) in Nicotiniana
tabaccum var Samsum.
[0104]FIG. 16:
[0105]Illustrates the constructs formed as a schematic representation of
pEGFP (pEGFP-C1 expression vector, Clonetech), pEGFPart5 and pDsRED
constructs. As indicated above, the plasmids were constructed by
inserting tandem 5'-splicing donor sites into construct pEGFP by
subcloning into the mammalian expression vector pEGFP using SacI
restriction site. The CMV promoter and the SV40 transcriptional
terminator are represented.
[0106]FIG. 17:
[0107]Epifluorescence and bright field merged microscopy images of HeLa
cells at 2 days post transfection with the constructs pEGFP+pDsRED (upper
left panel), pEGFPart5+pDsRED (upper right panel), pEGFP+pEGFPart5+pDsRED
(lower left panel), pEGFP+EGFP RNAi (co-transfected siRNA EGFP)+pDsRED
(lower right panel).
[0108]The scale bar represents 10 μm.
[0109]FIG. 18:
[0110]Downregulation of GFP expression by tandem insertion of 5'ss in HeLa
cells. Western blot analysis shows accumulation of green fluorescence
protein (panel A) or co-expressed red fluorescence protein (panel B);
line 1: pEGFP+pDsRED; line 2: pEGFPart5+pDsRED; line 3:
2×pEGFP+pDsRED; line 4: pEGFP+pEGFPart5+pDsRED. The blots were
probed with antibodies against green fluorescent protein (GFP) (panel A)
or against red fluorescent protein (RFP) used as an internal calibrator
[0111]The inventors have designated the present invention as "Aberrant RNA
Technology" or "ART" and references to constructs described in the
examples are to be construed accordingly
[0112]To evaluate the applicability as a gene knock-down approach and
study the molecular basis of this phenomenon we introduced a consensus
5'ss within the 3'-UTR of a reporter gene encoding the green fluorescent
protein (GFP) and analysed the effect on gene expression transiently
expressed in single cells and in plant leaf tissues.
5'ss Sequence in 3' UTR of GFP
[0113]To evaluate the applicability as a gene knock-down approach and
[0114]All T-DNA constructs were introduced into A. tumefaciens LBA 4404
(VirG) strain by electroporation as previously described (Koscianska et
al, 2003, Plant Mol Biol 59:647-661). Agrobacteria were grown overnight
in LBG medium supplemented with Kanamycin (50 μg/ml) and
Chloramphenicol (75 μg/ml). OD600 was adjusted to 0.1 by diluting
the bacteria in 10 mM MES, pH=5.6, 10 mM MgCl2, 150 μM
Acetosyringone and incubated for at least 1 hour at room temperature.
Infiltration of the diluted bacteria was done as previously described
(Johansen and Carrington, 2001, Plant Physiol 126:930-938). Plants were
kept in constant conditions in growth chamber at 22° C. with a 16
hour photoperiod, light intensity ranging from 400 to 1000
μmol.m-2 sec-1. GFP fluorescence was monitored under UV
illumination as previously described (Lacomme and Santa Cruz, 1999, Proc
Natl Acad Sci USA 96:7956-7961). Pictures were taken using an Olympus
C-2500L digital camera.
RT-PCR and Quantification of Gene Expression
[0115]First strand cDNAs were generated using oligo-dT or random hexamers
oligonucleotides (Qiagen, UK) as previously described (Lacomme et al.,
2003, Plant J 34:543-553).
[0116]Semi-quantitative RT-PCR, Real-time "Taqman" RT-PCR and statistical
analysis were as previously described (Lacomme et al., 2003, Plant J
34:543-553). Forward and reverse primers used for semi quantitative
RT-PCR of GFP expression were 5'-GGGCACAAATTTTCTGTCAG-3' (SEQ ID No: 5)
and 5'-GTTGTGGGAGTTGTAGTTGTATTC-3' (SEQ ID No: 6). Taqman primers for
ubiquitin and phytoene desaturase (pds) were previously reported (Lacomme
et al., 2003, Plant J 34:543-553). The assessment of transcriptional
read-through within the NOS terminator was performed on first strand cDNA
synthesized using either oligo-dT, REV1
(5'-AAATAACGTCATGCATTACATGTTAATTATT-3') (SEQ ID No: 7), or REV2
(5'-TTCTATCGCGTATTAAATGTATAATTG-3') (SEQ ID No: 8) primers the latest
located downstream the putative 3'-acceptor splicing site and the second
putative polyadenylation site AAUAAU as indicated in FIG. 9. Assessment
of the length of polyadenylated GFP mRNAs was performed using oligo-dT
primed cDNA further amplified using 5'-TCCACACAATCTGCCCTTTC-3' (SEQ ID
No: 9) and 5'-GCGAGCTCCGCGGCCTTTTTTTTTTTT-3' (SEQ ID No: 10) respectively
as forward and reverse primer.
[0117]The plasmids used for microprojectile bombardment of onion epidermal
cells were generated as follow. An expression cassette consisting of CaMV
35S::mgfp5-ER::3'-NOS derived from pBINmgfp5-ER plasmid (provided by Jim
Hasselhoff et al., 1997, Proc Natl Acad Sci USA 94:2122-2127) was
ampified by PCR using primers 5'-CCCAAGCTTTTTCAGAAAGAATGCTAACCC-3' (SEQ
ID No: 11) and 5'-CCCAAGCTTGATCTAGTAACATAGATGACACC-3' (SEQ ID No: 12),
digested by HindIII and cloned into a modified pBluescript
KS+(Stratagene) from which the SacI site was removed giving pSgfpex.
Into the SacI linearised pSgfpex vector annealed U1-1 or U1-2 DNA
fragments generated by self-annealing of primers
5'-CGAGMAGGTRAGTAGGCGCGCCGAGCT-3' (SEQ ID No: 13) and
5'-CGGCGCGCCTACTTACCTGCTCGAGCT-3' (SEQ ID No: 14) for U1-1 and
5'-CGAGMAGGTRAGTAGGCGCGCCMAGGTRAGTAGAGCT-3' (SEQ ID No: 15) and
5'-CTACTTACCTGGGCGCGCCTACTTACCTGCTCGAGCT-3' (SEQ ID No: 16) (M is A or C;
R is A or G) were ligated giving four plasmids: pSgfpU1-1s (U1-1 pair of
oligos in sense orientation, further referred to as 1×S),
pSgfpU1-1a (U1-1 in antisense, further referred to as 1×A),
pSgfpU1-2s (U1-2 in sense, further referred to as 2×S), and
pSgfpU1-2a (U1-2 in antisense, further referred to as 2×A). The
plasmids used for agroinfiltration of leaves were generated as follow.
Into the SacI linearised plasmid pBINmgfp5-ER. pBinmGFP5-ER sequence is
deposited in GenBank (accession number 1848288). GFP insert was cloned
into BamHI and SacI restriction sites of pBIN121 vector (GenBank
accession number 19569229). After removing the GUS insert from pBin121 by
BamHI-SacI digestion, annealed U1-1 or U1-2 pair of primers were ligated
giving four plasmids: GFP-5'ss (U1-1 pair of oligos in sense
orientation), GFP-5'ssas (U1-1 in antisense orientation),
GFP-2×5'ss (U1-2 in sense orientation), and GFP-2×5'ssas
(U1-2 in antisense orientation). Construction of
GFP-MCS-2×5'ss-OCS: the GFP ORF from pBINmgfp5-ER plasmid was
amplified by PCR using primers
5'-gtgtgtCTCGAGCCatgGCCAAGACTAATCTTTTTCTCTTTCTCA-3' (SEQ ID No: 17) and
5'-gtgtgtGGCGCGCCTACGTACCTAGGgttaaccAAGCTCATCATGTTTGTAT AGTTC-3' (SEQ ID
No: 18), digested by XhoI and AscI and cloned into pFGC5941 (provided by
Rich Jorgensen, University of Arizona, USA, GenBank accession number
32265027), resulting in plasmid pKWBi51. Then ChSA intron sequences were
excised by AscI and PacI and ligated with annealed oligonucleotide pair
5'-CGCGCCatta taaaTCTAGACAGGTAagtaCggatccGCAGGTAagtaGACGTCctctAGCC-3'
(SEQ ID No: 19) and 5'-CCGGGGCTagagGACGTCtacttacctgCggatcc
GtacttacctgTCTAGAtttataatGG-3' (SEQ ID No: 20) and blunted by T4 DNA
polymerase giving GFP-MCS-2×5'ss-OCS. The construct
GFP-MCS-2×5'ss-NOS was generated by subcloning of the blunted
XhoI-PacI GFP-MCS-2×5'ss from the GFP-MCS-2×5'ss-OCS
construct into the XhoI-SacI blunted pBin19 vector and PCR-screened and
verified by sequencing for the correct orientation. The constructs hpGFP
and GFP were generated as previously described (Koscianska et al., 2005,
Plant Mol Biol 59:647-661). The construct PDS-2×5'ss was generated
by subcloning into the BamHI-SacI digested pBin19 binary vector the
314-bp PCR-amplified PDS cDNA fragment using oligonucleotides
5'-ATGGGATCCATGAAGGAACTAGCGAAGCTTTTC-3' (SEQ ID No: 21) and
5'-TACGAGCTCTTAGTTCACTATGCTAACTACGCTTG-3' (SEQ ID No: 22) (respectively
forward and reverse primers) and digested by BamHI-SacI. The annealed
U1-2 oligonucleotides were cloned into the SacI site of the pBin PDS
[0118]The green fluorescence emission in the leaf tissue was measured
using a spectrofluorimeter (Spectramax M5, Molecular Devices, Excitation
wavelength: 468 nm, Emission wavelength: 503 nm, Cut-off: 495 nm). Leaf
discs were excised, and placed into a 96-well plate. Background
autofluorescence of leaf tissue was deduced from total fluorescence.
Background autofluorescence of leaf tissue was deduced from total
fluorescence giving an Arbitrary Units (AU) of fluorescence emission.
Microsoft Excel Software was used to perform statistical analyses and
graphical presentation. All data are expressed as means±SD. Each group
analysed consisted of three to eight independent samples, each sample
measured from 5 to 9 independent leaf discs. All experiments were
repeated at least twice.
[0119]Leaf discs of agroinfiltrated areas were harvested after 3 dpi.
Histochemical staining for β-glucuronidase activity was performed as
previously described (Jefferson et al, 1987, EMBO J 6:3901-3907; Swoboda
et al., 1994, EMBO J 13:484-489).
Micro Projectile Bombardment of Onion Epidermal Cells
[0120]Particle bombardment assays were performed on the abaxial side of
adaxial epidermal peels from onion bulb scales. The method was
essentially as described by Gal-On et al. (1997, Journal Virol Meth
64:103-110) and Haupt et al. (2005, Plant Cell 17:164-181). All
experiments were conducted with a home-made biolistics system (Gal-On et
al, 1997, Journal Virol Meth 64:103-110). Approximately 2 μg of
plasmid DNA was mixed with 1 mg of tungsten particles (M-25, DuPont no.
75056) in aqueous suspension and added 70 μl of 2.5M CaCl2 and 30
μl of 0.1M spermidine. The particles were mixed by shaking 30 min at
+4° C., spun down 2 min at 10,000 g and washed with 96% ethanol.
The pellet was resuspended in 45 μl of 96% ethanol. 10 μl of
DNA/tungsten mixture was loaded onto the grid of a discharge assembly and
left until the ethanol evaporated. Two bombardments were made before
reloading the grid. Bombarded epidermis was observed under the confocal
laser scanning microscope (Leica Microsystems, Heidelberg, Germany) after
[0121]Total RNA was extracted from 100 mg of leaf tissue from patch assay
as well as from systemically silenced tissues using RNEasy kit (Qiagen,
UK), TRI RNA Isolation Reagent (Sigma-Aldrich, UK) or TRIzol Reagents
(Invitrogen, UK) according to the manufacturers.
[0122]The purified total RNA was used for Northern blot analysis and first
strand cDNA synthesis.
[0123]Analysis of higher molecular weight RNAs was performed by Northern
blot hybridization as described previously (Dalmay et al., 1993, Virology
194:697-704). Six μg of total RNA was used for separation in
formaldehyde containing denaturing gel. Random priming DNA probe of GFP
construct was used for detecting the high molecular weight RNAs. After
hybridization signals were detected by X-ray film or phosphorimager
screen visualized by FLA-7000 Fluorescent Image Analyzing System
[0124]Insertion of Duplicated 5' Splicing Site within the 3'-UTR of a cDNA
Inhibits its Expression.
[0125]Delivery of DNA into the plant cells by agroinfiltration has been
used for the transient expression of genes in plants and the induction of
gene silencing (Johansen and Carrington, 2001, Plant Physiol
126:930-938). Chimeric construct GFP-5'ss-NOS harbouring a single 5'-ss
in the 3'-UTR followed by the nopaline synthase (NOS) terminator was
engineered and expressed in plant leaf using an Agrobacterium
infiltration assay. By three days post infiltration a bright GFP
fluorescence comparable to the GFP-NOS construct (FIG. 1, 1×S) was
observed indicating that no significant effect on GFP expression was
occurring. A construct harbouring a tandem insertion of the 5'-ss at the
same location (construct GFP-2×5'ss-NOS) did not display GFP
fluorescence (FIG. 1, 2×S). This behaviour was comparable to what
was observed with the RNAi assay where a GFP construct was co-infiltrated
with a hpGFP construct generating GFP dsRNA (FIG. 1, agromix hpGFP+GFP).
Insertion of the same tandem 5'-ss in antisense orientation (FIG. 1,
2×A) did not significantly affect GFP fluorescence and was
comparable to the GFP-NOS construct suggesting a strong effect of
sequence polarity in triggering inhibition of GFP fluorescence. Moreover,
transient expression in biolistically transfected single epidermal onion
cells triggered a similar down-regulation (FIG. 2D) demonstrating that
5'-ss mediated down-regulation is observed in a different transient
expression system using a different mode of DNA delivery. Assessment of
GFP accumulation both at the protein and mRNA levels confirmed that GFP
mRNA accumulation was strongly reduced in both GFP-2×5'ss and hpGFP
in comparison to GFP or GFP-2×5'ssas samples (FIGS. 3 A and
B). This demonstrates that 5'ss-mediated down-regulation is mediated by
the duplicated 5'ss located within the 3'-UTR. Expression of chimeric
constructs containing the bacterial uidA gene, which encodes the
β-glucuronidase (GUS) reporter gene (GUS-2×5'ss-NOS), resulted
in the absence of GUS staining in comparison to leaf discs infiltrated
with the GUS construct (FIG. 4). This shows that GUS expression was
strongly affected in the GUS-2×5'ss-NOS construct. This suggests
that the 5'ss-mediated inhibition of gene expression of mRNAs encoding
distinct reporter genes (GFP or GUS) is affected in a non-sequence
5'Splicing Site Mediates Inhibition of Gene Expression in trans
[0126]The previous results suggest that the modified genes are undergoing
mRNA degradation probably by being identified as aberrant and recruited
by the endogenous mRNA surveillance machinery for degradation. We further
investigated whether the 5'ss could mediate down-regulation of gene
expression in trans.
[0127]For this purpose we co-infiltrated the constructs; GFP-2×5'ss,
GUS-2×5'ss, PDS-2×5'ss (phytoene desaturase or PDS), or hpGFP
with GFP, and assessed GFP fluorescence by spectrofluorimetry. All
constructs harbour a NOS terminator with the exception of hpGFP where the
transcription terminator originates from the octopine synthase (OCS)
gene. Co-infiltration of GFP-2×5'ss and GFP resulted in inhibition
of both gene expression with a 7-10-fold reduction in fluorescence
emission (FIGS. 5 A and B). A similar effect was observed when
GUS-2×5'ss and PDS-2×5'ss were co-infiltrated with GFP.
Despite the limited sequence homology between them encompassing only the
NOS terminator (FIG. 5 A to D), an approximate 2-fold decrease in GFP
fluorescence was observed. It suggests, that the level of a gene
expression inhibition in trans is proportional to the length of
homologous sequences between the trigger and the target gene. Silencing
can be triggered by non-coding sequence including transcription
termination elements such as the NOS terminator (Canto et al, 2002, Molec
Plant-Microbe Interact 15:1137-1146). In order to rule out any silencing
effect due to the presence of the NOS terminator sequence,
co-infiltration of GUS or PDS constructs with GFP were analysed. No
significant GFP inhibition in trans was observed when GFP and GUS or GFP
and PDS were coinoculated (FIG. 5 A to D). This confirms that
5'ss-mediated inhibition triggers a strong inhibition in trans. This
suggests that stretches of sequence homology limited to the transcription
terminator element are sufficient to trigger inhibition of gene
expression in trans during 5'ss-mediated knock-down.
5'ss-Mediated Systemic Inhibition of Gene Expression is not Abolished by
Virus-Encoded Silencing Suppressor
[0128]Plant viruses encode suppressors of silencing as a part of their
counter-defense strategy to suppress host RNA-mediated defense mechanisms
(Voinnet et al, 1999, Proc Natl Acad Sci USA 96:14147-14152). The protein
p19 from Cymbidium ringspot virus (CymRSV) is a potent silencing
suppressor that acts by sequestering siRNA and therefore preventing their
incorporation into the RISC complex (Silhavy et al, 2002, EMBO J
21:3070-3080; Lakatos et al., 2004, EMBO J 23:876-884). We investigated
the effect of p19-silencing suppression on 5'ss-mediated gene expression
[0129]In the first instance we challenged N. benthamiana line 16c, which
carries a highly expressed GFP transgene (Voinnet et al., 2001, Trends
Genet 17:449-459), with constructs hpGFP and GFP-2×5'ss
co-infiltrated with or without p19. At 8 days post inoculation in the
absence of p19, the inoculated tissue had turned red due to silencing of
the transgene when observed under UV illumination (FIG. 6a). In the
presence of p19, the patch inoculated with the hpGFP was fluorescing
brightly under UV light, indicating that silencing was suppressed (FIG.
6A, middle upper panel). For GFP-2×5'ss, an incomplete suppression
of inhibition was observed and appeared as a thin border of GFP-silenced
cells at the margin of agro-infiltrated zones (FIG. 6a, middle lower
panel). Further, non cell autonomous RNA silencing initiated by a dsRNA
construct (hpGFP) could be observed in the 16c line where a systemic
inhibition of GFP fluorescence occurred by 8 dpi as shown in FIG. 6B and
as previously reported (Voinnet, 2001, Trends Genet 17:449-459).
GFP-2×5'ss triggers a faster systemic GFP down-regulation that was
obvious by 6 dpi, preceding the systemic silencing triggered by hpGFP
(FIG. 6B). When hpGFP and GFP-2×5'ss were co-infiltrated with p19,
systemic inhibition of GFP expression was observed only on 16c plants
co-inoculated with GFP-2×5'ss in a comparable fashion to plants
inoculated only with GFP-2×5'ss (FIG. 6B). A complete suppression
of GFP silencing by p19 was observed for hpGFP (FIG. 6B) as previously
reported (Silhavy et al, 2002, EMBO J 21:3070-3080). Northern blot
analysis of systemic leaves of 16c plants taken at the same time point
after challenge with GFP, hpGFP, GFP-2×5'ss and empty vector
revealed that the GFP mRNA accumulated to a lower level in the case of
GFP-2×5'ss in comparison to GFP or hpGFP (FIG. 6C). This
demonstrates that 5'ss-mediated inhibition is observed not only on a
co-delivered transgene but also on a stably expressed transgene without
being suppressed by viral-encoded silencing suppressor.
5'-ss Mediated Inhibition of Gene Expression is Influenced by the Nature
of the Transcription Terminator and Requires an AAUAAA Polyadenylation
[0130]Further mapping of the required genetic elements for
GFP-2×5'ss inhibition were effectuated. A previous report has
demonstrated that in a mammalian system, the presence of a canonical
polyadenylation signal (AAUAAA) is required for mRNA inhibition of
expression (Fortes et al, 2003, Proc Natl Acad Sci USA 100:8264-8269).
[0131]Chimeric constructs harbouring the duplicated 5'-ss were introduced
upstream of the octopine synthase (OCS) terminator (FIG. 7A). The OCS
terminator is widely used in plant expression systems and does not
contain the AAUAAA canonical polyadenylation signal which is present
within the NOS terminator. The OCS terminator harbours instead three
non-canonical polyadenylation signals (AAUGAA) (FIG. 7A). When
transiently expressed in agro-infiltrated leaves the construct
GFP-MCS-2×5'ss-OCS displayed bright fluorescence indicating that no
significant effect on gene expression was observed as opposed to the
GFP-2×5'ss-NOS (FIG. 7B). This suggests that the duplicated 5'-ss
element together with cis-elements from the terminator sequence are
required to mediate inhibition of gene expression. The introduction of a
multiple cloning site during the engineering of the duplicated 5'ss
upstream of the OCS terminator (FIG. 7A, construct
GFP-MCS-2×5'ss-OCS), resulted in a predicted secondary RNA
structure (Brodsky et al., 1992 Dimacs 8:127-139; Brodsky et al., 1995,
Biochemistry 60(8):923-928 folding as a hairpin. To investigate whether
this structure had the potential to counteract the effect of 5'ss, we
re-introduced the GFP-MCS-2×5'ss upstream of the NOS terminator
(construct GFP-MCS-2×5'ss-NOS). Inhibition of GFP expression in cis
and in trans was restored (FIG. 7B) in a similar fashion to that observed
with the original GFP-2×5'ss construct. This confirmed the relative
flexibility of nucleotidic sequence composition in the vicinity of the
tandem 5'-ss repeat and the NOS terminator. This demonstrates that the
inhibitory effect was not affected by the insertion of additional
nucleotides, which could form putative RNA secondary structures. This
suggests that in plants, mechanisms of mRNA expression inhibition are
operating in a comparable fashion to that in mammalian cells and suggests
that in plants the proximity of a AAUAAA polyadenylation signal is
required to mediate 5'ss mRNA degradation.
Alteration of Polyadenylation Status is Observed in GFP-2×5'ss mRNA
[0132]As the insertion of a 5'ss is likely to affect mRNA maturation and
more specifically mRNA polyadenylation, the polyadenylation status of
GFP-2×5'ss mRNA was analysed. The ratio of polyadenylated and
non-polyadenylated mRNA fractions for GFP and GFP-2×5'ss were
determined using semi-quantitative RT-PCR from agro-infiltrated leaf
tissues (FIG. 8).
[0133]Firstly, cDNA was generated for both GFP and GFP-2×5'ss
samples using oligodT or random hexamers primers, each allowing the
synthesis of poly(A)+and both poly(A)+and poly(A)respectively. As presented in FIG. 8A, a lower amount of
poly(A)+mRNA is observed for GFP-2×5'ss in comparison to the
GFP construct. However, by priming with random hexamers the signal of
total mRNA was comparable to poly(A)+for GFP (FIG. 8A, lower panels)
indicating that most of the GFP mRNA are correctly polyadenylated. This
was not the case for GFP-2×5'ss where a higher amount of RT-PCR
product was amplified if cDNA was synthesized using random hexamers
primers (FIG. 8A, bottom panels) suggesting that the ratio of
poly(A)+/total mRNA is lower for GFP-2×5'ss indicative of a
deficiency in polyadenylation. In all cases a similar level of ubiquitin
mRNA was detected (FIG. 8B).
[0134]Regulation of poly(A) tail addition typically involves the choice
between two or more poly(A) sites on a single pre-mRNA resulting in mRNAs
differing in their 3'UTR sequences. Sequence analysis of the 3'UTR of
randomly selected cDNA clones from GFP and GFP-2×5'ss constructs
revealed that the poly(A) tail is added at two different regions located
either after the first AAUAAA poly(A) signal or after another downstream
putative poly(A) signal AAUAAU (data not shown). Oligonucleotide primers
for first strand cDNA synthesis and RT-PCR were designed in order to
synthesize both polyadenylated and unpolyadenylated mRNA in order to
discriminate between mRNAs that use the first (REV1 primer) or the second
(REV2 primer) putative poly(A) signal (FIG. 9A).
[0135]As shown in FIG. 8B, poly(A) mRNA are accumulating at a lower level
in GFP-2×5'ss as previously observed (FIG. 9B). A similar level of
PCR product was obtained for the GFP construct for poly(A) and
REV1-primed cDNA (FIG. 9B, upper panel) indicating that the first
polyadenylation signal is preferentially used. A comparable level of
poly(A) and REV1-primed cDNA was obtained for GFP-2×5'ss indicating
that to some extent polyadenylation is still occurring in
GFP-2×5'ss (FIG. 9B, upper panel). Using the downstream primer REV2
for priming, cDNA synthesis resulted in a significantly higher
amplification level for GFP-2×5'ss in comparison to poly(A)
fraction (FIG. 9B, lower panel). The PCR product signals obtained for GFP
were much lower and of comparable intensity for the poly(A) and the
REV2-primed fractions (FIG. 9B, lower panel). Taken together this
suggests that GFP-2×5'ss generates a higher proportion of
unpolyadenylated mRNA most likely by interfering with the poly(A) signal
AAUAAA and therefore generating a higher proportion of longer mRNAs. The
alternative use of the second putative poly(A) signal occurs to a much
lower extent with the GFP construct (FIG. 9B). This was confirmed in FIG.
9C using oligodT and FWD primers for RT PCR. In the GFP-2×5'ss+p19
sample a greater proportion of the highest vs smallest molecular weight
mRNA was obtained as opposed to GFP.
The Inhibitory Effect Mediated by 5'ss is not Due to a Splicing Event
[0136]The 10 nt sequence is a 5'-splicing donor site and therefore can be
recognized by endogenous splicing factors to eventually mediate splicing,
providing a 3'-acceptor splicing site is located in the vicinity.
Sequence searches (Brendel et al., 2004, Bioinformatics 20(7):1157-1169;
Kleffe et al., 1996, Nucl Acids Res 24(23):4709-4718; Brendel et al.,
1998, J Mol Biol 276(1):85-104; Brendel and Kleffe, 1998, Nucl Acids Res
26(20):4728-4757; Usuka et al., 2000, J Mol Biol 297(5):1075-1085)
indicated that a putative 3' acceptor site is present within the NOS
terminator sequence (FIG. 9A). A possible explanation of down-regulation
of gene expression would be a consequence of a splicing event occurring
between the 5' donor site and the 3'-acceptor splicing site within the
NOS terminator and generating an aberrant mRNA. RT-PCR was performed
using primers annealing downstream of the putative acceptor site (REV2,
FIG. 9A). The data reveal that in comparison to GFP, GFP-2×5'ss
generates a larger PCR product of about the size of the inserted
2×5'ss SacI fragment, therefore confirming that no splicing event
has occurred and indicating that the inserted 5' donor sites behave as
unpaired 5'-donor splice sites.
5'ss-Mediated Inhibition of Expression of an Endogenous Gene
[0137]To evaluate the efficacy of the approach for gene knock-down in
plants, the effect on the endogenous phytoene desaturase (PDS) was
tested. For this purpose, N. benthamiana leaves were agroinfiltrated with
the PDS-2×5'ss construct and leaf samples were taken at 3 dpi and 7
dpi. Samples were taken from two separate leaves from three plants per
time-point. The effect of PDS-2×5'ss on PDS expression was analysed
at the mRNA level by monitoring PDS mRNA level by Real time "Taqman"
RT-PCR analysis by amplification of a portion of PDS upstream of the
region used in construct PDS-2×5'ss. The PDS mRNA levels were
normalised to ubiquitin mRNA in all samples as previously described
(Lacomme et al., 2003, Plant J 34:543-553). As presented in FIG. 10, the
level of PDS mRNA detected in leaf samples agroinfiltrated with the
PDS-2×5'ss construct were significantly lower than those detected
in the controls (infiltrated with a different construct or
non-infiltrated). The PDS mRNA levels in PDS-2×5'ss were down by
13-fold by 3 dpi and 20-fold by 7 dpi in comparison to control leaves.
This demonstrates that 5'ss-mediated inhibition can target efficiently
not only co-delivered genes or transgenes, but also endogenous genes such
as PDS.
[0138]Annealed U1-2 pair of primers were ligated into pBI121 plasmid
digested by SacI restriction endonuclease giving plasmid
pGUS-2×5'ss. The tandem of 5'ss was located in 3'UTR between stop
codon of GUS cDNA (upstream) and nopaline synthase terminator
(downstream). The same strategy was employed to construct plasmid
containing three tandem repeats of 5'ss (6 copies of 5'ss) using instead
phosphorylated oligonucleotides for annealing and ligation. A GUS
construct harbouring three copies of the 5'ss tandem repeat was generated
and the sense orientation of the repeats were confirmed by sequencing.
Introduction of spacer sequences into existing pGFP-2×5'ss vector
digested with restriction endonuclease AscI was done as follows. A pair
of oligonucleotides
(5'-cgcgccgatgcagatattcgtaattatgcgggcaacgtctggtatcagcgg-3' (SEQ ID No:
23) and 5'-cgcgccgctgatacCagacgttgcccgcataattacgaatatctgcatcgg-3') (SEQ
ID No: 24) was annealed and elongated using Taq polymerase. Resulting
dsDNA was digested by AscI restriction endonuclease and ligated into
pGFP-2×5'ss vector giving pGFP-5'ss-Spacer50-5'ss. A pair of
oligonucleotides (5'-aaggcgcgccgatgcagatattc-3' (SEQ ID No: 25) and
5'-aaggcgcgccgcgcttgctgagtttc-3' (SEQ ID No: 26)) was used for the
generation of a 1000 bp PCR product using GUS coding sequence (pos.
188-1185) as a template. The resulting PCR product was cloned into
pGFP-2×5'ss vector giving pGFP-5'ss-Spacer1000-5'ss plasmid.
[0139]GUS Assay
[0140]Collected leaf discs were homogenized in TissueLyser (Qiagen) in the
presence of 0.6 ml homogenization buffer (50 mM Na-Phosphate pH7, 10 mM
β-mercaptoethanol, 1 mM EDTA pH8, 0.1% sarcosyl, 0.1% Triton X-100).
The extract was spun-down in a microcentrifuge (2000 rpm, 15 min. at
4° C.). Then 0.5 ml of supernatant was transferred into 96-well
plates and assayed immediately according to FluorAce 6-glucuronidase
Reporter Assay Kit manual (BioRad cat No. 170-3151). The level of GUS
expression was measured as activity of β-glucuronidase enzyme per
minute per milligram of total protein. Total protein in the samples was
measured by Bradford assay.
Comparison of Level of Gene Expression Down-Regulation of Plasmids
Carrying One or Two Unpaired Copies of 5'ss Located in a 3'UTR.
[0141]Nicotiana benthamiana leaves were agroinfiltrated with the
constructs: pBINmgfp5-ER (GFP), pGFP-5'ss (GFPart1) or pGFP2×5'ss
(GFPart2). A relative fluorescence was measured as described in Material
and Methods section above. One copy of unpaired 5'ss is able to inhibit
gene expression in cis by 30% whereas a construct carrying two copies of
unpaired 5'ss down-regulates gene expression by up to 95%. The results
Effect of Spacer Length Inserted Between Two 5' Splicing Donor Sites on
Down-Regulation of Gene Expression in cis and trans.
[0142]Nicotiana benthamiana leaves were agroinfiltrated with constructs:
pBINmgfp5-ER (GFP), pGFP-2×5'ss (GFP-ART), pGFP-5'ss-spacer50-5'ss
(Spacer50) or pGFP-5'ss-spacer1000-5'ss (Spacer1000) in presence (white)
or absence (black) of GFP construct. Relative fluorescence was measured
as described in Material and Methods section above. The data are
expressed as Arbitrary Units of Relative fluorescence. No significant
difference in GFP downregulation both in cis and in trans were observed
in comparison to the original 6 nucleotides spacer present in the GFP-ART
construct. The results are shown in FIG. 12.
Influence of Additional Copies of 5'ss within 3'UTR on Expression of GUS
[0143]Construct of β-glucuronidase (GUS) containing 3 copies of a
tandem repeat of 5'ss in the 3'UTR (ie. a total of 6 copies of the 5'ss
sequence) was prepared and compared to pGUS-2×5'ss. Nicotiana
benthamiana leaves were agroinfiltrated with pBI121 (GUS),
pGUS-2×5'ss (GUS-ART), pGUS-6×5'ss (GUS-ART(3)) or
pGFP-2×5'ss (GFP-ART). To assess the level of GUS cDNA expression
β-glucuronidase assay was done. The results were shown as enzymatic
activity of β-glucuronidase in relative fluorescence units per
minute per milligram of total protein. Activity of GUS expressed from
pBI121 plasmid was set as 100%. No significant difference in
β-glucuronidase activity was observed between GUS-ART (one copy of
the tandem 5'ss sequence) and GUS-ART3x (3 copies of the tandem 5'ss
sequence). The results are shown in FIG. 13.
Influence of Additional Copies of 5'ss within 3'-UTR on Expression of GFP
[0144]A construct of β-glucuronidase containing 3 copies of a tandem
repeat of 5'ss (ie. 6 copies of the 5'ss sequence) in the 3'-UTR was
prepared and compared to pGUS-2×5'ss. Nicotiana benthamiana leaves
were co-agroinfiltrated with pBI121 (GUS), pGUS-2×5'ss (GUS-ART),
pGUS-6×5'ss (GUS-ART(3)) or pGFP-2×5'ss (GFP-ART) constructs
in presence of plasmid pGFP-2×5'ss. Constructs carrying
β-glucuronidase (GUS) cDNA contains an identical 3'-UTR (252
nucleotides long) to the construct carrying the GFP cDNA sequence. It was
previously shown that 3'-UTR can trigger down-regulation of gene
expression in trans by 50-60%. Relative fluorescence was measured as
described earlier in Example 1. Additional copies of the 5'ss within the
3'UTR (GUS-ART(3) construct, containing 3 copies of the tandem repeat
5'ss) did not significantly affect the downregulation of gene expression
in trans of a co-expressed GFP transgene. Data are expressed in arbitrary
units of relative fluorescence emission to a GFP construct. The results
5'ss-Mediated Knock Down in Different Plant Species.
[0145]Down-regulation of GFP expression in Nicotiana tabacum var Xanthii
(Panel a, left) and Nicotiana tabacum var. Samsun (Panel b, right). N.b.:
Down-regulation of gene expression in cis by unpaired 5'ss in onion
(Allium cepa) epidermal cells previously demonstrated in FIG. 2. The
Effect of 5'ss Sequence on Expression in Mammalian Cells
Plasmid DNA Transfection of Hela Cells in 24-Well Format Plates
[0146]1. Plate 0.5-2×105 cells in 500 μl of growth medium
without antibiotics and incubate cells at 37° C. for 24 hours
(reach 90-95% confluence).
[0147]2. For each transfection sample, prepare mixes as follows:
[0148]a. Dilute 150 ng of DNA in 50 μl of Opti-MEM® I Reduced
Serum Medium (Gibco) without serum (or other medium without serum). Mix
gently. [0149]b. Mix Lipofectamine® 2000 (Invitrogen) gently before
use, then dilute the 1 μl in 50 μl of Opti-MEM® I Medium.
Incubate for 5 minutes at room temperature. Note: Proceed to Step c
within 25 minutes. [0150]c. After the 5 minute incubation, combine the
diluted DNA with diluted Lipofectamine® 2000 (total volume=100 μl).
Mix gently and incubate for 20 minutes at room temperature.
[0151]3. Add 100 μl of the transfection mix to each well containing
approximately 2.5×105 cells in 5000 medium. Mix gently by
rocking the plate back and forth.
[0152]4. Incubate cells at 37° C. in a CO2 incubator for 18-48
hours prior to testing for transgene expression. Medium may be changed
[0153]FIG. 16 shows the constructs formed as a schematic representation of
Protein Extraction from Hela Cells [0154]1. Remove growth medium from the
cells by decantation or aspiration. [0155]2. Wash cells to remove
residual medium. Slowly add a volume of PBS, equal to the original medium
volume being careful not to dislodge cells. Mix gently and remove the
wash solution. Repeat the wash once in order to remove any other minor
contaminants. [0156]3. After removal of the final wash solution from the
cells, add 100 ul of RIPA Buffer (Pierce) into each of 24 well (1 ml for
0.5 to 5×107 cells). Incubate on ice or in a refrigerator
(2-8° C.) for five minutes. [0157]4. Rapidly scrape the plate to
remove and lyse residual cells. Transfer the cell lysate to a tube on
ice. The lysate can either be used immediately or flash-frozen in liquid
nitrogen and stored at -70° C. for future use. It is best to
freeze the lysate before clarification, since the freeze-thaw cycle may
cause some denatured protein to aggregate. [0158]5. Clarify the lysate by
centrifugation at 8,000 g for 10 minutes at 4° C. to pellet the
cell debris. Note: If a mucoid aggregate of denatured nucleic acids is
present, carefully remove it with a micropipette before centrifugation.
[0159]6. Carefully transfer the supernatant containing the soluble
protein to a tube on ice for further analysis. [0160]7. Total protein
concentration was quantified using Bradford assay. PAGE and
immunoblotting techniques were as previously described.
Assessment of EGFP Expression in HeLa Cells Using Epifluorescence
Microscopy and Western Blotting Techniques.
[0161]Experiments were effectuated in 24-wells plate format. In each well
approximately 2.5×105 HeLa cells were grown. Cells were
co-transfected with the following constructs combinations: pEGFP+pDsRED,
pEGFPart5+pDsRED, 2×pEGFP+pDsRED, pEGFP+pEGFPart5+pDsRED (See FIG.
16) and enumeration of cells expressing EGFP was effectuated using a
fluorescence microscope (Nikon Optiphot epifluorescence microscope) and
3CCD Color Video Camera (KY-F558 Photonic Science). Approximately 1% of
HeLa cells, transiently transfected with pEGFPart5 construct displayed
green fluorescence, whereas about 60% of cells transfected with pEGFP
construct showed fluorescence. Two days post transfection pictures were
taken using a Nikon Optiphot epifluorescence microscope using either a
green or a red filter to detect EGFP and DsRED fluorescence respectively
(see FIG. 17). The cells transfected with the mix pEGFP+pEGFPart5+pDsRED
showed similar number of EGFP fluorescent cells as for cells transfected
with pEGFP+pDsRED constructs (FIG. 17).
[0162]Bright field and fluorescent (green and red) merged images are
presented. Proliferating HeLa cells expressing EGFP appears bright green
under the fluorescence microscope, the bright field image allow the
observation of non-expressing cells (especially with constructs
pEGFPart5+pDsRED). Note that due to the slowest maturation process of
DsRED in comparison to EGFP the red fluorescence is barely detectable at
2 days post transfection as opposed to the green fluorescence.
[0163]Western blot analysis of the cells transfected with the
above-mentioned construct combinations was effectuated (see FIG. 18). A
significantly lower accumulation of EGFP was observed when cells were
transfected with the construct pEGFPart5 harbouring the tandem 5'ss
sequence for the samples pEGFPart5+pDsRED and pEGFP+pEGFPart5+pDsRED as
opposed to control samples pEGFP+pDsRED and 2×pEGFP+pDsRED
respectively. The DsRED is used as a marker of transfection and internal
calibrator for western blot analysis.
[0164]Construct pEGFPart5 was engineered to harbour a tandem insertion of
5'ss in the 3' UTR of EGFP. Their transient expression in HeLa cells
causes a knock-down of GFP expression in cis (line A2) but also in trans
(line A4) in comparison to control pEGFP constructs (A1 and A3). Western
blot analysis shows accumulation of green fluorescence protein (panel A)
or co-expressed red fluorescence protein (panel B); line 1: pEGFP+pDsRED;
line 2: pEGFPart5+pDsRED; line 3: 2×pEGFP+pDsRED; line 4:
pEGFP+pEGFPart5+pDsRED. The blots were probed with antibodies against
green fluorescent protein (GFP) (panel A) or against red fluorescent
protein (RFP) used as an internal calibrator (panel B).
[0165]These data suggests that the pEGFPart5 construct express EGFP to
significantly lower levels in HeLa cells both in cis and in trans by
affecting expression of the modified pEGFPart5 construct and a distinct
pEGFP construct.
[0166]Standard exemplary method for transfection of mammalian cells
Cell and DNA Preparation:
[0167]1. Plate cells 24 hours before transfection. Usual plating
density is 1×106 cells/100 mm dish/10 mls complete media.
[0168]Note: If transfecting a suspension culture, suspension cell
concentration should be 5×106/ml. Suspension cells grown in
RPMI may be difficult to transfect with this kit. It is recommended to
use a DMEM for suspension transfection in this case. [0169]2. Feed
cells fresh, complete media 3 hours before transfection. [0170]3. All
DNAs used should be phenol, phenol/chloroform/isoamyl alcohol extracted
ethanol precipitated and dissolved in sterile UltraPure water or a
[0170] [0171]1. 1 ml of calcium phosphate precipitate is needed for each
100 mm plate of cells to be transfected. [0172]2. Prepare 1×HBS
fresh for each experiment. 0.5 ml of 1×HBS is needed for each 100
mm plate. [0173]3. Formula for 1×HBS is as follows: [0174]Add 0.88
ml sterile UltraPure water to tube [0175]Add 0.1 ml 10×HBS and mix
well [0176]Add 150 NaOH Solution and mix well [0177](note: the pH will be
correct and need not be checked)
Formula for 1 ml Calcium Phosphate DNA Precipitate:
[0177] [0178]1. Set up two sterile polypropylene tubes for each DNA to
be precipitated, label tubes #1 and #2 along with the DNA to be used
[0179]2. Add to tube #1: 0.5 mls of 1×HBS and 100 of phosphate
solution. [0180]3. Add to tube #2: 0.43 mls of UltraPure water minus
volume of DNA. Total DNA should equal 20 μg. [0181]Note: If genomic
DNA is being used, the total DNA should equal 30 μg. Genomic DNA will
replace carrier and plasmid DNA's. [0182]4. Gently mix the DNAs into
the water. [0183]5. Add 600 of calcium solution and mix gently
Forming the Calcium Phosphate and DNA Precipitate:
[0183] [0184]1. Place a sterile 1 ml pipet into tube #1 and gently
bubble air through the solution so that it is slowly mixing. [0185]2.
Draw the contents of tube #2 up into an appropriately sized sterile
pipet. Add slowly, dropwise, to the gently bubbling and mixing solution
in tube #1. As the two solutions mix they will appear milky and then form
a white precipitate. Continue to bubble and add slowly until the entire
contents of tube #2 have been added. [0186]3. Allow the suspension to sit
at room temperature for 20 minutes before adding to the cells.
Adding the Precipitate to the Cells:
[0186] [0187]1. Mix the precipitate well by pipeting or vortexing,
making sure that any large clumps that may have formed on the bottom of
the tube are broken up and that the precipitate is evenly resuspended.
[0188]2. Add 1 ml of suspension to a 100 mm plate containing 10 mls of
complete media. The suspension must be added slowly, dropwise, while
gently rocking the media in the plate. [0189]3. Return the plates to the
incubator and leave the precipitate on for 12-24 hours.
Maintenance of Transfected Cells:
[0189] [0190]1. Remove the media containing the precipitate and add
fresh complete media leaving this media on for 24 hours. [0191]2. Remove
the media and add the appropriate selection media to select stable
colonies or add complete media for transient expression incubation.
26110DNAArtificial SequenceSynthetic embodiment of 5' splicing site
sequence 1maggtragta
10210DNAArtificialSynthetic embodiment of 5' splicing site
sequence 2caggtaagta
103308DNAArtificialSynthetic embodiment of nucleotidic
composition of the 3' UTR of GFP-2x5' splicing site 3taagagctcg
agcaggtaag taggcgcgcc caggtaagta gagctcgaat ttccccgatc 60gttcaaacat
ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga 120ttatcatata
atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga 180cgttatttat
gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga 240tagaaaacaa
aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt 300tactagat
3084855DNAArtificialSynthetic embodiment of nucleotidic composition
of the 3' UTR of GFP-MCS-2x5' splicing site-OCS 4taaccctagg tacgtaggcg
cgccattata aatctagaca ggtaagtacg gatccgcagg 60taagtagacg tcctctagct
taattaagac ccgggactag tccctagagt cctgctttaa 120tgagatatgc gagacgccta
tgatcctgct ttaatgagat atgcgagacg cctatgatcg 180catgatattt gctttcaatt
ctgttgtgca cgttgtaaaa aacctgagca tgtgtagctc 240agatccttac cgccggtttc
ggttcattct aatgaatata tcacccgtta ctatcgtatt 300tttatgaata atattctccg
ttcaatttac tgattgtacc ctactactta tatgtacaat 360attaaaatga aaacaatata
ttgtgctgaa taggtttata gcgacatcta tgatagagcg 420ccacaataac aaacaattgc
gttttattat tacaaatcca attttaaaaa aagcggcaga 480accggtcaaa cctaaaagac
tgattacata aatcttattc aaatttcaaa agtgccccag 540gggctagtat ctacgacaca
ccgagcggcg aactaataac gctcactgaa gggaactccg 600gttccccgcc ggcgcgcatg
ggtgagattc cttgaagttg agtattggcc gtccgctcta 660ccgaaagtta cgggcaccat
tcaacccggt ccagcacggc ggccgggtaa ccgacttgct 720gccccgagaa ttatgcagca
tttttttggt gtatgtgggc cccaaatgaa gtgcaggtca 780aaccttgaca gtgacgacaa
atcgttgggc gggtccaggg cgaattttgc gacaacatgt 840cgaggctgag cagga
855520DNAArtificialForward
primer used for semi-quantitative RT-PCR of GFP expression
5gggcacaaat tttctgtcag
20624DNAArtificialReverse primer used for semi-quantitative RT-PCR
of GFP expression 6gttgtgggag ttgtagttgt attc
24731DNAArtificialREV1 primer 7aaataacgtc atgcattaca
tgttaattat t 31827DNAArtificialREV2
primer 8ttctatcgcg tattaaatgt ataattg
27920DNAArtificialforward primer 9tccacacaat ctgccctttc
201027DNAArtificialreverse primer
10gcgagctccg cggccttttt ttttttt
271130DNAArtificialPrimer for amplification of CaMV
35s::mgfp5-ER::3'-NOS derived from pBINmgfp5-ER plasmid 11cccaagcttt
ttcagaaaga atgctaaccc
301232DNAArtificialPrimer for amplification of CaMV
35s::mgfp5-ER::3'-NOS derived from pBINmgfp5-ER plasmid 12cccaagcttg
atctagtaac atagatgaca cc
321327DNAArtificialself annealing primer 13cgagmaggtr agtaggcgcg ccgagct
271427DNAArtificialself annealing
primer 14cggcgcgcct acttacctgc tcgagct
271537DNAArtificialself annealing primer 15cgagmaggtr agtaggcgcg
ccmaggtrag tagagct 371637DNAArtificialself
annealing primer 16ctacttacct gggcgcgcct acttacctgc tcgagct
371745DNAArtificialPrimer used for amplification of GFP
ORF from pBINmgfp5-ER plasmid 17gtgtgtctcg agccatggcc aagactaatc
tttttctctt tctca 451857DNAArtificialPrimer used for
amplification of GFP ORF from pBINmgfp5-ER plasmid 18gtgtgtggcg
cgcctacgta cctagggtta accaagctca tcatgtttgt atagttc
571962DNAArtificialAnnealed primer used for ligating ChSA intron
sequences 19cgcgccatta taaatctaga caggtaagta cggatccgca ggtaagtaga
cgtcctctag 60cc
622062DNAArtificialAnnealed primer for ligating ChSA intron
sequences 20ccggggctag aggacgtcta cttacctgcg gatccgtact tacctgtcta
gatttataat 60gg
622133DNAArtificialForward primer for amplification of 314
bp PDS cDNA fragment 21atgggatcca tgaaggaact agcgaagctt ttc
332235DNAArtificialReverse primer for
amplification of 314 bp PDS cDNA fragment 22tacgagctct tagttcacta
tgctaactac gcttg
352351DNAArtificialoligonucleotide used for creation of spacer
sequences for pGFP-2x5' splicing site vector 23cgcgccgatg cagatattcg
taattatgcg ggcaacgtct ggtatcagcg g
512451DNAArtificialoligonucleotide used for creation of spacer
sequences for pGFP-2x5' splicing site vector 24cgcgccgctg ataccagacg
ttgcccgcat aattacgaat atctgcatcg g 512523DNAArtificialPrimer
1 for GUS amplification (1000 bp PCR product) 25aaggcgcgcc
gatgcagata ttc
232626DNAArtificialPrimer 2 for GUS amplification (1000 bp PCR
product) 26aaggcgcgcc gcgcttgctg agtttc
2011-02-24Digital analysis of gene expression
2011-05-19Antisense modulation of c-reactive protein expression
2011-02-10Rnai inhibition of influenza virus replication
2009-05-14Antisense modulation of ptp1b expression
2011-05-26Construction of genetically tractable industrial yeast strains