U.S. patent application number 15/904548 was filed with the patent office on 2018-11-22 for culicinae mosquito tra-2 rna interference technique to genetically produce maleness population.
The applicant listed for this patent is Duong Thanh Hoang, Kim Phuc Hoang. Invention is credited to Duong Thanh Hoang, Kim Phuc Hoang.
Application Number | 20180334675 15/904548 |
Document ID | / |
Family ID | 46879771 |
Filed Date | 2018-11-22 |
United States Patent
Application |
20180334675 |
Kind Code |
A1 |
Hoang; Duong Thanh ; et
al. |
November 22, 2018 |
Culicinae Mosquito TRA-2 RNA Interference Technique to Genetically
Produce Maleness Population
Abstract
This invention entails a method to make a Tra-2 RNAi kernel
sequence, and uses for this kernel sequence. The method includes
the steps of amplifying an RNA recognition motive (RRM) DNA
sequence from a Culcinae mosquito species, to obtain a first RRM
DNA sequence of 240 base pairs; then reversely connecting the first
RRM DNA sequence with a second RRM DNA sequence via an intron or a
linker DNA sequence, in such a way that the second RRM DNA sequence
forms an inverted sequence to the first RRM DNA sequence.
Transcription of the two DNA sequences produces single strands of
mRNA that can bind together to form a double strand hairpin mRNA
structure. Other steps include inserting a tetracycline repressible
transactivator, and an insect spermatogenesis promoter.
Inventors: |
Hoang; Duong Thanh; (Hanoi
City, VN) ; Hoang; Kim Phuc; (Oxford, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoang; Duong Thanh
Hoang; Kim Phuc |
Hanoi City
Oxford |
|
VN
GB |
|
|
Family ID: |
46879771 |
Appl. No.: |
15/904548 |
Filed: |
February 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14356978 |
May 8, 2014 |
9926558 |
|
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PCT/VN2011/000011 |
Dec 29, 2011 |
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15904548 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01K 2267/02 20130101;
C12N 2330/51 20130101; A01K 67/0339 20130101; A01K 2227/706
20130101; C12N 2310/14 20130101; C12N 15/113 20130101; A01K 67/0333
20130101; A01K 2217/058 20130101 |
International
Class: |
C12N 15/113 20100101
C12N015/113; A01K 67/033 20060101 A01K067/033 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 23, 2011 |
VN |
1-2011-00772 |
Claims
1. A method to make a Tra-2 RNAi kernel sequence, which method
comprises the steps of: i) amplifying an RNA recognition motive
(RRM) DNA sequence from a Culcinae mosquito selected from the group
consisting of Aedes aldopictus, Aedus aegypti, Aedes polynesiensis
and Culex quinquefasciatus, to obtain a first RRM DNA sequence of
240 base pairs, ii) repeating step i), to obtain a second RRM DNA
sequence of 240 base pairs; iii) reversely connecting the first RRM
DNA sequence with the second RRM DNA sequence via an intron or a
linker DNA sequence, which intron or linker DNA sequence is
connected to the end of the first RRM DNA sequence and the
beginning of the second RRM DNA sequence, so that the second RRM
DNA sequence forms an inverted sequence to the first RRM DNA
sequence, wherein the transcription of the first RRM DNA sequence
and the second RRM DNA sequences produces single strands of mRNA
with complementary sequences exposed at ends of the strands of
mRNA, which complementary sequences are of sufficient length so as
to be capable of binding together to form a double strand hairpin
mRNA structure having a loop portion, where the loop portion is
formed by the transcription product of the intron or linker DNA
sequence.
2. The method of claim 1, wherein the RRM DNA sequence is selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID
NO:3.
3. The method of claim 1, wherein the intron or linker DNA sequence
is connected to the end of the first RRM DNA sequence and the
beginning of the second RRMA sequence via an AG sequence.
4. A method to make a Tra-2 RNAi construct, which method comprises
the steps of: i) creating an RNAi kernel sequence by amplifying an
RNA recognition motive (RRM) DNA sequence from a Culcinae sequence
selected from the group consisting of Aedes aldopictus, Aedus
aegypti, Aedes polynesiensis and Culex quinquefasciatus, to obtain
a first RRM DNA sequence of 240 base pairs, amplifying an RNA
recognition motive (RRM) DNA sequence from a Culcinae sequence
selected from the group consisting of Aedes aldopictus, Aedus
aegypti, Aedes polynesiensis and Culex quinquefasciatus, to obtain
a second RRM DNA sequence of 240 base pairs, reversely connecting
the first RRM DNA sequence with the second RRM DNA sequence via an
intron or a linker DNA sequence, which intron or linker DNA
sequence is connected to the end of the first RRM DNA sequence and
the beginning of the second RRM DNA sequence, so that the second
RRM DNA sequence forms an inverted sequence to the first RRM DNA
sequence, wherein the transcription of the first RRM DNA sequence
and the second RRM DNA sequences produces single strands of mRNA
with complementary sequences exposed at ends of the strands of
mRNA, which complementary sequences are of sufficient length so as
to be capable of binding together to form a double strand hairpin
mRNA structure having a loop portion, where the loop portion is
formed by the transcription product of the intron or linker DNA
sequence, ii) inserting a tetracycline repressible transactivator
operably linked to and controlling expression of the RNAi kernel
sequence, and iii) inserting an insect spermatogenesis promoter
operably linked and controlling expression of the tetracycline
repressible transactivator.
5. The method of claim 4, wherein the RRM DNA sequence is selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID
NO:3.
6. The method of claim 4, wherein the intron or linker DNA sequence
is connected to the end of the first RRM DNA sequence and the
beginning of the second RRMA sequence via an AG sequence.
7. The method of claim 4, wherein the insect spermatogenesis
promoter is Drosophila-.beta.2.
8. The method of claim 4, which comprises the further step of
inserting a regulatory component, or a terminator component, or
both.
9. The method of claim 8, wherein the regulatory component is
tetOx7 and the terminator component is SV40 polyA.
10. A method for increasing genetic maleness offspring in a
Culicinae mosquito population, comprising the steps of: producing a
Tra-2 RNAi construct, which method comprises the steps of: a)
creating an RNAi kernel sequence by i) amplifying an RNA
recognition motive (RRM) DNA sequence from a Culcinae sequence
selected from the group consisting of Aedes aldopictus, Aedus
aegypti, Aedes polynesiensis and Culex quinquefasciatus, to obtain
a first RRM DNA sequence of 240 base pairs, ii) amplifying an RNA
recognition motive (RRM) DNA sequence from a Culcinae sequence
selected from the group consisting of Aedes aldopictus, Aedus
aegypti, Aedes polynesiensis and Culex quinquefasciatus, to obtain
a second RRM DNA sequence of 240 base pairs, iii) reversely
connecting the first RRM DNA sequence with the second RRM DNA
sequence via an intron or a linker DNA sequence, which intron or
linker DNA sequence is connected to the end of the first RRM DNA
sequence and the beginning of the second RRM DNA sequence, so that
the second RRM DNA sequence forms an inverted sequence to the first
RRM DNA sequence, wherein the transcription of the first RRM DNA
sequence and the second RRM DNA sequences produces single strands
of mRNA with complementary sequences exposed at ends of the strands
of mRNA, which complementary sequences are of sufficient length so
as to be capable of binding together to form a double strand
hairpin mRNA structure having a loop portion, where the loop
portion is formed by the transcription product of the intron or
linker DNA sequence, iv) inserting a tetracycline repressible
transactivator operably linked to and controlling expression of the
RNAi kernel sequence, and v) inserting an insect spermatogenesis
promoter operably linked and controlling expression of the
tetracycline repressible transactivator, b) stably transforming a
Culicinae mosquito with the Tra-2 RNAi DNA construct, c) allowing
stable expression of the Tra-2 RNAi DNA construct during
spermatogenesis in the Culicinae mosquito, so as to effect Tra-2
gene knockdown of X(m) chromosome-bearing sperm, d) allowing the
Culicinae mosquito of step ii) to mate and thereby stably pass on
the Tra-2 RNAi DNA construct to offspring, resulting in continuous
interruption of the development of X(m) chromosome-bearing sperm
and thereby effecting genetic male bias in progeny.
11. The method for increasing genetic maleness offspring in a
Culicinae mosquito population of claim 10, wherein genetic male
bias in progeny is at least 90%.
12. A Tra-2 RNAi DNA construct produced by the method of claim
4.
13. A Culicinae mosquito stably transformed with the Tra-2 RNAi DNA
construct of claim 12, wherein expression of the DNA construct
occurs during spermatogenesis so as to effect Tra-2 gene knockdown
of X(m) chromosome-bearing sperms.
Description
[0001] This application is a continuation application of Ser. No.
14/356,978, filed May 8, 2014, which is a national application of
PCT/VN2011/000014, filed Dec. 29, 2011, which claims priority from
Vietnamese application VN 1-2011-00772, filed Mar. 23, 2011. This
application claims priority from all these documents, and the
entire contents of these documents are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of molecular
biology to develop a novel method for controlling mosquito
populations. Culicinae mosquitoes carry one or more loci of
transformant Tra-2 RNAi constructs which target to mosquito
Transformer-2 locus in respective or none respective Culicinae
mosquitoes. Tra-2 sequences used to assemble Tra-2 RNAi recombinant
constructs are Tra-2 gene sequences of Culicinae mosquitoes and can
be derived from endogenous or exogenous sequences. The Tra-2 RNAi
expression is conditional, wherein the expression causing a
knockdown effect into the endogenous Tra-2 gene results in
mortality of X (m) chromosome bearing sperms and produces maleness
mosquito population in the nature environmental of the species.
BACKGROUND OF THE INVENTION
[0003] Nowadays, there are many biological control techniques have
been invented for controlling insects and plants (Smith and
Borstel, 1972. Science. 178. 1164-1174). A method invented to
control of insect populations is named the "sterile insect
technique" SIT. This method is also known with a different name as
"sterile insect release method" SIRM. A model of the SIRM based on
population parameters obtained from a large scale experiment to
eradicate melon fly was first developed by Ito (1977. Appl. Ent.
Zool. 12:310-312; 1979. Res. Popul. Ecol. 20. 216-226).The SIT
method creates sterile male insects and releases them into natural
habitats where the males would look for natural females to mate.
These females would be sterile or to produce offspring that cannot
develop up to the harmful stages. When a huge number of sterile
males released in a chronic time, it can cause a collapse of the
natural insect populations or even extinction. However, the insect
created by the SIT method need to be undergone a sexing step to
remove females. The reason is that in many insect species, even
sterile females, if being released, they would look for blood meals
and may transmit diseases (in mosquitoes) or damage fruits (in med
fly). As such, release of the female insects must be avoided.
[0004] Currently, there are different methods to separate male
insects from a total based on the differences of sexual traits as
body sizes or eclosion time. However, systematic errors of those
are high and more or less depending on each species, for instance,
our data indicated that 8-15% females of Aedes aegypti mosquitoes
can be misidentified as males (K. P. Hoang, unpublished data).
Alternatively, insects can be frozen down on ice to separate
females and males. However, this method is too high labor costs and
can damage small insects as male mosquitoes.
[0005] In another approach, X and gamma rays are used to
translocate a chromosome fragment which carries genes encoding for
different colors of silk worm male and female eggs (Strunnikov,
1979. Theor. Appl. Genet. 55, 17-21; Strunnikov. 1983. Control of
silkworm Reproduction, Development and Sex. MIR Publishers.
Moscow). However, the mutant strains created by radiation are
usually accompanied with a significant decline in male mating
competitiveness in comparison with its wild type males. This can
result in failures in vector control strategies if applying for the
release of insect males. Besides, irradiation method is not
specific to the certain target, the radiation not only causes big
mutations in chromosome systems of the target organisms but can
also be dangerous for producers. This is also an expensive method
with plenty of limitations. Asburner et al., (1998) disclose a
method by introduction of an exogenous DNA fragment into the insect
genetic system to create insect transgenic species (Insect
Molecular Biology, 7 (3), 201-213). This approach was lately
improved by a patent of Handler (2006. PN: U.S. Pat. No.
7,005,296B1).
[0006] DeVault disclose to use a female specific promoter to be
ligated into a lethal gene. The gene is only activated in females
and therefore males are uniquely remained in the selection. These
males are irradiated for sterilization before releasing into nature
(DeVault et al., 1996; Biotechnology, Vol 14; 46-49; DeVault et
al.,1996. Genome Res. 6: 571-579). This achievement gains a big
progress in the genetic sexing experiments, however the use of
radiation can severely damage for small insects with its
consequences of decreasing male mating competitiveness ability.
[0007] To avoid the radiation damages, a new method named RIDL
(Release of Insects carrying a dominant lethal) has been disclosed
in a patent (Alphey, 1999. PN: WO 01/39599 A2; Alphey, 2007.
Area-Wide Control of Insect Pests: From Research to Field
Implementation, Springer, Dordrecht, The Netherlands). The RIDL
offers a solution to many of the drawbacks of traditional SIT that
have limited its application in mosquitoes as mentioned above. RIDL
differs from conventional SIT in that the released insects are not
sterilized by irradiation but its sterility is resulted from a
homozygote for a dominant lethal gene. Highly efficient repressible
RIDL systems were first demonstrated in Drosophila models and
recently in the Mediterranean fruit fly (Thomas et al., 2000.
Science, 287: 2474-2476.; Gong et al., 2000. Nature Biotech., 23:
453-456.). This system exploits a tetracycline-repressible
transactivator (tTA) to control expression of the dominant lethal
(Gossen and Bujard, 1992. Proc Natl Acad Sci USA 1992,
89(12):5547-5551). The tetracycline (Tet) that to be mixed in
larval rearing medium or food can bind to tTA and preventing it
binding to tetO sequences and driving the effector gene. The tetO
sequences plays a role of an operator would be free to suppress the
lethal gene. In the absence of Tet, tTA protein binds to the
operator sequence and the effector gene would be free to express.
In natural environment where Tet is absent, released transgenic
males mates with wild type females and their offspring would be
killed by the effector gene activation. In Aedes mosquitoes, RIDL
has been proved to be efficient, of which the males created haven't
been declined their fitness when competing with wild type males.
(Phuc et al., 2007; BMC Biology
http://www.biomedcentral.com/1741-7007/5/11). However, the RIDL
method has a serious shortcoming that it still produces offspring
in both sexes. It therefore needs an addition step of sexing to
remove females before releasing.
[0008] Fu et al., (2010. PNAS, Vol. 107, No. 10, 4550-4554)
discloses a method in which a fusion between RIDL system and a
female sex-specific regulation based on an endogenous Actin-4
promoter that derived from Aedes aegypti females. The effector gene
is specifically activated only in the direct-flight muscle of
female mosquitoes and this expression makes females to be
flightless. These females after eclosion would be stuck on the
water surface and to be dead eventually. By this method, only 50%
of offspring becomes males which can continuously pass the
transformant genetic systems into next generation. However, this
method in practice has a shortcoming. This happens when plenty of
the flightless females staying on the water surface, their bodies
and leg movements can prevent other eclosion males to come up with
the water which may eventually drown males. The higher rearing
density is the higher "collateral damage" for males, but in
industrial insectary, rearing at high density is the only
option.
[0009] Transformer-2 gene has been seen as a key factor in
combination with Tra for sex determination in different eukaryotes
although it may involve differently in different taxa depending on
evolutionary divergence. Fortier and Belote (2000. Genesis 26(4):
240-244), Salvemini et al (2009. Int. J. Dev. Biol. 53: 109-120)
and Sarno et al (2010.
http://www.biomedcentral.com/1471-2148/10/140) used the RNAi method
to knock down the Tra-2 genes in Drosophila, Ceratitis Capitata and
Anastrepha, respectively. The knockdown effect can convert females
of these species into pseudo males carrying XX chromosomes. In
their studies, the RNA interference method is performed by
injection of Tra-2 double stranded RNA (dsRNA) into embryos after
an invitro synthesized step. No tra-2 orthologue has been
identified in Anopheles and the Tra-2 orthologue in Aedes aegypti
mosquitoes seems to involve in a different genetic mode. A full
length mRNA transcription of Tra-2 gene in Aedes aegypti is not
necessarily required for its downstream gene cascade, doublesex
(dsx) to be spliced. One female specific Dsx can be default spliced
to be females (Salvemini et al., 2011. BMC Evolutionary Biology
2011, 11:41 http://www.biomedcentral.com/1471-2148/11/41). It,
therefore, suggests that the wish to create all maleness offspring,
including 50% pseudo [XX (mm)] males by a conversion from females
is impossible if targeting Tra-2 in Culicinae. In fact, in our
experiments, Tra-2 dsRNA injection into Culicinae mosquito eggs
hasn't caused a significant bias in sex ratio.
[0010] The present invention sets out to overcome all the
shortcoming of the previous methods by using the common principles
of the RIDL method (Alphey, 1999. PN: WO 01/39599 A2; Alphey, 2007.
Area-Wide Control of Insect Pests: From Research to Field
Implementation, Springer, Dordrecht, The Netherlands) in
combination with a discovery of X (m) bearing sperm killing effect
due to Tra-2 RNAi genetic system. These transgenic Culicinae
mosquitoes are therefore to produce more than 90% genetic maleness
offspring.
BRIEF DESCRIPTION OF THE FIGURE
[0011] FIG. 1 describes RNAi kernel sequences controlled by a
regulatory element in a transactivator system within a PiggyBac
plasmid. This a is diagrammatic representation of a completed
Tra2-RNAi system, linearized at the 3' end of the piggyBac
transposon. There are three functional segments within the ends of
the transposon: the marker (3xP3 promoter-ECFP-SV40 poly A3') to
allow detection of transgenic individuals by fluorescence. tTA
protein is under the control of an insect spermatogenesis promoter
(Drosophila .beta.2) and terminated by a SV40 poly A 3' sequence.
The third segment is the Tra-2 Rnai cassette (tetO7-CMV minimal
promoter--two Tra2 mRNA inverted repeats joined by a fragment of a
linker or intron sequence. This is also terminated by a SV40 poly A
3' sequence. The Tra-2 Rnai cassette acts by a tTA protein binding
into the tet07 in the absence of Tetra cycline to free CMV minimal
promoter to drive.
SUMMARY AND TECHNIQUE PRINCIPLES OF THE INVENTION
[0012] We discovered that is not like the Tra-2 in Drosophila,
Ceratitis Capitata and Anastrepha, the Tra-2 gene in Culicinae
mosquitoes is involved in male specific, spermatogenesis processes
and the knockdown of Tra-2 gene hasn't resulted in a conversion
from females to males as occurred in the other Dipteran insects.
Because, a transient effect of dsRNA cannot last from eggs to
adults to consistently cause a knockdown effect into
spermatogenesis stages and following consequences in next
generation, therefore all attempts to use dsRNA injections to
obtain transient effects would be invalid.
[0013] To successfully repress the Tra-2 gene in mosquitoes, it is
necessary to create permanent transgenic lines with Tra-2 RNAi
constructs by which the interference effect will be stably
expressed during the spermatogenesis stages. We found that Tra-2
gene knockdown in Culicinae mosquitoes can cause lethality of X (m)
chromosome bearing sperms and therefore only Y-chromosome (M)
bearing sperms are survival and maleness offspring would be
produced. These males are genetic males which carry a Y chromosome.
Males created by this method are not sterile but they produce
healthy Y chromosome bearing sperms only. Theoretically, maleness
offspring would continuously pass the Tra-2 RNAi constructs into
natural populations until the populations gone extinct.
[0014] This invention discloses all of the methods to create Tra-2
RNAi DNA constructs, to transform it into mosquitoes and to observe
its expression.
[0015] The invention uses putative Transformer-2 encoding gene
sequences from Aedes albopictus, Aedes polynesiensis, Culex
quinquefasciatus or the other Culicinae mosquitoes as materials to
assemble Tra-2 RNAi genetic constructs by using DNA recombination
techniques. In Examples of this invention, parts or whole RRM (RNA
recognition motif) sequences which are belonging to putative
Transformer-2 encoding sequences from Aedes albopictus, Aedes
polynesiensis and Culex quinquefasciatus are used.
[0016] The Tra-2 of Aedes aegypti is identified by blasting against
the Aedes aegypti Genbank database with an input is the Drosophila
Tra-2A amino acid sequence. The outcome was AAEL004293-RA protein
belonging to supercontig 1.113 (Aedes aegypti-Vectorbase). Aedes
aegypti are closely related species with Aedes albopictus and Aedes
polynesiensis, therefore primers derived from the AAEL004293-RA
sequence can be used to amplify Tra-2 sequences of Aedes albopictus
and Aedes polynesiensis mosquitoes. The regions with highest
similarity among the orthologous Tra-2 genes are RRMs (RNA
recognition motives) with a length of 240 bp. For many other Aedes
spp these primers were tested and can successfully amplify these
240 bp regions. We found two RRMs loci (or allele), each exists in
both Aedes albopictus and Aedes polynesiensis. They are different
10% amino acid from each other and named as SEQ ID: No 1 (RRM1) and
SEQ ID: No 2(RRM2). To knock down these two loci (allele), it may
be required to transform two respective RNAi constructs into each
species to repress the respective RRM locus (allele).
[0017] A putative Tra-2 gene of Culex quinquefasciatus is
identified by blasting Culex quinquefasciatus database with the
RRM1 and RRM2 sequences. The name of the outcome is CPIJ016646;
supercontig 3.780:5008-5247. The RRMs of Culex orthologous Tra-2
gene is named as SEQ ID: No 3 (RRM3). For many other Culex spp,
primers derived from the first and the end of the RRM3 region have
been tested and can successfully amplify these 240 bp regions.
[0018] In order to knock down the Tra-2 genes in Aedes albopictus,
Aedes polynesiensis, Culex quinquefasciatus and the other Culicinae
mosquitoes by the RNAi technique, there are three solutions to be
disclosed in the invention. [0019] The first solution is to use SEQ
ID: No 1 (RRM1) as materials to invitro create an RNAi kernel
sequence 1. This is a recombinant DNA fragment combining two
identical sequences of RRM1 but in opposite directions. A
connection between the two RRM1 repeats is a straight intron or
linker DNA sequence. The RNAi kernel sequence 1 is then ligated
with transactivator and regulatory elements and a fluorescent
marker within a PiggyBack plasmid. This plasmid would then be
available for transforming into both Aedes albopictus and Aedes
polynesiensis to knock down their RRM1 locus.
[0020] The regulatory element for the kernel sequence is a minimal
promoter associated with operator sequences (tetOx7). The minimal
promoter plus tetOx7 can be conditional by using the commercial
transactivator regulation systems (Clontech). Gene expression is
activated as a result of binding of the Tet-Off protein (tTA) to
tetracycline response elements (TREs) located within the minimal
promoter.
[0021] For the best of expression, the invention suggests to use
insect spermatogenesis specific promoters to control tTA protein
gene. Besides, we also prefer to derive a minimal promoter from an
insect spermatogenesis specific promoter for controlling the RNAi
kernel sequence 1 which helps to eliminate all leakiness effect.
This solution may be applied for any other Aedes spp which has a
highly similar sequence of its RRM in comparison with the RMM1 or
after obtaining its own Tra-2 RRM sequences by the same pair of
primers. [0022] The second solution is to use SEQ ID: No 2 (RRM2)
as materials to invitro create an RNAi kernel sequence 2. This is a
recombinant DNA fragment combining two identical sequences of RRM2
but in opposite directions. A connection between the two RRM2
repeats is a straight intron or linker DNA sequence. The RNAi
kernel sequence 2 is then ligated with regulatory elements and a
fluorescent marker within a piggyBack plasmid. This plasmid would
then be available for transforming into both Aedes albopictus and
Aedes polynesiensis to knock down their RRM2 locus.
[0023] In the details, the regulatory elements in the second
solution are identical as those described in the first
solution.
[0024] This solution may be applied for any other Aedes spp which
has a highly similar sequence of its RRM in comparison with the
RMM2 or after obtaining its own Tra-2 RRM sequences by the same
pair of primers. [0025] The third solution is to use SEQ ID: No 3
(RRM3) as materials to invitro create a RNAi kernel sequence 3.
This is a recombinant DNA fragment combining two identical
sequences of RRM3 but in opposite directions. A connection between
the two RRM3 repeats is a straight intron or linker DNA sequence.
The RNAi kernel sequence 3 is then ligated with regulatory elements
and a fluorescent marker within a piggyback plasmid. This plasmid
would then be available for transforming into Culex
quinquefasciatus.
[0026] In the details, the regulatory elements in the third
solution are identical as those described in the first and second
solutions. This solution can be applied for any other Culex spp
which has a highly similar sequence of its RRM in comparison with
the RMM3 or after obtaining its own Tra-2 RRM sequences by a pair
of primers to be mentioned in Examples.
[0027] The connective intron or linker between two Tra-2 RRM
inverted repeats can be any eukaryote intron sequence. However,
introns from the respective species are preferred. The length of
connective linker or intron can be from few to few hundred
nucleotides. We prefer that two nucleotides of GT and AG need to be
inserted at the beginning and at the end of intron or linker,
respectively. These are the strengthening signals for spliceosomes
to recognize and to splice out the connective introns or
linkers.
[0028] Three DNA Tra-2 RNAi RRM-1, Tra-2 RNAi RRM-2, Tra-2 RNAi
RRM-3 kernel sequences are disclosed here as examples of using
specifically Tra-2 DNA sequences to produce an interference effect
into the respective species. The transcription of the DNA kernel
structure containing two identically inverted repeats would produce
single strands of mRNAs, which exposes two complementary sequences
at its two ends. The complementary sequences would bind together
forming a double strand hairpin mRNA structure (dsRNAi), in which
the looping part is formed by the intron or linker. Mosquito cells
recognize the abnormal structure and react by dicing the dsRNA
interference in a defend mechanism activity which is followed by
destroying intact single strand Tra-2 mRNAs from itself (Wang et
al., 2006. Cell 127, 803-815; Hammond et al., 2001. Nat Rev Genet.
2(2):110-9). The Tra-2 gene is, therefore, knocked down.
Illustrations and its Explanations.
[0029] The RNAI kernel seqence 1
TABLE-US-00001 Tra-2 RRM RNAi-1 Forward sequence Connection Reverse
sequence 5'AGTAAGTGCCTCGGTGTGTTCGGCCTAA
GCAGCTACACCAACGAAACCAGCCTGATGG ACGTTTTCGCACCGTACGGAACCATTGACA
AGGCGATGATTGTCTACGATGCCAAGACGA AGGTTTCCCG GGGTTCGGATTCGTGTACT
TCCAGGAGCAGAGTGCGGCCACCGAAGCCA AAATGCAGTG AATGG ATGATGCTGCATG
AGCGCACGATTAGAGTGGATTATTCGGTGA CC-3' (SEQ ID NO: 1) GT-Intron-AG
(or linker) 5'GGTCACCGAATAATCCACTCTAATC GTGCGCTCATGCAGCATCAT CCATT
CACTGCATTTTGGCTTCGGTGGCCGCA CTCTGCTCCTGGAAGTACACGAATCCG AACCC
CGGGAAACCTTCGTCTTGGCA TCGTAGACAATCATCGCCTTGTCAATG
GTTCCGTACGGTGCGAAAACGTCCATC AGGCTGGTTTCGTTGGTGTAGCTGCTT
AGGCCGAACACACCGAGGCACTTACT-3' (SEQ ID NO: 4) The respective amino
acid sequence 1
-S--K--C--L--G--V--F--G--L--S--S--Y--T--N--E--T--S--L--M--D- 21
-V--F--A--P--Y--G--T--I--D--K--A--M--I--V--Y--D--A--K--T--K- 41
-V--S--R--G--F--G--F--V--Y--F--Q--E--Q--S--A--A--T--E--A--K- 61
-M--Q--C--N--G--M--M--L--H--E--R--T--I--R--V--D--Y--S--V--T- (SEQ
ID NO: 5) Note: 3' end of the forward sequence is connected with an
intron or linker sequence by GT. 5' end of reverse sequence is
connected with the intron or linker sequence by AG. The bold
italicized characters indicate substitutions. Y, S, N, K etc show
different possibilities of changed nucleotides at the same
position. The substitutions may or may not change amino acid code.
The RNAi kernel sequence 2.
TABLE-US-00002 Tra-2 RRM RNAi-2 Forward sequence Connection Reverse
sequence 5'AGTAAGTGCCTCGGTGTGTTCGGCCT AG AGCTA ACCA CGAA CCA CCTGAT
GGA GT TTC C CCGT CGG ACCAT G ACAAGGC ATGATTGTCTACGATGCCAAG ACGAAGG
TCCCG GGGTT GG TTCGT GTA TTCCAGGAGCAGAGT CGGCCAC G AGCCAAA TGCAGTG
AA GGAATG CTGCA GAGCG ACGATTAGAGTGGATTA TTCGGTGACC-3' (SEQ ID NO:
2) GT-Intron- AG (or linker) 5'GGTCACCGAATAATCCACTCTAATCGT CGCTC
TGCAG CATTCC TT CACTGCA TTTGGC T C GTGGCCG ACTCTGCTCCTGGAA TACACGAA
CC AACCC CGGGA CCTTCGTCTTGGCATCGTA GACAATCAT GCCTTGTC ATGGT CCG
ACGG G GAA AC TCCATCAGG TGG TTCG TGGT TAG CT CT
AGGCCGAACACACCGAGGCACTTACT-3' (SEQ ID NO: 6) The respective amino
acid sequence 1 -S--K--C--L--G--V--F--G--L--S--S--Y--T-- --E--T--
--L--M--D- 21 -V--F--S--P--
--G--T--I--D--K--A--M--I--V--Y--D--A--K--T--K- 41
-A--S--R--G--F--G--F--V--Y--F--Q--E--Q--S-- --A--T--E--A--K- 61 -
--Q--C--N--G--M-- --L--H--E--R--T--I--R--V--D--Y--S--V--T- (SEQ ID
NO: 7) Note: 3' end of the forward sequence is connected with an
intron or linker sequence by GT. 5' end of reverse sequence is
connected with the intron or linker sequence by AG. The bold
italicized characters indicate substitutions. Y, S, N, K etc show
different possibilities of changed nucleotides at the same
position. The substitutions may or may not change amino acid
code.
TABLE-US-00003 Tra-2RRMRNAi-3 Forward sequence Connection Reverse
sequence 5'CGTAACGGAATAGTCCACCCGGATG GTTCGCTCGTGCATTACCATTCCGTTG
CACTGCACCTTGGCTGCGGAAGCGTCC TCCAGGTTGACAAAGTACACGAATCCG
AACCCGCGGGACGCCTTCGTCTTGGCA TCGTACACGATCTGCACCTTCTCGATC
AATCCGAACCGGCCAAACACGGTCCTC AGGTCCGCCTCCTGGGTGTAATTGCTG
AGGCCAAACACGCCGAGGCAGGTCGA-3' (SEQ ID NO: 3) GT-Intron-AG (or
linker) 5'TCGACCTGCCTCGGCGTGTTTGGCCTC AGCAATTACACCCAGGAGGCGGACCTGAG
GACCGTGTTTGGCCGGTTCGGATTGATCG AGAAGGTGCAGATCGTGTACGATGCCAAG
ACGAAGGCGTCCCGCGGGTTCGGATTCGT GTACTTTGTCAACCTGGAGGACGCTTCCG
CAGCCAAGGTGCAGTGCAACGGAATGGTA ATGCACGAGCGAACCATCCGGGTGGACTA
TTCCGTTACG-3' (SEQ ID NO: 8) The respective amino acid sequence 1
-S--T--C--L--G--V--F--G--L--S--N--Y--T--Q--E--A--D--L--R--T- 21
-V--F--G--R--F--G--L--I--E--K--V--Q--I--V--Y--D--A--K--T--K- 41
-A--S--R--G--F--G--F--V--Y--F--V--N--L--E--D--A--S--A--A--K- 61
-V--Q--C--N--G--M--V--M--H--E--R--T--I--R--V--D--Y--S--V--T- (SEQ
ID NO: 9) Note: 3' end of the forward sequence is connected with an
intron or linker sequence by GT. 5' end of reverse sequence is
connected with the intron or linker sequence by AG. The bold
italicized characters indicate substitutions. Y, S, N, K etc show
different possibilities of changed nucleotides at the same
position. The substitutions may or may not change amino acid
code.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The Tra-2 RNAi system in the present invention may be any
part of Tra-2 encoding sequences (mRNA) of Tra-2 genes originated
from Aedes albopictus, Aedes polynesiensis, Culex quinquefasciatus
or the other Culicinae mosquitoes which are capable of producing a
knockdown (interference) effect to the Tra-2 gene of the respective
species. We not rule out the possibilities that a Tra-2 RNAi system
containing Tra-2 recombinant sequences from a certain Culicinae
mosquito species can also cause a knockdown (interference) effect
to the other closely related mosquito species within Culicinae.
Definitions:
[0031] Culicinae mosquitoes refer to mosquito species which have a
pair of chromosome (chromosome I) that are similar in size but are
distinguishable in many species by the presence in the X (m) or
absence in the Y(M) of C-banding intercalary heterochromatin
(Knudson et al., 1996. 175-214. The Biology of Disease vectors.
University Press of Colorado). [0032] Tra-2 gene sequences from
Culicinae mosquitoes refer to mRNA coding sequences only (Latchman,
1998. Gene regulation. A eukaryotic perspective. 3.sup.rd edition.
Stanley Thornes Publishers). [0033] The RNAi kernel sequences refer
to any recombinant DNA sequence which includes two inverted repeats
(IR) in conjunction by a linker or intron sequence. Sequence of the
IR is derived from any part of Culicinae Tra-2 mRNA sequences.
[0034] We definite that Tra-2 RNAi kernel sequences is an RNAi
encoding sequence, its expression is under the control of a
repressible transactivator protein system.
[0035] As mentioned above, we look for an existence of the Tra-2
genes in Aedes albopictus, Aedes polynesiensis and Culex
quinquefasciatus mosquitoes which may contain a highly conserved
region with a length of 240 bp (80 amino acids). This region has
been identified in Drosophila, Ceratitis Capitata as Tra-2 RRM
specific region (RNA recognition motif)
http://www.expasy.orq/cqi-bin/prosite-search-ac?PDOC00030.
[0036] Using a sequence from 1221182 to 1220943 of Aedes aegypti
Tra-2 gene (GenBank accession number: AAEL004293-RA), two primers
have been designed. A forward is CLF, 26 bp at beginning of the RRM
region (AGTAAGTGCCTCGGTGTGTTCGGCCT) (SEQ ID NO:10) and a reverse is
CLR, 23 bp at the end of the RRM region (CCGGTCACCGAATAATCCACTCAA)
(SEQ ID NO:11). PCR products amplified on DNA templates from Aedes
albopictus and Aedes polynesiensis were sequenced. It revealed that
in each species there are two different RRM sequences, RRM1 and
RRM2. RT-PCR has shown expression from both of those RRMs. RRM1 is
identical with the Tra-2 RRM in Aedes aegypti (AAEL004293-RA) but
RRM2 has 10% amino acid differences. These pair of primers can be
used to amplify ortholog Tra-2 RRM 240 bp regions from the other
Aedes spp, even the distance species as Aedes niveus, Aedes
annandalei or Aedes pseudoalbopictus. The amplification condition
is similar.
[0037] A Tra-2 sequence of Culex quinquefasciatus is available from
Genbank (GenBank accession number: CPIJ016646) when using the RRM1
and RRM2 sequence as inputs to blast. Its RRM sequence belongs to
the supercontig 3.780, from 5008 to 5247. We named it as RRM3. 24
bp at beginning and 22 bp at the end of RRM3 are used to create a
pair of primers to amplify it from Culex quinquefasciatus DNA.
These pair of primers can be used to amplify ortholog Tra-2 RRM 240
bp regions of the other Culex spp, even the distance species as
Culex visnue, Culex pipiens. The amplification condition is
similar.
[0038] For convenience in designing primers whole or a part of the
RRM regions (RRM1, RRM2 and RRM3) can be used to assemble the Tra-2
RNAi constructs by DNA recombinant techniques. However, in this
invention, it doesn't limit to use other Tra-2 encoding parts
outside the RRM region of these mosquitoes to build other Tra-2
RNAi constructs.
[0039] The elements that regulate the RNAi kernel sequences should
be located on the same chromosome as the RNAi kernel sequences. In
FIG. 1 shows the RNAi kernel sequences and Tetracycline (Tet)
transactivator system. An insect spermatogenesis promoter, for
instance Drosophila .beta.2, controls the tTA protein gene. In the
presence of Tet in larva rearing medium, tTA protein binds to Tet
and the operator sequence (tetO7) would bind to the minimal
promoter which regulates the transcription of the kernel structure.
The promoter is inactivated and no RNAi product is transcribed. In
the absence of Tet, this artificial protein binds to operator
tetOx7 in the absence of Tetracycline (Tet) and the minimal
promoter would be free to transcribe the RNAi strand. In the same
plasmid, a reporter gene as ECFP, Dsred2 or EGFP can be ligated to
a 3xP3 or Actin5C promoter. We can trace the plasmid by following
this fluorescent marker. The entire packet is ligated into a
PiggyBac plasmid. This complex can be transformed into mosquito
genetic background in one or more loci which can be in the same or
different chromosomes.
[0040] We suggest that a single locus of the transgene in a
transgenic line can be used as a background for another
transformation. A second transformant locus which occurs in the
same chromosome with the first one, would be particularly
preferred. Transformants occurred in the same chromosome would
prevent them to be segregated in the next generations and
especially in the case where the two Tra-2 loci (or allele) exist
in same species as Aedes albopictus and Aedes polynesiensis, in
which two respective RNAi transformants are necessary to repress
two loci (or allele).
[0041] The expression of the RNAi kernel sequences would knock down
the Tra-2 gene in the transgenic species. The knockdown effect
results in lethal X chromosome bearing sperms and only male
offspring is outcome.
Examples
[0042] Components: 1/RRM Tra-2 sequences: In this examples, we used
three types of RRMs (RRM1, RRM2 and RRM3) to create Tra-2 RNAi
kernel constructs. These sequences are obtained from sequencing the
target species or blasting from (http://www.vectorbase.orq/). It
doesn't limit to use a different part of the Tra-2 gene sequences
which are belonging to Aedes albopictus, Aedes polynesiensis, Culex
quinquefasciatus or the other Culicinae moquitoes in the invention.
All the other components of plasmids are identical. 2/Drosophila
.beta.2 tubulin promoter (or other insect spermatogenesis
promoter): PCR from Drosophila DNA. 3/Transactivator component
(tTA): Clontech. 4/Regulator element (tetOx7): Clontech. 5/Reporter
gene: http://piggybac.bio.nd.edu/.6/Piggybac
plasmids:http://piagybac.bio.nd.edu/.7/Helper
plasmid:http://piqqybac.bio.nd.edu/.
I. RRMs from Aedes Albopictus and Aedes Polynesiensis.
[0043] These examples show how the RRM sequences of Tra-2 were
identified from Aedes albopictus and Aedes polynesiensis. It also
shows the way to create the RNAi kernel sequence by using the RRM
sequences from Aedes albopictus and Aedes polynesiensis.
[0044] As mentioned above, RRM regions of Aedes albopictus and
Aedes polynesiensis have been amplified by a PCR used a pair of
primers of 26 bp at beginning and 23 bp at the ending of Aedes
aegypti supercontig 1.113 (1221182-1220943).
TABLE-US-00004 (SEQ ID NO: 10) CLF AGTAAGTGCCTCGGTGTGTTCGGCCT (SEQ
ID NO: 11) CLR CCGGTCACCGAATAATCCACTCAA
DNA from Aedes albopictus and Aedes polynesiensis are extracted
using a QIAGEN kit. PCR is carried out in 25 .mu.l reaction in a
condition of 2.5 .mu.l PCR buffer; 1.5 .mu.l MgCL (25 mM); 0.5
.mu.l dNTPs (10 mM); each primer 0.5 .mu.l (10 pmol/.mu.l); 0.15
.mu.l Taq DNA polymerase (5U/.mu.l); 10-40 ng DNA template. Thermal
profile of PCR is [94oC/4; (94oC/30''; 59oC/30'';
72oC/45'').times.3; (94oC/30''; 57oC/30''; 72oC/45'').times.3;
(94oC/30''; 54oC/30''; 7200/45'').times.35; 72oC/10]. PCR products
are then purified and sequenced with the same primers. Two 240 bp
sequences of RRMs are obtained below.
TABLE-US-00005 RRM1 DNA sequence (SEQ ID NO: 1)
5'AGTAAGTGCCTCGGTGTGTTCGGCCTAAGCAGCTACACCAACGAAACCAGCCTGATGGACGTTTTCGCACCG-
TACG GAACCATTGACAAGGCGATGATTGTCTACGATGCCAAGACGAAGGTTTCCCG
GGGTTCGGATTCGTGTACTTCCAGG AGCAGAGTGCGGCCACCGAAGCCAAAATGCAGTG AATGG
ATGATGCTGCATGAGCGCACGATTAGAGTGGATTATT CGGTGACC-3'. Underlined
regions disclosed as SEQ ID NOS 10, 12 and 13, respectively, in
order of appearance. RRM2 DNA sequence (SEQ ID NO: 2)
5'AGTAAGTGCCTCGGTGTGTTCGGCCT AG AGCTA ACCA CGAA CCA CCTGATGGA GT
TTC C CCGT CGG ACCAT GACAAGGC ATGATTGTCTACGATGCCAAGACGAAGG TCCCG
GGGTT GG TTCGTGTA T TCCAGGAGCAGAGT CGGCCAC GA GCCAAA TGCAGTG AA
GGAATG CTGCA GAGCG ACGATTAGAG TGGATTATTCGGTGACC-3' Underlined
regions disclosed as SEQ ID NOS 10, 14 and 13, respectively, in
order of appearance. RRM1 amino acid sequence (SEQ ID NO: 5) 1
-S--K--C--L--G--V--F--G--L--S--S--Y--T--N--E--T--S--L--M--D- 21
-V--F--A--P--Y--G--T--I--D--K--A--M--I--V--Y--D--A--K--T--K- 41
-V--S--R--G--F--G--F--V--Y--F--Q--E--Q--S--A--A--T--E--A--K- 61
-M--Q--C--N--G--M--M--L--H--E--R--T--I--R--V--D--Y--S--V--T- RRM2
amino acid sequence (SEQ ID NO: 7) 1
-S--K--C--L--G--V--F--G--L--S--S--Y--T-- --E--T-- --L--M--D- 21
-V--F-- --P-- --G--T--I--D--K--A--M--I--V--Y--D--A--K--T--K- 41 -
--S--R--G--F--G--F--V--Y--F--Q--E--Q--S-- --A--T--E--A--K- 61 -
--Q--C--N--G--M-- --L--H--E--R--T--I--R--V--D--Y--S--V--T-
(Underlines indicate the region selected for primers. Bold italics
indicate nucleotide and amino acid substitutions).
Beside, these pair of primers can be used to amplify this Tra-2 RRM
240 bp region of the other Aedes spp, even from the distance
species as Aedes niveus, Aedes annandalei or Aedes
pseudoalbopictus. Using the same PCR condition, an exact 240 bp
band would be amplified among other bands. An agarose gel
extraction step is performed for the 240 bp band by Qiagen columns.
The DNA elution is diluted between 10-20 times in water and 1 .mu.l
to be used as template for the same PCR. A 240 bp specific band
would be amplified and can be used to assemble Tra-2 RNAi
constructs for the respective species.
[0045] Two fragments of 135 bp from the bottom parts of these RRM1
and RRM2 regions are used to assemble Tra-2 RNAi constructs.
Because the sequences of RRM1 and RRM2 are only different in some
parts, therefore the primers derived outside of those parts can be
used for amplifying both RRMs. PCR is carried out in 25 .mu.l
reaction in a condition of 2.5 .mu.l PCR buffer; 1.5 .mu.l MgCL (25
mM); 0.5 .mu.l dNTPs (10 mM); each primer 0.5 .mu.l (10
pmol/.mu.l); 0.15 .mu.l Taq DNA polymerase (5U/.mu.l); 10-40 ng DNA
template. Thermal profile of PCR is [94oC/4; (94oC/30''; 59oC/30'';
72oC/45'').times.3; (94oC/30''; 57oC/30''; 72oC/45'').times.3;
(94oC/30''; 54oC/30''; 72oC/45'').times.35; 72oC/10']
TABLE-US-00006 1-(BA-EX1F) (SEQ ID NO: 15)
5'CGATCTCGGATCCATGCCAAGACGAAGGTTTCCCGAG 3' 2-(X-Ex1R) (SEQ ID NO:
16) 5'CGGCAATGACCTCGAGACCGGTCACCGAATAATCCACTCAA 3' 3-(SAL-EX1F)
(SEQ ID NO: 17) 5'GGCGTCAATGTCGACATGCCAAGACGAAGGTTTCCCGAG 3'
4-(ECORI-EX1R) (SEQ ID NO: 18)
5'CGGACGTTGGAATTCGACGGTCACCGAATAATCCACTCAA 3'
[0046] Primers 1 & 3 or 2 & 4 are similar forward and
reverse primers. A combination between 1 & 2 would produce the
same PCR product as that of 3 & 4. The differences in those PCR
products are endonuclease restriction enzyme sequences inserted in
the front parts of the primers (underline). This allows the PCR
products can be ligated to an intron or linker that contains the
same restriction sites in a desired direction. If a connection
between the two inverted repeats is a linker about 10 bp, PCRs to
amplify these fragments can use the same reverse primer (2 or 4)
and therefore products would contain the same restriction sites at
3' end (XhoI or EcoRI). Two PCR products would be easily inversely
connected after an XhoI or EcoRI enzyme treatments. However, if we
want to insert an intron between the two inverted repeats, it needs
to use both inverse primers. Two PCR products would then have
different sticky ends at 3' (XhoI and EcoRI) and can be easily
ligated with an intron that ends by XhoI and EcoRI restriction
sites. In this invention, any linker or intron sequence from other
insects can be used in conjunction with the two inverted repeats,
provided that two nucleotides GT and AG would be inserted at the
first and the end of those sequences, respectively. These are
recognition signals for intron splicing sites.
[0047] After two identical DNA fragments are reversely connected
via an intron or linker, these RNAi kernel sequences (1 & 2)
can be easily ligated into the transactivator system in a desired
direction provided that the transactivator plasmids contain the
same restriction sites.
II. RRM from Culex Quinquefasciatus
[0048] Genomic sequences of Culex quinquesfaciatus are available in
the Vectorbase.org website (http://www.vectorbase.org/). We used
two RRMs (RRM1 and RRM2) as queries to blast against the database.
Outcome is a 240 bp sequence which is highly similar with RRM1 and
RRM2 in its helix structure as well as phylogenic relationship.
RRM3 contains up to 69% and 73% amino acid similarity with RRM1 and
RRM2, respectively. The annotation of Tra-2 Culex quinquesfaciatus
is CPIJ016646; supercont3.780:5008-5247.(RRM3)
TABLE-US-00007 RRM3 DNA sequence (SEQ ID NO: 3)
CGTAACGGAATAGTCCACCCGGATGGTTCGCTCGTGCATTACCATTCCGTTGCACTGCACCTTGGCTG
CGGAAGCGTCCTCCAGGTTGACAAAGTACACGAATCCGAACCCGCGGGACGCCTTCGTCTTGGCATCG
TACACGATCTGCACCTTCTCGATCAATCCGAACCGGCCAAACACGGTCCTCAGGTCCGCCTCCTGGGT
GTAATTGCTGAGGCCAAACACGCCGAGGCAGGTCGA. Underlined regions disclosed
as SEQ ID NOS 19 and 20, respectively, in order of appearance. RRM3
amino acid sequence (SEQ ID NO: 9) 1
-T--C--L--G--V--F--G--L--S--N--Y--T--Q--E--A--D--L--R--T- 21
-V--F--G--R--F--G--L--I--E--K--V--Q--I--V--Y--D--A--K--T--K- 41
-A--S--R--G--F--G--F--V--Y--F--V--N--L--E--D--A--S--A--A--K- 61
-V--Q--C--N--G--M--V--M--H--E--R--T--I--R--V--D--Y--S--V--T-
(Underlines indicate the region selected for primers).
[0049] In Culex quinquesfaciatus whole RRM3 sequence can be used to
create an RNAi kernel sequence as its nucleotide sequences at
beginning and at the end are suitable to design good primers. 24 bp
at beginning and 22 bp at the end of RRM3 (underline) are used to
create a pair of primers. These pair of primers can be used to
amplify this Tra-2 RRM 240 bp region of the other Culex spp, even
the distance species as Culex vishnue, Culex pipiens or Culex
tritaeniorhynchus by a similar condition. Using the same KR
condition, an exact 240 bp band would be amplified among other
bands. A gel extraction step is performed for the 240 bp band by
Qiagen columns. The DNA elution is diluted between 10-20 times in
water and 1 .mu.l to be used as template for the same PCR. A 240 bp
specific band would be amplified and can be used to assemble Tra-2
RNAi constructs for the respective species.
TABLE-US-00008 7-(BA-EX1F) (SEQ ID NO: 21)
5'CGATCTCGGATCCCGTAACGGAATAGTCCACCCGGAT 3' 8-(X-Ex1R) (SEQ ID NO:
22) 5'CGGCAATGACCTCGAGACTCGACCTGCCTCGGCGTGTTTG 3' 9-(SAL-EX1F) (SEQ
ID NO: 23) 5'GGCGTCAATGTCGACCGTAACGGAATAGTCCACCCGGAT 3'
10-(ECORI-EX1R) (SEQ ID NO: 24)
5'CGGACGTTGGAATTCGATCGACCTGCCTCGGCGTGTTTG 3'
[0050] PCR is carried out in 25 .mu.l reaction in a condition of
2.5 .mu.l PCR buffer; 1.5 .mu.l MgCL (25 mM); 0.5 .mu.l dNTPs (10
mM); each primer 0.5 .mu.l (10 pmol/.mu.l); 0.15 .mu.l Taq DNA
polymerase (5U/.mu.l); 10-40 ng DNA template. Thermal profile of
PCR is [94oC/4; (94oC/30''; 59oC/30''; 7200/45'').times.3;
(94oC/30''; 57oC/30''; 72oC/45'').times.3; (94oC/30''; 54oC/30'';
7200/45'').times.35; 72oC/10']. Afterward, these PCR products are
also performed in the same manner with those have been done in
Aedes albopictus and Aedes polynesiensis. Whatever, these fragments
are connected by a linker or intron, after this RNAi kernel
sequence (3) is constructed, it would be available to ligate into
the transactivator plasmids to transform Culex quinquesfaciatus
embryos.
III. Connection of the RNAi Kernel Structures with Tre
Repressor.
[0051] The pTre-tight plasmid (Cat. No. 631059) from Clontech is
mixed with the RNAi kernel sequence (1 or 2 or 3) in 1:3 molar
ratio in a 30 .mu.l reaction in the presence of BamHI and SaII
restriction enzymes. After digestion, ligation is performed by
adding T4 ligation into the denatured restriction enzyme mixture.
The circle plasmid is transformed into competent cells
(DH5.alpha..TM. derivative, New England Biolabs), isolated and
cultured overnight to harvest a larger amount of plasmid DNA from
each clone. The size of new plasmid would be 2.6 kb plus the size
of the RNAi kernel sequences. In the case of RRM1 and RRM2 from
Aedes albopictus and Aedes polynesiensis, only 135 bp of each RRM
are used, the plasmid size would be about 3070 bp if using an
intron of 200 bp. If a linker of 10 bp is used, the plasmid is
about 2870 bp. In the case of Culex quinquesfaciatus, whole 240 bp
is used, if it is accompanied with 200 bp intron, the fragment size
would be 3280 bp. If a linker of 10 bp is used, the plasmid is
about 3090 bp.
[0052] A fragment includes the Tre operator and the RNAi kernel
sequence (tetOx7+PminCMV+RNAi kernel sequence +SV40 polyA) can now
be amplified by two primers which contain HindIII and Acc65I
restriction sites. These pending sites are available for ligation
with Piggybac plasmid and the other parts of the construct.
TABLE-US-00009 (Tre-HindIII) (SEQ ID NO: 25)
CGATCTAAGCTTCTCGAGTTTACTCCCTATCAGTGA (Tre-Acc65I) (SEQ ID NO: 26)
CGATCTGGTACCAGTCAGTGAGCGAGGAAGCTCGAG
[0053] PCR is carried out in 25 .mu.l reaction in a condition of
2.5 .mu.l PCR buffer; 1.5 .mu.l MgCL (25 mM); 0.5 .mu.l dNTPs (10
mM); each primer 0.5 .mu.l (10 pmol/.mu.l); 0.15 .mu.l Taq DNA
polymerase (5 U/.mu.l); 10-40 ng DNA template. Thermal profile of
PCR is [94oC/4; (94oC/90''; 54oC/60''; 72oC/2 min 30'').times.35;
72oC/10]. The PCR products amplified from the RRMs of Aedes
albopictus and Aedes polynesiensis with a 10 bp linker would be 875
bp, meanwhile a 200 bp intron would produced 1065 bp products. The
PCR products amplified from the RRM of Culex quinquesfaciatus would
be 1085 bp and 1275 bp, which are respectively to a 10 bp linker or
200 bp intron. The PCR products are digested by Acc65I and HindIII
and purified by Qiagen columns. The product is available for a
final ligation.
IV. Connection of the Drosophila .beta.2 Tubulin Promoter with a
Transactivator Sequence.
[0054] Drosophila .beta.2 tubulin promoter sequence is obtained
from GenBank or http://flybase.org/reports/FBqn0003889.html. Two
primers which contain EcoRI and Apa I are designed from the
sequence. These primers amplify 230 bp of 5'UTR of .beta.2 tubulin
gene from Drosophila genomic DNA. Thermal profile of PCR is
[94oC/4; (94oC/30''; 55oC/30''; 72oC/45'').times.35; 72oC/10].
TABLE-US-00010 .beta.2-ApaI-F (SEQ ID NO: 27)
CGATCTGGGCCCGGAAATCGTAGTAGCCTATTTGTGA .beta.2-EcoRI-R (SEQ ID NO:
28) CGGACGTTGGAATTCCCTGAATGTGTACAATTTCACGCAT
[0055] The pTet-Off-Advanced plasmid (Clontech, Catalog Nos.
630934) is digested by two restriction enzymes EcoRI and HindIII
producing a band of 1222 bp. This DNA fragment is then ligated to
the .beta.2 tubulin promoter sequence via the EcoRI restriction
site to produce a fragment of 1458 bp. tTA protein is now
controlled by .beta.2 tubulin promoter. The ligation product is
digested by ApaI and purified by Qiagen columns. The product is
available for a final ligation.
V. Whole Plasmid Assembles.
[0056] pXL-BacII-ECFP plasmid from http://piggybac.bio.nd.edu/ is
used to assemble all the above fragments into completed Tra-2 RNAi
constructs. The pXL-BacII-ECFP plasmid carries a 3xP3 promoter
which drives ECEP reporter gene. This reporter gene would be tissue
specific expressed under the promoter. When mosquitoes are
transformed with this marker, mosquito eyes would be fluorescently
cyan color.
[0057] The pXL-BacII-ECFP plasmid is digested by ApaI and Acc65I
and purified by Qiagen columns. The linear plasmid is 5390 bp. The
plamid is then mixed with Tre fragments (III), .beta.2 +tTA
fragment (IV) in 1:3:3 molar ratio. T4 ligation is added into a 30
.mu.l reaction.
[0058] Ligation product is used to transform into competent cells.
Ligation products are expected in a range of different sizes as
follow:
[0059] For Aedes albopictus and Aedes polynesiensis, two plasmid
containing 10 bp linker or 200 bp intron are 7723 bp and 7913 bp,
respectively. Meanwhile, plasmids of Culex quinquesfaciatus would
be 7933 bp and 8123 bp for 10 bp linker and 200 bp intron,
respectively.
VI. Plasmid Injection and Transformant Selection.
[0060] The Tra-2 RNAi plasmids is mixed with a pBSII-1E1-orf
(http://piggybac.bio.nd.edu/) helper plasmid. The helper produces
transposase enzyme which helps Piggybac in the Tra-2 RNAi plasmids
jumping into mosquito genome. A good concentration of the injection
mixture would be 600 ng of the Tra-2 RNAi plasmid plus 400 ng of
the helper per micro liter (.mu.l) of 1.times. phosphate buffer.
Mosquito eggs would be injected within 2 hrs after oviposition into
egg posterior ends. After 4 days post injection, the eggs are
submerged into tetracycline solution (0.008 g per litter). Go
survivors would be kept to cross with wild type males or females.
G1 larvae are screened under a stereo fluorescent microscope. Any
fluorescent larva found that would be the transformant one and to
be crossed to build transformant lines. These lines would be tested
in Tet-on and Tet-off conditions to check sex ratio. Any line
having maleness skew over 80% in Te-off condition would be kept for
further analysis and for vector control applications.
Invention Effects
[0061] The method exposed in this invention would help to produce
one sex (maleness) in Culicinae mosquitoes. Males created by this
invention would pass on the Tra-2 RNAi genetic system into natural
population when being released. If the number of released males is
big enough, it can result in a collapse of natural vector
population, even gone extinct of whole population in a certain
time.
Sequence CWU 1
1
281240DNAAedes sp.modified_base(129)..(129)a, c, t, g, unknown or
othermodified_base(195)..(195)a, c, t, g, unknown or other
1agtaagtgcc tcggtgtgtt cggcctaagc agctacacca acgaaaccag cctgatggac
60gttttcgcac cgtacggaac cattgacaag gcgatgattg tctacgatgc caagacgaag
120gtttcccgng ggttcggatt cgtgtacttc caggagcaga gtgcggccac
cgaagccaaa 180atgcagtgya atggnatgat gctgcatgag cgcacgatta
gagtggatta ttcggtgacc 2402240DNAAedes sp.modified_base(27)..(27)a,
c, t, g, unknown or othermodified_base(63)..(63)a, c, t, g, unknown
or othermodified_base(69)..(69)a, c, t, g, unknown or
othermodified_base(78)..(78)a, c, t, g, unknown or
othermodified_base(93)..(93)a, c, t, g, unknown or
othermodified_base(123)..(123)a, c, t, g, unknown or
othermodified_base(129)..(129)a, c, t, g, unknown or
othermodified_base(138)..(138)a, c, t, g, unknown or
othermodified_base(171)..(171)a, c, t, g, unknown or
othermodified_base(213)..(213)a, c, t, g, unknown or other
2agtaagtgcc tcggtgtgtt cggcctnagy agctayacca mcgaarccar cctgatggay
60gtnttckcnc cgtwcggnac cathgacaag gcnatgattg tctacgatgc caagacgaag
120gyntcccgng ggttyggntt cgtgtayttc caggagcaga gtkcggccac
ngargccaaa 180mtgcagtgya ayggaatgrw rctgcaygag cgnacgatta
gagtggatta ttcggtgacc 2403240DNACulex quinquefasciatus 3cgtaacggaa
tagtccaccc ggatggttcg ctcgtgcatt accattccgt tgcactgcac 60cttggctgcg
gaagcgtcct ccaggttgac aaagtacacg aatccgaacc cgcgggacgc
120cttcgtcttg gcatcgtaca cgatctgcac cttctcgatc aatccgaacc
ggccaaacac 180ggtcctcagg tccgcctcct gggtgtaatt gctgaggcca
aacacgccga ggcaggtcga 2404240DNAAedes sp.modified_base(46)..(46)a,
c, t, g, unknown or othermodified_base(112)..(112)a, c, t, g,
unknown or other 4ggtcaccgaa taatccactc taatcgtgcg ctcatgcagc
atcatnccat tycactgcat 60tttggcttcg gtggccgcac tctgctcctg gaagtacacg
aatccgaacc cncgggaaac 120cttcgtcttg gcatcgtaga caatcatcgc
cttgtcaatg gttccgtacg gtgcgaaaac 180gtccatcagg ctggtttcgt
tggtgtagct gcttaggccg aacacaccga ggcacttact 240580PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Ser Lys Cys Leu Gly Val Phe Gly Leu Ser Ser Tyr Thr Asn Glu Thr 1
5 10 15 Ser Leu Met Asp Val Phe Ala Pro Tyr Gly Thr Ile Asp Lys Ala
Met 20 25 30 Ile Val Tyr Asp Ala Lys Thr Lys Val Ser Arg Gly Phe
Gly Phe Val 35 40 45 Tyr Phe Gln Glu Gln Ser Ala Ala Thr Glu Ala
Lys Met Gln Cys Asn 50 55 60 Gly Met Met Leu His Glu Arg Thr Ile
Arg Val Asp Tyr Ser Val Thr 65 70 75 80 6240DNAAedes
sp.modified_base(28)..(28)a, c, t, g, unknown or
othermodified_base(70)..(70)a, c, t, g, unknown or
othermodified_base(103)..(103)a, c, t, g, unknown or
othermodified_base(112)..(112)a, c, t, g, unknown or
othermodified_base(118)..(118)a, c, t, g, unknown or
othermodified_base(148)..(148)a, c, t, g, unknown or
othermodified_base(163)..(163)a, c, t, g, unknown or
othermodified_base(172)..(172)a, c, t, g, unknown or
othermodified_base(178)..(178)a, c, t, g, unknown or
othermodified_base(214)..(214)a, c, t, g, unknown or other
6ggtcaccgaa taatccactc taatcgtncg ctcytgcagr wrcattccyt tycactgcam
60tttggcrtcn gtggccgkac tctgctcctg gaaytacacg aanccyaacc cncggganyc
120cttcgtcttg gcatcgtaga caatcatngc cttgtchatg gtnccgwacg
gngkgaanac 180ytccatcagg rtggrttcgm tggtytagct yctnaggccg
aacacaccga ggcacttact 240780PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 7Ser Lys Cys Leu Gly Val
Phe Gly Leu Ser Ser Tyr Thr Thr Glu Thr 1 5 10 15 Asn Leu Met Asp
Val Phe Ser Pro Phe Gly Thr Ile Asp Lys Ala Met 20 25 30 Ile Val
Tyr Asp Ala Lys Thr Lys Ala Ser Arg Gly Phe Gly Phe Val 35 40 45
Tyr Phe Gln Glu Gln Ser Ser Ala Thr Glu Ala Lys Leu Gln Cys Asn 50
55 60 Gly Met Glu Leu His Glu Arg Thr Ile Arg Val Asp Tyr Ser Val
Thr 65 70 75 80 8240DNACulex quinquefasciatus 8tcgacctgcc
tcggcgtgtt tggcctcagc aattacaccc aggaggcgga cctgaggacc 60gtgtttggcc
ggttcggatt gatcgagaag gtgcagatcg tgtacgatgc caagacgaag
120gcgtcccgcg ggttcggatt cgtgtacttt gtcaacctgg aggacgcttc
cgcagccaag 180gtgcagtgca acggaatggt aatgcacgag cgaaccatcc
gggtggacta ttccgttacg 240980PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 9Ser Thr Cys Leu Gly Val
Phe Gly Leu Ser Asn Tyr Thr Gln Glu Ala 1 5 10 15 Asp Leu Arg Thr
Val Phe Gly Arg Phe Gly Leu Ile Glu Lys Val Gln 20 25 30 Ile Val
Tyr Asp Ala Lys Thr Lys Ala Ser Arg Gly Phe Gly Phe Val 35 40 45
Tyr Phe Val Asn Leu Glu Asp Ala Ser Ala Ala Lys Val Gln Cys Asn 50
55 60 Gly Met Val Met His Glu Arg Thr Ile Arg Val Asp Tyr Ser Val
Thr 65 70 75 80 1026DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 10agtaagtgcc tcggtgtgtt cggcct
261124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11ccggtcaccg aataatccac tcaa 241224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(23)..(23)a, c, t, g, unknown or other
12atgccaagac gaaggtttcc cgng 241323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13ttagagtgga ttattcggtg acc 231423DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(17)..(17)a, c, t, g, unknown or
othermodified_base(23)..(23)a, c, t, g, unknown or other
14atgccaagac gaaggyntcc cgn 231537DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 15cgatctcgga tccatgccaa
gacgaaggtt tcccgag 371641DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 16cggcaatgac ctcgagaccg
gtcaccgaat aatccactca a 411739DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 17ggcgtcaatg tcgacatgcc
aagacgaagg tttcccgag 391840DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 18cggacgttgg aattcgacgg
tcaccgaata atccactcaa 401924DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 19cgtaacggaa
tagtccaccc ggat 242022DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 20caaacacgcc
gaggcaggtc ga 222137DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 21cgatctcgga tcccgtaacg gaatagtcca
cccggat 372240DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 22cggcaatgac ctcgagactc gacctgcctc
ggcgtgtttg 402339DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 23ggcgtcaatg tcgaccgtaa cggaatagtc
cacccggat 392439DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 24cggacgttgg aattcgatcg acctgcctcg
gcgtgtttg 392536DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 25cgatctaagc ttctcgagtt tactccctat cagtga
362636DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 26cgatctggta ccagtcagtg agcgaggaag ctcgag
362737DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 27cgatctgggc ccggaaatcg tagtagccta tttgtga
372840DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28cggacgttgg aattccctga atgtgtacaa tttcacgcat
40
* * * * *
References