First published online September 23, 2003
Gene vector and transposable element behavior in mosquitoes
David A. O'Brochta1,*,
Nagaraja Sethuraman1,
Raymond Wilson2,
,
Robert H. Hice3,
Alexandra C. Pinkerton3,
,
Cynthia S. Levesque3,
Dennis K. Bideshi3,
Nijole Jasinskiene4,
Craig J. Coates5,
Anthony A. James4,
Michael J. Lehane2 and
Peter W. Atkinson3
1 Center for Biosystems Research, University of Maryland Biotechnology
Institute, College Park, MD 20742-4450, USA,
2 School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57
2UW, UK,
3 Department of Entomology, University of California, Riverside, CA 92521,
USA,
4 Department of Molecular Biology and Biochemistry, University of
California, Irvine, CA 92697, USA
5 Department of Entomology, Texas A&M University, College Station, TX
77843-2475, USA
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Fig. 1. Comparison of Hermes B5, B6 and B6mut elements. The black
arrows represent the inverted terminal repeats (ITRs), and the actual
sequences of the ITRs are shown. The terminal nucleotide of the right ITR is
highlighted in bold to show the difference between the ends of B5 and
B6. The Musca domestica genomic DNA flanking the B5
and B6 elements is different. In the B6mut element, the
terminal nucleotide of B6 was changed to a G.
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Fig. 2. (A) The structure of plasmid Hermes QH7011 used to genetically
transform Aedes aegypti. The plasmid pBSKS contains an autonomous
Hermes element with the Hermes transposase gene under the
control of the hsp70 promoter of Drosophila melanogaster as
well as EGFP (enhanced green fluorescent protein) under the control
of the D. melanogaster actin5C promoter (not drawn to scale). M.
domestica genomic DNA flanking the ends of Hermes are relics of
the original cloning of Hermes and are indicated by boxes. (B)
Structure of Hermes QH7011 in the germ line of A. aegypti as
deduced by Southern blots and PCR analysis of the breakpoints (data not
shown). The entire element has integrated along with the Musca
flanking sequences and the pBSKS vector DNA. Rearrangements towards the ends
of the entire integrated sequence are shown and consist of a partial
duplication of the Musca sequences flanking the right end. In
addition, a rearrangement of pBSKS vector DNA in the form of an inversion
occurred during the integration process (broken line).
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Fig. 3. Summary of the transposable element display method. Genomic DNA is digested
with a restriction enzyme (RE) that results in a junction fragment, including
the terminal sequences of the element and flanking genomic DNA. Specific
adapters are added followed by two rounds of PCR. The first PCR results in the
preliminary amplification of the junction fragment, and the second reaction
further amplifies the fragments of interest using an element-specific primer
labeled with Cy5. Fragments are size fractionated by denaturing acrylamide gel
electrophoresis and visualized in a phosphoimager. Each band represents a
unique junction fragment. Band intensity reflects template abundance. The most
abundant products (darkest bands) are from elements that were inherited
vertically, while lighter bands are elements transposing in the somatic tissue
of the insect, resulting in clones of cells with the element in a new
location. Template abundance of somatic transposition events varies depending
on the point in development when transposition occurs. Samples 1-3 represent
three Drosophila melanogaster individuals with different genotypes
with respect to the location of the autonomous Hermes element
inherited through the germ line (arrowheads).
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Fig. 4. (A) Transposable element display analysis visualizing Hermes
right-end junction fragments in individual Aedes aegypti containing
the autonomous Hermes QH7010 element. c1 and c2 are controls: c1 is a
non-transgenic wild-type mosquito and c2 is a transgenic mosquito containing a
non-autonomous Hermes element also containing the
actin5C:EGFP gene (Pinkerton et
al., 2000 ). Bands in c1 and c2 are considered non-specific PCR
products. Multiple, intensely labeled fragments were observed only from DNA
prepared from the individuals containing the autonomous element. Molecular
size markers, in base pairs, are shown. Bands isolated, reamplified and
sequenced from this experiment are indicated (R2, R7, R10 and R11). (B)
Labeled fragments were excised from transposable element display gels
containing left- and right-hand Hermes ends (left-hand analysis not
shown here), and their sequences were determined. Hermes inverted
terminal repeat (ITR) sequences are indicated by the black arrows, and
flanking A. aegypti sequences are shown with the proposed 8 bp target
site duplications underlined. Only partial flanking sequences are shown, i.e.
those immediately adjacent to the Hermes ITRs.
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Fig. 5. Southern blot analysis of a line derived from a germ-line transposition of
a cn-carrying Mos1 vector. Line 16 was started from one
individual whose parent was from line 128 and injected with a helper plasmid
expressing before blastoderm formation. One of the resulting progeny had an
eye color different from the parental insects and was used to establish line
16. Genomic DNA was isolated from adults and cut with SacI, which
cuts twice within the gene vector, and transferred to a nylon membrane. The
filter was hybridized with a radiolabeled cn+ gene fragment (see
fig. 1 of
Coates et al., 1998 for details
of the analysis). An internal 2.5-kb fragment is present in lines 128 and 16.
Additional hybridizing fragments are diagnostic of independent insertion sites
within the genome. The difference in pattern between 128 and 16 indicates the
presence of a transposition event.
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Fig. 6. (A) Germ-line and somatic transpositions of Mos1 in Aedes
aegypti (based on data reported by
Wilson et al., 2003). Black
arrows represent the terminal sequences at the left end of Mos1. The
sequences of the integration sites and, where it is known, the name of the
locus into which the element integrated are shown. Four of the integration
events were into the original Mos1 vector, and the location of these
transposition events within the vector is indicated by open triangles below
the diagram of the Mos1 vector. Mar L and Mar R refer to the inverted
terminal repeats (ITRs) of the Mos1 vector. Cinnabar and
DsRed are transgenes contained on the vector. Cinnabar was
used as a transformation marker and DsRed was part of an
enhancer-reporter system.
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Fig. 7. Transposable element display analysis of progeny arising from a cross
between individuals heterozygous for a non-autonomous piggyBac
element and a Mos1 vector containing the piggyBac
transposase gene under the regulatory control of the hsp70 promoter
of Drosophila melanogaster and individuals homozygous for
khw. These progeny were selected for analysis
because their eye color phenotype was different from the parental phenotype,
suggesting that a transposition resulting in a position-dependent alteration
in the phenotype had occurred. Progeny from two parental lines are shown (40D,
40L). The piggyBac element inherited through the germ line yields an
intensely labeled PCR product (arrow). P refers to the parental insect.
Numbered lanes contain results from individual progeny. No evidence of
germ-line movement is present. Some of the progeny analyzed were themselves
heterozygous for the non-autonomous element and the transposase-expressing
Mos1 vector (h). In these heterozygotes, there was no evidence for
somatic transposition of the elements. Molecular size markers, in base pairs,
are shown.
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© The Company of Biologists Ltd 2003
