Download High efficiency, site-specific excision of a marker gene by the phage

Nucleic Acids Research, 2003, Vol. 31, No. 22 e147
DOI: 10.1093/nar/gng148
High ef®ciency, site-speci®c excision of a marker
gene by the phage P1 cre±loxP system in the yellow
fever mosquito, Aedes aegypti
Nijole Jasinskiene, Craig J. Coates1, Aurora Ashikyan and Anthony A. James*
Department of Molecular Biology and Biochemistry, University of California, 3205 McGaugh Hall, Irvine,
CA 92697-3900, USA and 1Center for Advanced Invertebrate Molecular Sciences, Department of Entomology,
Texas A&M University, College Station, TX 77843-2475, USA
Received as resubmission September 30, 2003; Accepted October 1, 2003
ABSTRACT
The excision of speci®c DNA sequences from integrated transgenes in insects permits the dissection
in situ of structural elements that may be important
in controlling gene expression. Furthermore,
manipulation of potential control elements in the
context of a single integration site mitigates against
insertion site in¯uences of the surrounding
genome. The cre±loxP site-speci®c recombination
system has been used successfully to remove a
marker gene from transgenic yellow fever mosquitoes, Aedes aegypti. A total of 33.3% of all fertile
families resulting from excision protocols showed
evidence of cre±loxP-mediated site-speci®c excision. Excision frequencies were as high as 99.4%
within individual families. The cre recombinase was
shown to precisely recognize loxP sites in the
mosquito genome and catalyze excision. Similar
experiments with the FLP/FRT site-speci®c recombination system failed to demonstrate excision of
the marker gene from the mosquito chromosomes.
INTRODUCTION
An important feature of a transformation system is its utility in
the analysis of isolated genes and control DNA sequences.
Molecular dissection of the primary structure of control DNA
sequences relies on the ability to assay functionally altered
promoters. While it is possible to evaluate promoter fragments
in a number of assays in vitro, genes whose functions are
manifest in complex physiological or developmental processes, or involve more than one type of cell or tissue often can
be studied only in the background of the whole organism. This
requires the ability to put an altered promoter back into the
genome of the organism. However, few transformation
systems allow targeted integration of a gene into the exact
chromosomal environment from which it was derived.
Targeted gene replacement in insects was achieved ®rst in
Drosophila melanogaster (1), and techniques developed by
Golic and colleagues have been adapted widely for use in this
species (2). However, these techniques are best suited for gene
knockout experiments that result in lines lacking the function
of a speci®c gene.
Insertion of a transgene into random or non-homologous
chromosome regions introduces experimental variation due to
position effects. The local environment of the insertion site
may contain enhancer or silencing DNA sequences (3) or
chromatin domains (4) that modify the expression of the
introduced transgene. To account for this variability, transformation experiments in D.melanogaster are designed to
recover ®ve to 10 independent insertion events and establish
them as separate lines. Independent lines could come from
primary transformation experiments in which a transgene is
inserted into a previously untransformed recipient strain, or
from mobilizations and reinsertions of pre-existing, integrated
transgenes (5). The activity of the inserted transgene is
evaluated in each line and an average picture of the gene
function is derived. It is presumed that this averaged function
represents the properties of the inserted element.
Position effects on inserted transgenes also confound direct
comparisons among different promoter constructs. Often a
number of different promoter-reporter genes will be made by
varying the amount of putative control DNA contained in the
constructs. The intent of these experiments is to answer
questions about the function of speci®c DNA sequence
domains on the expression of the reporter gene. However,
since these constructs most likely will integrate into different
sites in the chromosomes, the analyses must include efforts to
account for these effects. Subtle effects of introduced sequence
difference most likely will not be observed because they are
below the background variation imposed by the position
effects. Matrix or scaffold-attachment DNA can insulate the
expression of a gene from the effects of the surrounding
chromatin (6,7). DNA sequences that insulate transgenes from
local insertion site effects have been used in analyses of gene
expression in D.melanogaster (8,9). However, primary transformation experiments in mosquitoes are not as ef®cient as
those for D.melanogaster and signi®cant effort would be
required to demonstrate rigorously that insulating sites function in the mosquito as they do in the fruit ¯y. Therefore, we
used a technique based on the cre±loxP and FLP/FRT sitespeci®c recombination systems derived from bacteriophage P1
and the yeast Saccharomyces cerevisiae, respectively (10,11),
*To whom correspondence should be addressed. Tel: +1 949 824 5930; Fax: +1 949 824 2814; Email: [email protected]
Nucleic Acids Research, Vol. 31 No. 22 ã Oxford University Press 2003; all rights reserved
e147 Nucleic Acids Research, 2003, Vol. 31, No. 22
PAGE 2 OF 5
that will permit comparisons of different promoter constructs
integrated into the same site of the genome.
We tested a modi®cation of the `coplacement' technique
developed in D.melanogaster (12) in which DNA ¯anked by
loxP or FRT sites can be excised following expression of the
homologous recombinase. Here we show that a marker gene, a
wild-type copy of the D.melanogaster cinnabar gene, can be
excised with the cre±loxP system at a high frequency from
mosquito chromosomes restoring the mutant white-eye
phenotype of the recipient strain. Constructs expressing FLP
did not catalyze the excsion of FRT-¯anked DNA, and thus the
coplacement scheme is not feasible with existing tools.
However, the applications of cre±loxP techniques for manipulating the mosquito genome will help in both basic and applied
problems in vector biology.
MATERIALS AND METHODS
Homozygous mosquitoes of the khw strain (13) were used in
these experiments. This strain, originally designated white-eye
(14), has a mutation in the gene encoding kynurenine
monooxygenase and this results in a recessive white-eye
color phenotype (15). Mosquitoes were reared using standard
protocols (16).
The donor plasmid used in this study was derived by
ligation of a BglI±EcoRI fragment of pMS109 (17) carrying
loxP and FRT recognition sites for the cre and Flp site-speci®c
recombinases, respectively, into the BglI±EcoRI site of
pSL1180 (Amersham-Pharmacia). The nucleotide sequence
of the minimal loxP recombination site consists of 13 nt
inverted repeat sequences ¯anking an 8 nt asymmetric middle
region, 5¢-ATAACTTCGTATA GCATACAT TATACGAAGTTAT-3¢ (10).
This cloning results in a plasmid with the site-speci®c
recognition sequences inserted into a multiple cloning site
(polylinker). In a separate reaction, pBSMos1, containing the
mariner transposable element, Mos1 (18,19), was digested
with SacI and the vector backbone fragment containing the
Mos1 right-hand region with the terminal inverted repeat
(TIR) was puri®ed by electrophoresis in agarose, as was the
1.2 kb fragment containing the Mos1 left-hand TIR. The two
fragments were ligated, thus removing much of the 5¢-end
¯anking genomic region. The EcoRI and HindIII sites in the
original multiple cloning site were destroyed by digestion,
blunt-ending and religation (consecutively for each site) to
generate p-D-M. The EcoRI±SacII fragment from the
pMS109-pSL1180 product was blunt-end ligated into the
SalI site of p-D-M to create p-D-M-SL. In a separate reaction,
an XbaI±SacII fragment from pH[Cn] (13,20) was ligated into
the XbaI±SacII site of pBCKS+ (Stratagene, San Diego, CA).
The SacII-Blunt±XhoI fragment from this construct was
excised and ligated into the PmlI±XhoI site of p-D-M-SL.
The XhoI±NotI fragment from pHSHH1.9 (21), which contains the Hermes transposase under control of a heat-shock
promoter, was ligated into the XhoI±NotI sites of pBCKS+.
This sequence was included for a set of experiments not
related to this study. Finally, the KpnI±NotI fragment from
this construct was ligated into the KpnI±NotI site of p-D-MSL, to yield the donor plasmid, p[loxP/FRT-cn-Hsphermes]
(Fig. 1). This plasmid along with the Mos1 transposase helper
plasmid, pKhsp82MOS (22), was used to produce primary
Figure 1. Structure of the donor element, p[loxP/FRT-cn-Hsphermes]
before and after excision. (A) Schematic representation of the plasmid. The
boxed ®gures represent DNA sequences contained in the transformation
construct. The left- (ML) and right-hand (MR) Mos1 TIRs have arrowheads
to denote the polarity of the repeat sequences, and ¯ank a sequence containing two adjacent loxP and FRT recognition sites (loxP and FRT, respectively), each arranged in the same respective orientation (also denoted by
arrowheads). The recognition sites ¯ank a wild-type copy of the
D.melanogaster cinnabar gene (CN). The Hermes transposase gene
(Hsphermes) is also shown. The location of diagnostic restriction endonuclease cleavage sites, BamHI, SacI and XhoI, are shown as vertical
arrows with labels, and the lengths of the anticipated fragments are listed.
The extent of the DNA fragments used as probes in Southern analyses are
indicated as triple horizontal lines and labeled (cn+ probe and Hermes
probe). (B) Schematic representations of the transformation construct integrated into a mosquito chromosome and the expected products following
site-speci®c excision. All labels are the same as in (A). The dotted heavy
line represents A.aegypti chromosomal DNA. Excision of the fragment
between the arrows (top) leads to a product that is missing the marker gene
(bottom). Animals with the intact construct have purple eyes, and those
with the excised construct have white eyes.
transformed lines. The helper plasmid, pMLS104 (a gift
from M. L. Siegal, Stanford University), encoding the cre
recombinase under the control of the D.melanogaster heat
shock protein 70 (hsp70) gene was used for the excision
experiments.
Microinjection procedures to produce the primary transformed lines containing the p[loxP/FRT-cn-Hsphermes]
transgene were essentially the same as in Jasinskiene et al.
(20). Crosses with individual microinjected males and multiple untreated females were used to establish single-founder
families (G0 generations). Following standard procedures
(20), G0 females were mated in pools with untreated males.
Positive (colored eyes) G1 progeny were used to establish
transgenic families. Genomic DNA was isolated from these
individuals, prepared for Southern analysis and probed with
radiolabeled DNA complementary to the D.melanogaster cn+
gene (cn+ probe) and/or with sequences complementary to the
Hermes transposase (Hermes probe).
Excision experiments were performed by injecting embryos
transformed with p[loxP/FRT-cn-Hsphermes] with the cre
helper plasmid, pMLS104, at 0.8 mg/ml in transformation
buffer (20). Animals were heat-shocked 18±24 h after
injection at 41°C for 1 h to induce recombinase expression.
These procedures maximize embryo survival and permit
recombination and transposition in germ cells (20,23).
Surviving adults were mated individually to multiple khw
members of the opposite sex to produce a G0. Adult progeny
Nucleic Acids Research, 2003, Vol. 31, No. 22 e147
PAGE 3 OF 5
Table 1. G0 families producing G1 progeny with colored eyesa
G0 family
Total no. of G1
progeny screened
No. of G1 progeny
with colored eyes (%)
128
P3b
P3.1
P11
P10
431
375
375
188
190
32
1
3
4
10
(7.4)
(0.3)
(0.8)
(2.0)
(5.2)
aThese
b`P'
lines have insertions of the p[loxP/FRT-cn-Hsphermes] construct.
indicates a line derived from pooled G1 females (20).
resulting from these matings were scored for the presence of a
white-eye phenotype. White-eye siblings were mated to
produce a G1 and to establish a line. Gene ampli®cation of
control and putative excised DNA was carried out using
primers ML4 (5¢-GGGAATGTCGGTTCGAACAT-3¢), cnF
(5¢-ATCCCATTTGGTCTACGAG-3¢) and hspout (5¢-TTATACTCCGGCGCTCTTTT-3¢), and the following reaction
conditions: one cycle at 94°C for 3 min; 29 cycles at 94°C for
40 s, 63°C for 45 s and 72°C for 1 min; followed by one cycle
at 72°C for 7 min. Samples and marker DNA were resolved on
agarose gels and photographed. Similar experiments were
performed using the FLP helper plasmid, pP[ry+ hsFLP],
which has the FLP recombinase under the control of the
D.melanogaster hsp70 gene promoter (22,24). All injection,
rearing, mating and screening protocols were identical to the
cre±loxP experiments.
RESULTS
Transformation of Aedes aegypti with p[loxP/FRT-cnHsphermes]
A total of 1382 khw/khw A.aegypti embryos were injected with
donor plasmid, p[loxP/FRT-cn-Hsphermes] and the Mos1
helper plasmid, pkhsp82MOS. Of the injected animals, 295
matured to the adult stage, and 73 single-male founder
families and 12 pooled lines of females were set up from the
survivors. Five independent primary transformed lines were
recovered (Table 1) for an absolute transformation frequency
of 1.3% (1/73) and an adjusted transformation frequency of
5.8% (5/85) (23). Southern blot analyses revealed that all ®ve
lines had integrated the transformation construct (data not
shown). Analysis of line P11 showed that it likely contained an
integration of a single transformation construct (Fig. 2). In
addition to the 1.9 and 2.8 kb fragments of the BamHI
digestion, a large ~9.0 kb fragment in the XhoI digestion is
evident and this results from hybridization of the cn+ probe to
a fragment produced by a restriction site within the plasmid
and one in the genome. The D.melanogaster cn+ probe does
not cross-hybridize with the endogenous orthologous mosquito gene. The P11 line was made homozygous and used for
further analysis.
Excision of the D.melanogaster wild-type cinnabar gene
from line P11
A total of 443 homozygous P11 embryos were microinjected
with the recombinase-encoding plasmid, pMLS104. Following individual matings of the 39 surviving adults to 15
members of the opposite sex of the khw recipient strain, all
progeny were checked for eye color. Seven of 21 (33.3%)
Figure 2. The transformed line, P11, has a single insertion of the transformation construct, p[loxP/FRT-cn-Hsphermes]. Genomic DNA from line P11
was digested with either XhoI or BamHI and probed with the cn+ gene.
Genomic DNA from the recipient strain, khw was used as a control. In
addition to the diagnostic fragments at 1.9 and 2.8 kb in the BamH1 pattern,
an additional fragment of ~9.0 kb in the XhoI digestion is consistent with a
single insertion of the transgene.
Table 2. Distribution of putative excision progeny in individual families
Family
Phenotypic
class
Number of
animals
Percentage of
white-eye
phenotypes
24
Purple-eye
White-eye
Purple-eye
White-eye
Purple-eye
White-eye
Purple-eye
White-eye
Purple-eye
White-eye
Purple-eye
White-eye
Purple-eye
White-eye
3
499
28
23
4
3
8
2
16
5
4
2
2
3
99.4
6
25
15
18
29
30
45
48
20
24
50
60
fertile families produced some progeny with white eyes.
Furthermore, analysis of the number of progeny in the
individual lines showing white-eyes revealed that the excision
ef®ciency within a line could be as low as 20% and as high as
99.4% (Table 2). All seven families were set up as sublines of
the original P11 line and designated P1, 6, 15, 18, 24, 25
and 29.
Molecular analysis of putative excision events
Three types of analyses were performed to con®rm the
molecular structure of a putative cre-mediated excision event.
Gene ampli®cation with oligonucleotide primers, ML4 and
hspout, ¯anking the putative excised region showed that
white-eyed individuals from all seven sublines produced an
~600 bp fragment whose size is consistent with the loss of the
cn+ gene and ¯anking loxP site (Fig. 3). Furthermore,
Southern analysis of two of the P11 sublines, 6 and 24 using
the cn+ probe and Hermes probe, revealed that they lack a
2.9 kb fragment that is diagnostic in a SacI digestion of
genomic DNA for the presence of a portion of the cn+ gene
e147 Nucleic Acids Research, 2003, Vol. 31, No. 22
PAGE 4 OF 5
Figure 4. Southern blot analysis of excision lines. Genomic DNA was prepared from G1 animals, digested with SacI and probed with a mixture of the
cn+ and Hermes probes (Fig. 1). Fragments of 2.9 and ~9.0 kb are expected
if no excision occurs (P11; Fig. 1).
Figure 3. Gene ampli®cation analysis of genomic DNA from putative excision events. Genomic DNA from G1 individuals was used as a template for
ampli®cation with speci®c primers, ML4, cnF and hspout (small horizontal
arrows). A 1.5 kb DNA fragment is expected from the primers cnF and
hspout in the P11 parental line. A 0.6 kb fragment is anticipated in excised
lines, 24, 6, 25, 15, 18, 29 and P1, using the primers ML4 and hspout. The
ML4-hspout fragment in the parental line, P11 is too large to amplify
ef®ciently in these reactions.
and ¯anking loxP site (Fig. 4). Two of the larger fragments,
6.0 and 9.0 kb, in the control P11 digestion likely represent the
adjacent genomic fragments while the origin of the third
fragment is unknown. The presence of the 9.0 kb fragment in
lines 24 and 6 support the conclusion that it is the fragment
containing Hsphermes and MR (Fig. 1). Finally, genomic
DNA ampli®ed from line 24 was sequenced to determine the
primary structure of the product of the putative excision event
(Fig. 5). This analysis veri®ed that the Mos1 left-hand repeat
sequence was adjacent to a loxP site that was contiguous with
DNA that adjoined the Hsphermes sequences. The recombination event leading to excision is precise with no loss or
addition of any DNA other than what would be predicted
based on the function of the cre±loxP system.
FLP/FRT site-speci®c excision experiments
Three separate experiments were performed to determine if
the FLP/FRT site-speci®c recombination system could function as ef®ciently as the cre±loxP system in excising DNA
from mosquito chromosomes. In the ®rst experiment, 6539
progeny derived from 28 fertile matings of line P3 mosquitoes
injected with the FLP helper plasmid and mated with the khw/
khw recipient line yielded no progeny with white eyes. In a
second experiment, 16 270 progeny from 88 fertile matings of
injected P11 animals were screened, and four white-eyed
adults were recovered. However, none of these animals bred
true, and preliminary molecular analyses failed to demonstrate
any evidence of excision (data not shown). A new isolate of
the helper plasmid was used in the third experiment and 12 892
progeny from 69 families derived from injections of line P11
were screened. None of these mosquitoes had white eyes.
Sequence analyses of the construct DNA inserted in line P11
con®rmed that the FRT sites were intact structurally, and did
not contain any mutations, additions or deletions (data not
Figure 5. Sequence analyses of the excision site in line 24. The DNA ¯anking the remaining loxp site in line 24 was sequenced. Sequence corresponding to the Mos1 left-hand TIR repeat domain and D.melanogaster Hsp70
promoter now ¯ank a single loxP site (bold). The adjacent BamHI site is
shown in italics.
shown). We interpret these experiments to indicate that FLPmediated excision of a marker gene ¯anked by FRT sites is not
as ef®cient as the cre±loxP system in the conditions used here.
DISCUSSION
Site-speci®c excision of the D.melanogaster cn+ gene from
transgenic A.aegypti is relatively ef®cient with the cre±loxP
system and procedures used here. The ef®ciencies, 33% of all
lines showing excision with as many as 99.4% of individuals
within a line having an excised fragment, although lower than
the 100% reported for D.melanogaster (12), still qualify this as
a powerful tool for gene analysis in A.aegypti. Indeed,
excision ef®ciencies likely could be increased if an inducible
source of cre recombinase was integrated into the mosquito
genome as was done with the fruit ¯y experiments.
Furthermore, stable lines of mosquitoes expressing the
recombinase controlled by constitutive or tissue-speci®c
promoters could provide powerful tools for dissecting important gene functions. The lower levels of ef®ciency of excision
within lines came from those crosses in which for convenience
only a few progeny were counted. However, in each case,
suf®cient progeny were obtained to establish an excised line
by mating siblings. This recovery rate coupled with the
relatively low number of embryos injected make this a
technique that could be used routinely for this species. It
should be noted that coplacement methods can only compare
PAGE 5 OF 5
two different constructs at a time and that several different
transformed lines are needed to attribute signi®cance to small
differences in expression (25).
It remains to be demonstrated whether loxP sites in
mosquito chromosomes can serve as `docking sites' for sitespeci®c integration following direct injection of a host strain
with integrated loxP sites with an integration construct
carrying a loxP site (23). Efforts to develop this speci®c
strategy in D.melanogaster have yet to be successful [although
other strategies for targeted gene replacement have been
developed (2)], but the biology of the mosquito maybe be
different enough to make this possible.
The survival of embryos in the primary and excision
injection portions of the experiments, 21.3 and 8.8%,
respectively, are within the range seen with other microinjection experiments with A.aegypti (20,22,26,27). In addition,
fertility of surviving G0 adults is consistent with previous
experiments (26). The transformation frequency of the
primary integration experiment is consistent with what is
seen with Mos1 transposition (22,26). Thus, the application of
the cre±loxP system appears not to increase the dif®culty of
handling transgenic A.aegypti. These techniques may be
considered straightforward and routine, although they still
remain labor intensive and time consuming.
The failure to detect site-speci®c excision with the FLP/
FRT system is puzzling. There is clear evidence that there are
no inhibitory factors in mosquito embryos that would interfere
with FLP function (23). Furthermore, integrated copies of
marker genes ¯anked by FRT sites could be moved from one
location in the D.melanogaster genome to a target FRT site
present at another location in the genome (24). Perhaps the
differences in results between the work done here and the
other experiments have to do with using integrated or nonintegrated donor elements and the speci®c timing of initiating
the recombinant event. It is also possible that the FLP protein
is not as stable as the cre recombinase. FLP-expressing
constructs based on conditional promoters derived from the
mosquito may also mitigate some of these effects. However, in
the ®nal analysis, the robust activity of the cre±loxP system
makes this general type of tool available to mosquito
researchers.
ACKNOWLEDGEMENTS
The authors thank their colleagues for comments and Lynn
Olson for helping in preparing the manuscript. The research
was supported by an award from the National Institutes of
Health (AI-44238) to A.A.J.
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