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First published online September 28, 2007
doi: 10.1242/10.1242/dev.005686
Review |
1 Program in Developmental Biology, Baylor College of Medicine, Houston, TX
77030, USA.
2 Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA.
3 Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030,
USA.
4 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX
77030, USA.
e-mails: kv134369{at}bcm.tmc.edu; hbellen{at}bcm.tmc.edu
SUMMARY
Drosophila melanogaster is a highly attractive model system for the study of numerous biological questions pertaining to development, genetics, cell biology, neuroscience and disease. Until recently, our ability to manipulate flies genetically relied heavily on the transposon-mediated integration of DNA into fly embryos. However, in recent years significant improvements have been made to the transgenic techniques available in this organism, particularly with respect to integrating DNA at specific sites in the genome. These new approaches will greatly facilitate the structure-function analyses of Drosophila genes, will enhance the ease and speed with which flies can be manipulated, and should advance our understanding of biological processes during normal development and disease.
Introduction
During the past few years, Drosophila melanogaster has gained in
popularity because of the availability of its genome sequence
(Adams et al., 2000
), its rapid
life cycle, the relative ease with which it can be handled and the multitude
of genetic tools that are available for its study
(Greenspan, 2004). The fly's
genome permits the most sophisticated manipulations of any of the known
eukaryotes. Indeed, the number of existing and recently developed
technological improvements, such as genome-wide transposon tagging and gene
targeting (Venken and Bellen,
2005) and the availability of numerous resources, including online
databases such as FlyBase and stocks from fly stock centers (see
Box 1 for links to some of
these resources) (Matthews et al.,
2005), greatly facilitate research in the field and move it
forward at a relentless pace. These technologies and resources further the
study of various aspects of developmental biology, genetics, cell biology,
neuroscience and behavior. Indeed, the identification of novel genes and their
functional characterization in vivo greatly depends on these available tools.
Moreover, as most human disease genes have a counterpart in the
Drosophila genome, including those involved in genetic disorders and
cancer (Bier, 2005;
Vidal and Cagan, 2006), the
fly is also becoming increasingly popular for studying the molecular
mechanisms of human disease. Much of this research relies on an efficient and
reliable transgenesis system.
Transgenesis in general can be defined as a group of technologies that
allow DNA to be introduced into an organism of choice. The main goal of
transgenesis is to integrate a foreign piece of DNA - a transgene - into an
organism's genome to result in germ line transmission (see
Fig. 1), in order to study gene
function. Insect transgenesis, in general, has been dominated by
transposon-mediated integration (Handler
and James, 2000). In Drosophila, transgenesis mainly
relies on the P element transposon and this has been the foundation
for most of the innovative developments within the fly field
(Ryder and Russell, 2003).
However, various improvements in fly transgenic techniques have been recently
reported that predominantly employ the site-specific integration of transgenes
at specific genomic docking sites (see glossary,
Box 2) via the use of different
recombinases and integrases (Groth et al.,
2004; Oberstein et al.,
2005; Horn and Handler,
2005; Bateman et al.,
2006; Venken et al.,
2006; Bischof et al.,
2007). Many of these advances have their origins in mouse
molecular genetics (Seibler and Bode,
1997; Bethke and Sauer,
1997; Bouhassira et al.,
1997; Groth et al.,
2000; Thyagarajan et al.,
2001) and have been very useful for developing new fly transgenic
techniques, as discussed below.
Here, we summarize many of the current methods that are used to generate transgenic flies. We first review classical transposon-mediated transgenesis and site-specific integration methods, before describing a plethora of recent improvements that have their basis in site-specific integration systems.
Transposon-mediated transgenesis in Drosophila
Transgenesis can be performed through various techniques. In
Drosophila, transgenesis mainly relies on the P element
transposon, the introduction of which
(Rubin and Spradling, 1982)
has been one of the most important breakthroughs in germ line transgenesis in
Drosophila. As such, Drosophila research has been highly
dependent on P element-mediated transgenesis, even though it has two
major drawbacks: the size of the DNA that can be integrated is limited and the
insertion sites cannot be controlled.
| Box 1. Relevant websites Drosophila Genomics Resource Center: plasmid resource center for fly transgenesis. DrosDel: docking site stock center for FLP remobilization. FlyBase: general online fly resource.
FlyC31: http://www.frontiers-in-genetics.org/flyc31
P(acman): recombineering and the http://flypush.imgen.bcm.tmc.edu/lab/pacman.html
http://flystocks.bio.indiana.edu/Browse/misc-browse/phiC31.htm
http://genepath.med.harvard.edu/WuLab/RMCE Recombineering website: resource for public available recombineering reagents. http://recombineering.ncifcrf.gov Gensat Database: resource for RecA assisted modification. Vienna Drosophila RNAi Center: transgenic RNAi fly lines. National Institute of Genetics (Japan) RNAi Fly Stocks: transgenic RNAi fly lines. http://www.shigen.nig.ac.jp/fly/nigfly
|
P elements are transposable elements, or transposons, which were
originally identified within the fly's own genome
(Castro and Carareto, 2004).
P elements, like other transposons, contain two terminal repeats,
including inverted repeat sequences and other internally located sequence
motifs absolutely required for their mobilization or transposition (see
Fig. 2 and
Table 1)
(Beall and Rio, 1997). Mobile
or autonomous P element transposons encode a functional enzymatic
protein called P transposase that catalyzes transposition through
both terminal repeats of the transposon. P element-mediated
transgenesis requires the separation of the P transposase and the
P element transposon backbone
(Rubin and Spradling, 1982).
A plasmid that encodes P transposase, a so-called helper plasmid, is
provided in trans with another plasmid (the transgene) that contains the
transposon backbone, the sequence of interest and a marker (see
Fig. 2B)
(Karess and Rubin, 1984). In
vitro synthesized mRNA that encodes the transposase or purified transposase
protein itself (Kaufman and Rio,
1991) can also be co-injected with modified P elements.
Co-injections limit transposase activity, which is often advantageous.
Alternatively, the transposase can be expressed from a genomic source
(Cooley et al., 1988),
allowing the injection of a P element without a helper plasmid.
Transgene expression can be rendered constitutive or inducible through the
inclusion of a heat-shock promoter. A hyperactive form of P
transposase has been isolated that results in increased transposition rates
(Beall et al., 2002). In
general, transposons are injected into fly strains that are devoid of the same
transposon, avoiding unwanted mobilization events of transposons present in
the genome, thereby ensuring the stable integration and maintenance of the
injected transgene. The unbiased identification of integration events is
crucial for transgenesis and predominantly relies on the incorporation of
dominant markers, which are identified through screening or selection (see
Table 2 and
Box 3 for more
information).
|
|
|
| Box 2. Glossary of specialized terms Acceptor site: A genomic site that receives in vivo DNA mobilized from a different location - the donor site. This occurs through FLP remobilization or P element replacement. Docking site: Alternatively called a landing site. A genomic site that receives injected DNA during embryo microinjection. Donor site: A genomic site that contains DNA sequence that will be donated for integration at another location, through FLP remobilization, P element replacement or gene targeting. Episomal fragment: An independent DNA element, such as a plasmid, that can exist extrachromosomally or that can be maintained by integrating into the genome of the host. Gal4/UAS system: Based on the yeast transcriptional activator GAL4 and its high-affinity binding site, the upstream activating sequence (UAS), this system is generally used to ectopically express a gene of interest. When a tissue-specific GAL4 line is crossed to an effector line that carries the UAS fused to a gene of interest, progeny with both the GAL4 and UAS components express the gene of interest in an activator (and often tissue)-specific manner. Insulator: A DNA sequence that blocks the interaction between cis-acting regulatory elements. These sites are sometimes used to protect transgenes from genomic position effects.
MARCM: MARCM (mosaic analysis with a repressible cell marker) allows
mutant clones generated by mitotic recombination to be identified in an
otherwise wild-type unlabeled background
(Lee and Luo, 1999 Mitotic recombination: A cross-over between two homologous double-stranded DNA molecules. This recombination occurs frequently during meiosis, but is relatively rare during mitosis. Position effects: The effect of the local chromosomal environment on the level or pattern of transgene expression, owing to local chromatin configuration or nearby cis-acting regulatory elements. Position effect variegation: A phenomenon discovered in Drosophila that occurs when genes placed close to large heterochromatic regions are repressed. This repression is metastable in that the silenced state can be occasionally released, giving rise to derepressed cells and a variegated phenotype. Rescue: A condition achieved by introducing a wild-type DNA fragment that can complement a genomic mutation by producing the functional or missing protein.
|
Transposition occurs by the excision or replication of the transposon from
the injected plasmid and its insertion into the host genome. Different
transposons have unique insertion site characteristics. Integration events of
P elements are strongly biased towards the 5' end of genes. Hot
spots - insertion sites that attract P elements at a much higher
frequency than others - also exist within the Drosophila genome
(Spradling et al., 1995;
Bellen et al.,2004). Moreover,
P elements have a narrow taxonomic activity and are non-functional
outside of the Drosophilidae (Handler et
al., 1993) owing to a host-specific factor that is required for
transposition (Rio and Rubin,
1988). To circumvent these limitations, several other transposons
with a different insertional specificity and a broader host range have been
identified that are suitable for germ line transformation in
Drosophila (see Table
1). These include piggyBac
(Handler and Harrell, 1999),
identified in the cabbage looper moth Trichoplusia ni
(Cary et al., 1989;
Handler, 2002); the
Tc1/mariner-like transposons Minos
(Loukeris et al., 1995) and
Mariner (Lidholm et al.,
1993), isolated from Drosophila hydei
(Franz and Savakis, 1991) and
Drosophila mauritiana, respectively
(Jacobson et al., 1986); and
the hobo, Ac, Tam3 (hAT) family members Hermes
(O'Brochta et al., 1996) and
hobo (Blackman et al.,
1989; Smith et al.,
1993), isolated from the house fly Musca domestica
(Warren et al., 1994) and
Drosophila melanogaster, respectively
(McGinnis et al., 1983). These
transposons function in a variety of organisms, but their use in
Drosophila transgenesis has been limited
(O'Brochta and Atkinson,
1996; Ryder and Russell,
2003). piggyBac and Minos have been used as
alternative mutagens because they have a different insertional specificity to
P elements (Hacker et al.,
2003; Horn et al.,
2003; Thibault et al.,
2004; Metaxakis et al.,
2005). As Mariner elements do not remobilize efficiently
(Lozovsky et al., 2002), and
because hobo is present in most laboratory stocks, neither is
commonly used. Finally, hobo and Hermes have been shown to
cross-mobilize (Sundararajan et al.,
1999). These features have limited the use of these transposable
elements.
|
The transposon-mediated integration of transgenes has been used for numerous experiments in the fly field. These experiments can be broadly subdivided into two main groups: gene disruption methods and transgenic technologies. Gene disruption occurs when a transposon insertion interferes with the function of a gene. Transgenic technologies usually involve introducing the different components of novel techniques (see below) or performing rescue experiments.
Almost all technological progress in flies depends on our ability to
transform them. Indeed, P element-mediated enhancer detection
(O'Kane and Gehring, 1987;
Bellen et al., 1989;
Bier et al., 1989), the use of
the FLP/FRT system to create mutant clones by inducing mitotic
recombination (see glossary, Box
2) (Xu and Rubin,
1993), the gene knockout methods in flies
(Rong and Golic, 2000;
Gong and Golic, 2003), the
creation of molecularly defined deletions throughout the genome
(Thibault et al., 2004;
Ryder et al., 2004), the
generation of marked mutant clones by MARCM (see glossary,
Box 2)
(Lee and Luo, 1999), and many
other technological advances have relied on transgenesis. The recent
availability of a genome-wide library of RNAi transgenic insertions that
allows the knockdown of most fly genes
(Dietzl et al., 2007) will
also provide an invaluable tool to study gene function.
In addition, transposon-mediated phenotypic rescue of a mutation is
considered to be the best and most convincing evidence that a piece of DNA
contains a gene of interest. Unfortunately, traditional high-copy-number
plasmids, including the P element-containing plasmids, have a limited
cargo capacity of 
20-25 kb of DNA owing to plasmid instability in
bacteria. To circumvent this, P elements were engineered in a
medium-copy-number cosmid backbone
(Haenlin et al., 1985;
Steller and Pirrotta, 1985),
providing a higher cargo capacity of up to 40-50 kb. Unfortunately, the
difficulties associated with obtaining integration of 30-50 kb P
element-based cosmids did not promote the use of this methodology. As a
result, a transgenic cDNA rescue based on the GAL4/UAS system (see
glossary, Box 2)
(Fischer et al., 1988;
Brand and Perrimon, 1993) or
heat-shock induction (Basler and Hafen,
1989) became more popular.
Neutralization of position effects
One of the major drawbacks of P element-mediated transgenesis is
that P elements most often integrate into the 5' regulatory
regions of genes (Bellen et al.,
2004), thereby causing two unwanted consequences. First, the
insertion often disrupts another gene that may or may not be relevant (e.g.
within the same pathway) to the gene that is being studied
(Norga et al., 2003). Second,
the gene within the transposon may be subject to unwanted position effects or
to position effect variegation (see glossary,
Box 2) dictated by the
surrounding genomic environment. Insertions in the regulatory region of a
gene, on which nearby cis-acting elements typically act, bring the gene into
an environment that is almost certainly subject to unwanted regulation.
Indeed, position effects and position effect variegation were observed early
on for markers such as white
(Hazelrigg et al., 1984;
Levis et al., 1985) and were
eventually exploited in different kinds of enhancer-trap screens to identify
temporally and spatially restricted expression patterns of developmentally
regulated genes (O'Kane and Gehring,
1987; Bellen,
1999).
Position effects can be partially neutralized through the incorporation of
insulator sequences (Roseman et al.,
1995). Insulators (see glossary,
Box 2) tend to shield the
transgene from regulatory influences imposed by the surrounding genome.
Insulators, such as gypsy, have been used in some P element
vectors because they are more mutagenic than other P elements that do
not contain insulators (Roseman et al.,
1995). They were also incorporated into P element
reporter transposons developed to analyze gene regulatory sequences
(Barolo et al., 2000;
Barolo et al., 2004).
Insulators allow for a better comparison of different transgene insertions at
different loci. Yet insulators may also influence the expression of the gene
that they flank within the construct and are still somewhat subject to
position effects in the genome.
There are at least four alternative genetic strategies to neutralize
position effects when different transgenes are being compared at the same
locus. The simplest method is transgene coplacement
(Siegal and Hartl, 1996),
which allows any two transgenes, such as a rescue fragment and its mutant
version, to be compared in the same orientation at the same locus
(Fig. 3). Both transgenes are
integrated into a P element that contains the site-specific
recognition sites FRT and loxP, the targets of FLP and Cre
recombinases, respectively (see Box
4 for more information on these recombinases). After integration
of the P element, FLP can remove one transgene and Cre can remove the
other. Recognition sites are oriented such that either recombination event
results in an identical configuration for either transgene. This method also
introduced the use of Cre recombinase into the Drosophila field
(Siegal and Hartl, 1996;
Siegal and Hartl, 2000). One
drawback of the technique is that only two transgenes can be compared at the
same locus.
| Box 3. Dominant marker genes for Drosophila transgenics Identifying transgene integration events is crucial for transgenesis and relies on the incorporation of dominant markers, which are identified through screening or selection (see Table 2). The former relies on the rescue (see glossary, Box 2) of a visible mutant phenotype that minimally affects viability.
Two popular markers are the adult eye color marker white and body
color marker yellow (see Table
2). The mini-white gene is one of the most widely used
white markers (Pirrotta,
1988
Recently, fluorescent protein-based markers have been developed, which are
also used in other insects and organisms
(Horn et al., 2002
|
A second method is based on FLP recombinase-mediated transgene
remobilization (Golic et al.,
1997) (Fig. 4A).
First, a `donor' P element (see glossary,
Box 2), containing a transgene
together with the white+ marker flanked by FRT
sites, is integrated into the fly genome using P transposition,
resulting in a donor site. Second, the transgene with the
white+ marker, flanked by FRT sites, is
remobilized through FLP excision. This episomal fragment (see glossary,
Box 2) can integrate into a
second single FRT-containing `acceptor' transposon (see glossary,
Box 2), which also carries
another dominant marker and is located elsewhere in the genome. Successful
mobilization events can be identified through screening, as relocalization
usually results in changes in white+ marker expression
owing to position effects. This strategy is facilitated if a split
white+ marker strategy is integrated into the system
(Fig. 4B): the
white+ marker is separated into 5' and 3'
fragments, and only becomes functional after the reconstitution of these
fragments through site-specific recombination within an intron located between
both fragments (Golic et al.,
1997). In FLP recombinase-mediated transgene remobilization,
white expression is only obtained after correct mobilization and
site-specific integration (Fig.
4C), facilitating the screening procedure of integration events.
Interestingly, thousands of P element insertions obtained by the
DrosDel project (see Box 1)
were generated by the mobilization of the P{RS5} and P{RS3}
transposons (Golic and Golic,
1996) and were subsequently used for the generation of precise
deletions (Ryder et al.,
2004). Both transposons can be used as acceptor elements for in
vivo FLP-mediated DNA mobilization using the split white+
marker strategy, and they provide numerous docking sites that are dispersed
all over the fly genome (Fig.
4D,E). A drawback of FLP recombinase-mediated transgene
remobilization in general is that a second round of crossings for
remobilization and screening has to be performed after an initial P
element-mediated transformation to obtain the required integration events of
donor elements.
|
|
;
Lankenau and Gloor, 1998). An
`acceptor' P element inserted at one location is replaced by a second
`donor' P element, integrated at another location, through in vivo
gap repair (Fig. 5). The
technique requires homologous recombination between the 10-20 bp footprints of
the 31 bp inverted terminal repeat that remain after excision of the acceptor
element and the homologous counterpart of the donor element. The homologous
recombination event is promoted owing to a double-stranded gap that is
generated after the excision of the acceptor element. Various donor P
elements can be targeted to the same locus, allowing different transgenes to
be directly compared with each other. A drawback of the technique is that
replacement can occur in both directions, requiring additional molecular
verification. The technique has not been used to perform structure-function
analysis of differently mutagenized transgenes but has been proven useful for
converting existing lacZ enhancer-detectorP elements into
GAL4 drivers (Sepp and Auld,
1999; de Navas et al.,
2006) that allow more versatile reporter analysis using the
GAL4/UAS system (Fischer et al.,
1988; Brand and Perrimon,
1993). Box 4. Site-specific recombinases and integrases
Site-specific recombinases and integrases (SSRIs) often require only two
components: a site-specific enzyme, which, preferentially, functions without
additional proteins, and a pair of DNA recombination sites (RSs)
(Sorrell and Kolb, 2005
A commonly used serine recombinase is the integrase from the
Streptomyces bacteriophage
Recombination between two RSs can lead to an inversion,
integration/excision or recombinase-mediated cassette exchange (RMCE),
depending on the orientation and types of RS (see figure). A translocation can
also occur if the RSs are on two different chromosomes (not shown). The
presence of two compatible RSs results in a recombination event, which in the
case of FRT or loxP leads to the reformation of a still
functional RS, potentially resulting in additional recombination events. This
problem can be overcome using RS inverted repeat variants, such as
lox71 and lox66, which contain mutations in the left and
right inverted repeat, respectively. Recombination between lox71 and
lox66 results in wild-type loxP and a double-mutant
lox72, two sites that do not recombine with each other
(Albert et al., 1995
Integration using a single RS results in the integration of the entire
plasmid, including the vector backbone. This can be avoided through RMCE
(Schlake and Bode, 1994
|
The best but most labor-intensive way to eliminate position effects is in
vivo gene targeting through homologous recombination. Gene targeting in
Drosophila can be performed using two strategies: `ends-in' or
insertional gene targeting (Rong and
Golic, 2000) and `ends-out' or replacement gene targeting
(Gong and Golic, 2003)
(Fig. 6). Insertional gene
targeting results in the insertion of the entire targeting sequence into the
region of homology. This results in a duplication that can be resolved during
a second round of homologous recombination
(Fig. 6A)
(Rong et al., 2002).
Replacement gene targeting results in the substitution of an endogenous DNA
sequence with exogenous DNA through a double-reciprocal recombination event
between two stretches of homologous sequence
(Fig. 6B). Both strategies
require the introduction of a `donor' element, which contains the
gene-targeting cassette, through transgenesis prior to in vivo homologous
recombination, and require extensive screening. Although the techniques have
not been used to compare the phenotypic outcome of different transgenes at the
same locus, they are gaining in popularity for creating targeted mutations
(O'Keefe et al., 2007).
Recent efforts have focused on making gene targeting more efficient in
Drosophila through the use of site-specific
zinc-finger-nuclease-stimulated gene targeting
(Bibikova et al., 2003;
Beumer et al., 2006).
Zinc-finger nucleases are protein fusions between the Fok1 nuclease and
(generally) three zinc-finger DNA-binding domains that introduce sequence
specificity. Because each zinc finger recognizes 3 bp, zinc-finger nucleases
can be designed to bind to a unique segment of 9 bp. As these nucleases need
to dimerize at the target site before they can cut the target DNA, a
recognition site of 18 bp is effectively required, a sequence that is likely
to be unique in the fly genome. Thus, cutting by zinc-finger nucleases can be
directed to specific target sites to create a double-stranded break, resulting
in increased gene-targeting efficiency when a linearized donor targeting
element is introduced.
|
Although all the strategies of site-specific integration described above are elegant and useful, they have not been used extensively. The main drawbacks are that they allow only a limited number of transgenes to be compared and are too labor-intensive, as they require transgenesis of a donor construct prior to extensive genetic screening to obtain the required site-specific transgenic insertion event. Hence, the primary goal of true-targeted transgenesis is to achieve efficient site-specific integration upon injection of the DNA without the need for further manipulations.
This strategy was pioneered in the fly field using the bacteriophage
C31 integrase, which can integrate transgenic constructs at defined
docking sites (Groth et al.,
2004). Moreover, C31 integrase-mediated transgenesis allows
large DNA fragments to be integrated into the fly genome, well beyond the
fragment sizes that can be introduced by P element-mediated
integration (Venken et al.,
2006). As discussed in more detail below, this approach has also
introduced a user-friendly DNA modification platform, called recombineering,
into Drosophila research.
C31 integrase catalyzes the recombination between the phage attachment
(attP) site present in its own bacteriophage genome and a bacterial
attachment (attB) site present within the bacterial host genome
(Thorpe and Smith, 1998) (see
Box 4). Previous work has shown
that the C31 integrase can catalyze the site-specific integration of
attB-containing plasmids into so-called attP-containing
`docking' or `landing' sites that have been introduced into mammalian cell
lines (Groth et al., 2000;
Thyagarajan et al., 2001).
Interestingly, attB-containing plasmids integrate more readily into
attP-containing genomic docking sites than do attP sites in
the reciprocal reaction, indicating that the integration reaction is
asymmetric in nature (Thyagarajan et al.,
2001; Belteki et al.,
2003). This phenomenon was recently confirmed in
Drosophila (Nimmo et al.,
2006).
In Drosophila, recombination is mediated via C31 integrase,
provided through an mRNA source, between an attP docking site,
previously integrated with a transposon into the fly genome, and an
attB site present in an injected plasmid
(Groth et al., 2004)
(Fig. 7A). Three so-called
pseudo-attP docking sites have been identified within the
Drosophila genome. As one of these pseudo-sites is located in the
endogenous transposable element copia, the true number of available
pseudo-sites is likely to be high
(Kaminker et al., 2002).
Fortunately, these pseudo-sites were shown not to be receptive to
attB plasmids, as all integration events were at the desired
attP sites (Groth et al.,
2004). However, rare non-specific integrations have been
documented in Drosophila (Venken
et al., 2006; Nimmo et al.,
2006; Bischof et al.,
2007). The C31 integrase-mediated transformation technique
has also recently been introduced successfully in the yellow fever mosquito
Aedes aegypti (Nimmo et al.,
2006).
After the original report describing two attP P element docking
sites (Groth et al., 2004),
numerous additional docking sites have been created. One set is embedded in a
piggyBac backbone (Venken et
al., 2006), whereas a second set is embedded in a Mariner
backbone (Bischof et al.,
2007). Venken et al. (Venken
et al., 2006) observed that one out of seven docking sites tested
was not receptive, suggesting that the genomic position of the docking site
can affect integration efficiency. This was not observed for the 19 sites
tested by Bischof et al. (Bischof et al.,
2007). A detailed characterization and comparison of all the
available docking sites will allow us to determine which ones are the most
useful for specific purposes, such as cDNA overexpression, RNAi, genomic
rescue or promoter/enhancer analysis.
Although the first reports used mRNA-encoded C31 integrase to
integrate the DNA (Groth et al.,
2004; Bateman et al.,
2006; Venken et al.,
2006), Bischof et al. (Bischof
et al., 2007) recently reported an efficient germ line C31
integrase source that is driven by nanos or vasa regulatory
elements. Interestingly, through C31 integrase-mediated transgenesis,
different C31 integrase sources have been incorporated at the same
docking sites. Additionally, the same C31 integrase source was integrated
into different docking sites, allowing the most efficient genomic C31
integrase source to be selected. In the same study, a Drosophila
codon-optimized C31 integrase was described that performs better than the
non-optimized version (Bischof et al.,
2007).
Site-specific integration using a single recombination site results in the
integration of the vector backbone, which may interfere with transgene
expression (Chen et al.,
2003). This can be minimized through marker genes strategically
positioned between transgene and vector backbone
(Venken et al., 2006).
Alternatively, appropriately engineered recombinase sites in both the docking
site and integration plasmid can be used to remove unwanted vector backbone
sequence after correct integration events are isolated
(Bischof et al., 2007).
Finally, the integration of the backbone can be directly avoided through
recombinase-mediated cassette exchange (RMCE)
(Baer and Bode, 2001).
In RMCE, both docking site and transgene are flanked by a recombination
site (see Box 4 and
Fig. 7). Double reciprocal
cross-over results in the integration of a transgene without its vector
backbone. However, two sets of directly oriented loxP or FRT
sites will favor deletion over RMCE. This problem can be overcome with sites,
called spacer variants, that support recombination between themselves but not
with others (see Box 4 for more
information). The use of RMCE with spacer variants was initially utilized in
the mouse in Cre- (Bethke and Sauer,
1997; Bouhassira et al.,
1997) and FLP- (Seibler et
al., 1998) based genetic engineering. This approach has been
recently exploited in Drosophila for both recombinases
(Oberstein et al., 2005;
Horn and Handler, 2005)
(Fig. 7B). For example, RMCE
has been used elegantly to perform structure-function analysis of the
eve2 (eve - FlyBase) enhancer with a lacZ reporter
(Oberstein et al., 2005). An
alternative way to ensure that RMCE avoids the deletion or integration of
plasmid backbone when employing FLP or Cre, is to use inverted recombination
sites (as shown for C31 integrase in
Fig. 7C), which was pioneered
in the mouse using Cre (Feng et al.,
1999). This strategy eliminates the deletion problem but causes
inversions.
|
C31 integrase
system, as these recombination reactions are unidirectional. Pioneered in the
yeast Schizosaccharomyces pombe
(Thomason et al., 2001) and
in mouse (Belteki et al.,
2003), a modification of this system using inverted att
recombination sites was recently described for Drosophila
(Bateman et al., 2006)
(Fig. 7C). This study
demonstrated that unmarked constructs can be integrated through RMCE, as
site-specific integration events are identified by loss of the marker
(Bateman et al., 2006). Recombineering: BAC transgenesis for Drosophila
Transposons are generally characterized by a low cargo capacity, limiting
the amount of DNA that can be integrated and mobilized. Transgene size
limitation can be overcome by the incorporation of a site-specific integration
system, such as C31 integrase, as shown in a chicken cell culture system
(Dafhnis-Calas et al., 2005),
or by gene targeting at the Hprt1 locus in mouse embryonic stem (ES)
cells (Heaney et al., 2004).
Unfortunately, an intermediate cell culture system supporting both in vitro
gene manipulation and subsequent germ line transmission, similar to mouse ES
cells, is not available for Drosophila. Moreover, there is a strong
negative correlation between the upper size limit of the insert and plasmid
copy number: large DNA fragments are unstable when present in high-copy-number
vectors in bacteria. Therefore, low-copy-number plasmids, such as P1
bacteriophage (Sternberg,
1990), bacterial artificial chromosomes (BACs)
(Shizuya et al., 1992) and P1
artificial chromosomes (PACs) (Ioannou et
al., 1994), were developed to maintain large inserts.
Unfortunately, these plasmids interfere with both cloning and microinjection
procedures, which require high DNA concentrations. This can be circumvented by
the use of a specialized plasmid backbone - the conditionally amplifiable BAC
- that has two origins of replication
(Wild et al., 2002): an
oriS for low-copy propagation and an oriV for high-copy
induction. Importantly, the manipulation of large DNA fragments in these
vectors has been facilitated through recent developments in in vivo
recombination-mediated genetic engineering, also known as recombineering
(Copeland et al., 2001;
Heintz, 2001;
Muyrers et al., 2001;
Sawitzke et al., 2007).
|
C31 integrase-mediated
transgenesis - have recently been integrated into a single transformation
system (Venken et al., 2006).
This system provides an efficient recombineering platform for
Drosophila, permitting the integration of large DNA fragments into
the fly genome. Selected fragments that encode the gene of interest are
obtained in the amplifiable BAC backbone through recombineering-mediated gap
repair, which can be performed at low copy number
(Fig. 8A). Gap repair in
high-copy and medium-copy plasmids has an upper size limit of 30 and 80
kb, respectively (Lee et al.,
2001). However, by maintaining the plasmid at low copy number,
large fragments of up to 102 kb can be efficiently gap-repaired
(Venken et al., 2006), as
observed by others (Kotzamanis and Huxley,
2004). Interestingly, one 133 kb fragment that encodes one of the
largest genes in the fly genome, Tenascin major, was reconstituted
from two different BACs, each containing part of the gene
(Venken et al., 2006).
Gap-repaired DNA was induced to high copy number, isolated, and integrated
into the fly genome using both P transposase and C31 integrase:
P transposase was used to integrate gap-repaired fragments of up to
39 kb, whereas C31 integrase was used to integrate gap-repaired fragments
of up to 133 kb.
A similar gap-repair approach was recently used to generate transgenes for
Drosophila in vivo (Takeuchi et
al., 2007). The gap-repaired constructs were obtained in flies
through homologous recombination into the endogenous locus after the in vivo
linearization of the transgene between both homology arms using the
meganuclease I-SceI (Fig. 8B).
The technique relies on endogenous fly enzymes, rather than on bacterial
enzymes, to mediate the gap repair. The authors observed an upper size limit
of 28 kb for correct gap repair.
An important reason for the development of recombineering is the ease with
which DNA can be modified. Indeed, restriction enzymes and DNA ligase are not
user-friendly when handling large DNA fragments, as the occurrence of unique
cutting sites decreases with increasing plasmid size. Recombineering does not
suffer from those limitations and allows BACs to be modified more rapidly
using PCR products or oligonucleotides that contain the desired mutation as
recombination templates (Copeland et al.,
2001; Court et al.,
2002) (Fig. 9A).
This strategy can easily be combined with positive/negative selectable
markers, such as galK and thyA
(Warming et al., 2005;
Wong et al., 2005).
Positive/negative selectable markers are targeted to the desired site for
mutagenesis during a first round of recombineering using selection, and are
then replaced by the desired mutation or tag during a second round of
recombineering using counterselection
(Fig. 9B). An alternative way
to modify DNA constructs uses the recombinogenic protein RecA, also known as
RecA-assisted modification (Yang et al.,
1997; Gong et al.,
2002), a methodology that is somewhat different from traditional
recombineering. In a first recombination step, a modifying plasmid is
integrated into the target plasmid, resulting in a co-integrate that becomes
resolved during a second round of recombination
(Fig. 9C). The technique
allows the integration and deletion of fragments within a genomic fragment
(Misulovin et al., 2001) and
has been applied on a high-throughput level in the mouse field to create an
atlas of gene expression in the mouse central nervous system
(Gong et al., 2003). BAC
modification was pioneered in the mouse field because most mouse genes tend to
have multiple distant regulatory regions and are therefore too large to be
handled using high-copy plasmid backbones
(Heintz, 2001). The efficient
recombineering-mediated tagging of genes in a genomic context has also been
recently demonstrated in Caenorhabditis elegans and C.
briggsae (Dolphin and Hope,
2006; Sarov et al.,
2006).
|
The advent of site-specific integration combined with recombineering and
other methodologies will impact the fly field in numerous ways. These
techniques make it possible to carry out structure-function studies at a
higher resolution with fewer transgenes, as position effects can be mitigated
using some of these approaches. Moreover, we are no longer confined to the
study of single genes but can now tackle entire gene complexes that play key
roles in development. Through repeated rounds of mutagenesis via
recombineering, one can dissect the in vivo role of each gene and each
regulatory region within a gene complex. Similar manipulations are now also
possible for larger genes and for loci that have previously had no available
mutations to study. These loci can now be identified and studied through the
introduction of small deletions by FLP/FRT recombination
(Parks et al., 2004;
Ryder et al., 2004).
Combinations of the different methodologies described here should also
greatly enhance our ability to manipulate the fly genome. For example,
P replacement with an attP-containing P element
could be used to convert many of the existing P elements into a
useful docking site for C31 integrase-mediated transgenesis or RMCE.
Alternatively, gene targeting of recombination sites at desired locations
might allow the site-specific integration of any DNA fragment. Finally,
C31 integrase-mediated transgenesis can be used to insert the donor
constructs that are required for gene targeting. These are just a few examples
of possible future fly manipulations.
In another vein, these technologies will also help to improve genome-wide
studies of Drosophila. For example, one could try to identify optimal
genomic sites for the integration of all RNAi constructs. These sites should
permit the optimal expression of RNA hairpin loops in all tissues at all
developmental stages to allow the efficient RNAi-mediated knockdown of any
gene. This approach may alleviate some of the potential drawbacks that are
associated with P transposase-mediated transgenesis of RNAi
constructs, such as poor transgene expression or misregulation
(Dietzl et al., 2007).
Furthermore, many different genomic DNA fragments containing cis-regulatory
elements that drive GAL4 expression could be integrated at the same docking
site to allow the labeling and manipulation of specific cell populations.
Finally, the integration of overlapping duplications of defined areas of the X
chromosome into the same docking site would be a useful way to map essential
genes on the X chromosome.
|
).
Moreover, at high doses, Cre has been demonstrated to result in undesired
effects in both vertebrates (Schmidt et
al., 2000; Loonstra et al.,
2001) and Drosophila
(Heidmann and Lehner, 2001).
Detrimental side effects have been observed for C31 integrase in
mammalian cell culture (Ehrhardt et al.,
2006; Liu et al.,
2006), although ectopic expression in vivo in mice and
Drosophila indicates that it has minimal side effects in these
organisms (Belteki et al.,
2003; Raymond and Soriano,
2007; Bischof et al.,
2007). Even gene targeting in Drosophila has not been
spared from artifacts, and second-site mutations have been shown to cause
interference with phenotypic characterization
(O'Keefe et al., 2007).
Interestingly, so far no such observations have been documented for FLP, which
is widely used within the fly community
(Blair, 2003).
Unfortunately, the solution to an important problem such as efficient
site-specific integration, immediately results in the creation of new
challenges: the handling of thousands of fly strains associated with typical
high-throughput projects, as well as the maintenance of thousands of new
stocks. Recent methods for the automated microinjection of fly embryos for
high-throughput in vivo RNAi experiments have been developed
(Zappe et al., 2006), and it
should now be possible to adapt this technology for DNA microinjections.
However, no solution has yet been developed to maintain numerous additional
fly stocks, except through the expansion of existing or new stock centers.
ACKNOWLEDGMENTS
We apologize to those whose work could not be cited owing to space limitations. We thank Karen Schulze for comments on the manuscript. K.J.T.V. and H.J.B. are supported by the NIH and the Howard Hughes Medical Institute.
REFERENCES
Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A.,
Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A.,
Galle, R. F. et al. (2000). The genome sequence of Drosophila
melanogaster. Science
287,2185
-2195.
Albert, H., Dale, E. C., Lee, E. and Ow, D. W. (1995). Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J. 7, 649-659.[CrossRef][Medline]
Baer, A. and Bode, J. (2001). Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for the targeted integration of transgenes. Curr. Opin. Biotechnol. 12,473 -480.[CrossRef][Medline]
Barolo, S., Carver, L. A. and Posakony, J. W. (2000). GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila. Biotechniques 29,726 , 728, 730, 732.[Medline]
Barolo, S., Castro, B. and Posakony, J. W. (2004). New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP. Biotechniques 36, 436-440, 442.[Medline]
Basler, K. and Hafen, E. (1989). Ubiquitous
expression of sevenless: position-dependent specification of cell fate.
Science 243,931
-934.
Bateman, J. R., Lee, A. M. and Wu, C. T.
(2006). Site-specific transformation of Drosophila via 
C31
integrase-mediated cassette exchange. Genetics
173,769
-777.
Beall, E. L. and Rio, D. C. (1997). Drosophila
P-element transposase is a novel site-specific endonuclease. Genes
Dev. 11,2137
-2151.
Beall, E. L., Mahoney, M. B. and Rio, D. C.
(2002). Identification and analysis of a hyperactive mutant form
of Drosophila P-element transposase. Genetics
162,217
-227.
Bellen, H. J. (1999). Ten years of enhancer
detection: lessons from the fly. Plant Cell
11,2271
-2281.
Bellen, H. J., O'Kane, C. J., Wilson, C., Grossniklaus, U.,
Pearson, R. K. and Gehring, W. J. (1989). P-element-mediated
enhancer detection: a versatile method to study development in Drosophila.
Genes Dev. 3,1288
-1300.
Bellen, H. J., Levis, R. W., Liao, G., He, Y., Carlson, J. W.,
Tsang, G., Evans-Holm, M., Hiesinger, P. R., Schulze, K. L., Rubin, G. M. et
al. (2004). The BDGP gene disruption project: single
transposon insertions associated with 40% of Drosophila genes.
Genetics 167,761
-781.
Belteki, G., Gertsenstein, M., Ow, D. W. and Nagy, A.
(2003). Site-specific cassette exchange and germline transmission
with mouse ES cells expressing C31 integrase. Nat.
Biotechnol. 21,321
-324.[CrossRef][Medline]
Benedict, M. Q., Salazar, C. E. and Collins, F. H. (1995). A new dominant selectable marker for genetic transformation; Hsp70-opd. Insect Biochem. Mol. Biol. 25,1061 -1065.[CrossRef][Medline]
Berghammer, A. J., Klingler, M. and Wimmer, E. A. (1999). A universal marker for transgenic insects. Nature 402,370 -371.[CrossRef][Medline]
Bethke, B. and Sauer, B. (1997). Segmental
genomic replacement by Cre-mediated recombination: genotoxic stress activation
of the p53 promoter in single-copy transformants. Nucleic Acids
Res. 25,2828
-2834.
Beumer, K., Bhattacharyya, G., Bibikova, M., Trautman, J. K. and
Carroll, D. (2006). Efficient gene targeting in Drosophila
with zinc-finger nucleases. Genetics
172,2391
-2403.
Bibikova, M., Beumer, K., Trautman, J. K. and Carroll, D.
(2003). Enhancing gene targeting with designed zinc finger
nucleases. Science 300,764
.
Bier, E. (2005). Drosophila, the golden bug, emerges as a tool for human genetics. Nat. Rev. Genet. 6, 9-23.[Medline]
Bier, E., Vaessin, H., Shepherd, S., Lee, K., McCall, K.,
Barbel, S., Ackerman, L., Carretto, R., Uemura, T. and Grell, E.
(1989). Searching for pattern and mutation in the Drosophila
genome with a P-lacZ vector. Genes Dev.
3,1273
-1287.
Bischof, J., Maeda, R. K., Hediger, M., Karch, F. and Basler,
K. (2007). An optimized transgenesis system for Drosophila
using germ-line-specific C31 integrases. Proc. Natl. Acad.
Sci. USA 104,3312
-3317.
Blackman, R. K., Koehler, M. M., Grimaila, R. and Gelbart, W. M. (1989). Identification of a fully-functional hobo transposable element and its use for germ-line transformation of Drosophila. EMBO J. 8,211 -217.[Medline]
Blair, S. S. (2003). Genetic mosaic techniques
for studying Drosophila development. Development
130,5065
-5072.
Bouhassira, E. E., Westerman, K. and Leboulch, P.
(1997). Transcriptional behavior of LCR enhancer elements
integrated at the same chromosomal locus by recombinase-mediated cassette
exchange. Blood 90,3332
-3344.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118,401 -415.[Abstract]
Cary, L. C., Goebel, M., Corsaro, B. G., Wang, H. G., Rosen, E. and Fraser, M. J. (1989). Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172,156 -169.[CrossRef][Medline]
Castro, J. P. and Carareto, C. M. (2004). Drosophila melanogaster P transposable elements: mechanisms of transposition and regulation. Genetica 121,107 -118.[CrossRef][Medline]
Chen, Z. Y., He, C. Y., Ehrhardt, A. and Kay, M. A. (2003). Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol. Ther. 8,495 -500.[CrossRef][Medline]
Cooley, L., Kelley, R. and Spradling, A.
(1988). Insertional mutagenesis of the Drosophila genome with
single P elements. Science
239,1121
-1128.
Copeland, N. G., Jenkins, N. A. and Court, D. L. (2001). Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2, 769-779.[Medline]
Court, D. L., Sawitzke, J. A. and Thomason, L. C. (2002). Genetic engineering using homologous recombination. Annu. Rev. Genet. 36,361 -388.[CrossRef][Medline]
Dafhnis-Calas, F., Xu, Z., Haines, S., Malla, S. K., Smith, M.
C. and Brown, W. R. (2005). Iterative in vivo assembly of
large and complex transgenes by combining the activities of C31
integrase and Cre recombinase. Nucleic Acids Res.
33, e189.
de Navas, L., Foronda, D., Suzanne, M. and Sanchez-Herrero, E. (2006). A simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in the bithorax complex of Drosophila. Mech. Dev. 123,860 -867.[CrossRef][Medline]
Dietzl, G., Chen, D., Schnorrer, F., Su, K. C., Barinova, Y., Fellner, M., Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S. et al. (2007). A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448,151 -156.[CrossRef][Medline]
Dolphin, C. T. and Hope, I. A. (2006).
Caenorhabditis elegans reporter fusion genes generated by seamless
modification of large genomic DNA clones. Nucleic Acids
Res. 34,e72
.
Egli, D., Yepiskoposyan, H., Selvaraj, A., Balamurugan, K.,
Rajaram, R., Simons, A., Multhaup, G., Mettler, S., Vardanyan, A., Georgiev,
O. et al. (2006). A family knockout of all four Drosophila
metallothioneins reveals a central role in copper homeostasis and
detoxification. Mol. Cell. Biol.
26,2286
-2296.
Ehrhardt, A., Engler, J. A., Xu, H., Cherry, A. M. and Kay, M.
A. (2006). Molecular analysis of chromosomal rearrangements
in mammalian cells after C31-mediated integration. Hum. Gene
Ther. 17,1077
-1094.[CrossRef][Medline]
Feng, Y. Q., Seibler, J., Alami, R., Eisen, A., Westerman, K. A., Leboulch, P., Fiering, S. and Bouhassira, E. E. (1999). Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange. J. Mol. Biol. 292,779 -785.[CrossRef][Medline]
Fischer, J. A., Giniger, E., Maniatis, T. and Ptashne, M. (1988). GAL4 activates transcription in Drosophila. Nature 332,853 -856.[CrossRef][Medline]
Franz, G. and Savakis, C. (1991). Minos, a new
transposable element from Drosophila hydei, is a member of the Tc1-like family
of transposons. Nucleic Acids Res.
19, 6646.
Fridell, Y. W. and Searles, L. L. (1991).
Vermilion as a small selectable marker gene for Drosophila transformation.
Nucleic Acids Res. 19,5082
.
Gloor, G. B., Nassif, N. A., Johnson-Schlitz, D. M., Preston, C.
R. and Engels, W. R. (1991). Targeted gene replacement in
Drosophila via P element-induced gap repair. Science
253,1110
-1117.
Goldberg, D. A., Posakony, J. W. and Maniatis, T. (1983). Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line. Cell 34,59 -73.[CrossRef][Medline]
Golic, K. G. and Golic, M. M. (1996). Engineering the Drosophila genome: chromosome rearrangements by design. Genetics 144,1693 -1711.[Abstract]
Golic, M. M., Rong, Y. S., Petersen, R. B., Lindquist, S. L. and
Golic, K. G. (1997). FLP-mediated DNA mobilization to
specific target sites in Drosophila chromosomes. Nucleic Acids
Res. 25,3665
-3671.
Gong, S., Yang, X. W., Li, C. and Heintz, N.
(2002). Highly efficient modification of bacterial artificial
chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin
of replication. Genome Res.
12,1992
-1998.
Gong, S., Zheng, C., Doughty, M. L., Losos, K., Didkovsky, N., Schambra, U. B., Nowak, N. J., Joyner, A., Leblanc, G., Hatten, M. E. et al. (2003). A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425,917 -925.[CrossRef][Medline]
Gong, W. J. and Golic, K. G. (2003). Ends-out,
or replacement, gene targeting in Drosophila. Proc. Natl. Acad.
Sci. USA 100,2556
-2561.
Greenspan, R. J. (2004). Fly Pushing: The Theory and Practice of Drosophila Genetics (2nd edn). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Groth, A. C., Olivares, E. C., Thyagarajan, B. and Calos, M.
P. (2000). A phage integrase directs efficient site-specific
integration in human cells. Proc. Natl. Acad. Sci. USA
97,5995
-6000.
Groth, A. C., Fish, M., Nusse, R. and Calos, M. P.
(2004). Construction of transgenic Drosophila by using the
site-specific integrase from phage C31. Genetics
166,1775
-1782.
Hacker, U., Nystedt, S., Barmchi, M. P., Horn, C. and Wimmer, E.
A. (2003). piggyBac-based insertional mutagenesis in the
presence of stably integrated P elements in Drosophila. Proc. Natl.
Acad. Sci. USA 100,7720
-7725.
Haenlin, M., Steller, H., Pirrotta, V. and Mohier, E. (1985). A 43 kilobase cosmid P transposon rescues the fs(1)K10 morphogenetic locus and three adjacent Drosophila developmental mutants. Cell 40,827 -837.[CrossRef][Medline]
Handler, A. M. (2002). Use of the piggyBac transposon for germ-line transformation of insects. Insect Biochem. Mol. Biol. 32,1211 -1220.[CrossRef][Medline]
Handler, A. M. and Harrell, R. A. (1999). Germline transformation of Drosophila melanogaster with the piggyBac transposon vector. Insect Mol. Biol. 8, 449-457.[CrossRef][Medline]
Handler, A. M. and James, A. A. (2000). Insect Transgenesis: Methods and Applications. Boca Raton, FL: CRC Press.
Handler, A. M., Gomez, S. P. and O'Brochta, D. A. (1993). A functional analysis of the P-element gene-transfer vector in insects. Arch. Insect Biochem. Physiol. 22,373 -384.[CrossRef][Medline]
Hazelrigg, T., Levis, R. and Rubin, G. M. (1984). Transformation of white locus DNA in drosophila: dosage compensation, zeste interaction, and position effects. Cell 36,469 -481.[CrossRef][Medline]
Heaney, J. D., Rettew, A. N. and Bronson, S. K. (2004). Tissue-specific expression of a BAC transgene targeted to the Hprt locus in mouse embryonic stem cells. Genomics 83,1072 -1082.[CrossRef][Medline]
Heidmann, D. and Lehner, C. F. (2001). Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation. Dev. Genes Evol. 211,458 -465.[CrossRef][Medline]
Heintz, N. (2001). BAC to the future: the use of bac transgenic mice for neuroscience research. Nat. Rev. Neurosci. 2,861 -870.[CrossRef][Medline]
Hoess, R. H., Ziese, M. and Sternberg, N.
(1982). P1 site-specific recombination: nucleotide sequence of
the recombining sites. Proc. Natl. Acad. Sci. USA
79,3398
-3402.
Hoess, R. H., Wierzbicki, A. and Abremski, K.
(1986). The role of the loxP spacer region in P1 site-specific
recombination. Nucleic Acids Res.
14,2287
-2300.
Horn, C. and Wimmer, E. A. (2000). A versatile vector set for animal transgenesis. Dev. Genes Evol. 210,630 -637.[CrossRef][Medline]
Horn, C. and Handler, A. M. (2005).
Site-specific genomic targeting in Drosophila. Proc. Natl. Acad.
Sci. USA 102,12483
-12488.
Horn, C., Jaunich, B. and Wimmer, E. A. (2000). Highly sensitive, fluorescent transformation marker for Drosophila transgenesis. Dev. Genes Evol. 210,623 -629.[CrossRef][Medline]
Horn, C., Schmid, B. G., Pogoda, F. S. and Wimmer, E. A. (2002). Fluorescent transformation markers for insect transgenesis. Insect Biochem. Mol. Biol. 32,1221 -1235.[CrossRef][Medline]
Horn, C., Offen, N., Nystedt, S., Hacker, U. and Wimmer, E.
A. (2003). piggyBac-based insertional mutagenesis and
enhancer detection as a tool for functional insect genomics.
Genetics 163,647
-661.
Ioannou, P. A., Amemiya, C. T., Garnes, J., Kroisel, P. M., Shizuya, H., Chen, C., Batzer, M. A. and de Jong, P. J. (1994). A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nat. Genet. 6, 84-89.[CrossRef][Medline]
Jacobson, J. W., Medhora, M. M. and Hartl, D. L.
(1986). Molecular structure of a somatically unstable
transposable element in Drosophila. Proc. Natl. Acad. Sci.
USA 83,8684
-8688.
Kaminker, J. S., Bergman, C. M., Kronmiller, B., Carlson, J., Svirskas, R., Patel, S., Frise, E., Wheeler, D. A., Lewis, S. E., Rubin, G. M. et al. (2002). The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective. Genome Biol. 3, RESEARCH0084.
Karess, R. E. and Rubin, G. M. (1984). Analysis of P transposable element functions in Drosophila. Cell 38,135 -146.[CrossRef][Medline]
Kaufman, P. D. and Rio, D. C. (1991). Germline
transformation of Drosophila melanogaster by purified P element transposase.
Nucleic Acids Res. 19,6336
.
Klemenz, R., Weber, U. and Gehring, W. J.
(1987). The white gene as a marker in a new P-element vector for
gene transfer in Drosophila. Nucleic Acids Res.
15,3947
-3959.
Kotzamanis, G. and Huxley, C. (2004). Recombining overlapping BACs into a single larger BAC. BMC Biotechnol. 4,1 .[CrossRef][Medline]
Lai, S. L. and Lee, T. (2006). Genetic mosaic with dual binary transcriptional systems in Drosophila. Nat. Neurosci. 9,703 -709.[CrossRef][Medline]
Langer, S. J., Ghafoori, A. P., Byrd, M. and Leinwand, L.
(2002). A genetic screen identifies novel non-compatible loxP
sites. Nucleic Acids Res.
30,3067
-3077.
Lankenau, D. H. and Gloor, G. B. (1998). In vivo gap repair in Drosophila: a one-way street with many destinations. BioEssays 20,317 -327.[CrossRef][Medline]
Le, T., Yu, M., Williams, B., Goel, S., Paul, S. M. and Beitel, G. J. (2007). CaSpeR5, a family of Drosophila transgenesis and shuttle vectors with improved multiple cloning sites. Biotechniques 42,164 , 166.[Medline]
Lee, E. C., Yu, D., Martinez de Velasco, J., Tessarollo, L., Swing, D. A., Court, D. L., Jenkins, N. A. and Copeland, N. G. (2001). A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73,56 -65.[CrossRef][Medline]
Lee, G. and Saito, I. (1998). Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216,55 -65.[CrossRef][Medline]
Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22,451 -461.[CrossRef][Medline]
Levis, R., Hazelrigg, T. and Rubin, G. M.
(1985). Effects of genomic position on the expression of
transduced copies of the white gene of Drosophila.
Science 229,558
-561.
Lidholm, D. A., Lohe, A. R. and Hartl, D. L. (1993). The transposable element mariner mediates germline transformation in Drosophila melanogaster. Genetics 134,859 -868.[Abstract]
Liu, J., Jeppesen, I., Nielsen, K. and Jensen, T. G. (2006). Phi c31 integrase induces chromosomal aberrations in primary human fibroblasts. Gene Ther. 13,1188 -1190.[CrossRef][Medline]
Lockett, T. J., Lewy, D., Holmes, P., Medveczky, K. and Saint, R. (1992). The rough (ro+) gene as a dominant P-element marker in germ line transformation of Drosophila melanogaster. Gene 114,187 -193.[CrossRef][Medline]
Loonstra, A., Vooijs, M., Beverloo, H. B., Allak, B. A., van
Drunen, E., Kanaar, R., Berns, A. and Jonkers, J. (2001).
Growth inhibition and DNA damage induced by Cre recombinase in mammalian
cells. Proc. Natl. Acad. Sci. USA
98,9209
-9214.
Loukeris, T. G., Arca, B., Livadaras, I., Dialektaki, G. and
Savakis, C. (1995). Introduction of the transposable element
Minos into the germ line of Drosophila melanogaster. Proc. Natl.
Acad. Sci. USA 92,9485
-9489.
Lozovsky, E. R., Nurminsky, D., Wimmer, E. A. and Hartl, D.
L. (2002). Unexpected stability of mariner transgenes in
Drosophila. Genetics
160,527
-535.
Matthews, K. A., Kaufman, T. C. and Gelbart, W. M. (2005). Research resources for Drosophila: the expanding universe. Nat. Rev. Genet. 6, 179-193.[CrossRef][Medline]
McGinnis, W., Shermoen, A. W. and Beckendorf, S. K. (1983). A transposable element inserted just 5' to a Drosophila glue protein gene alters gene expression and chromatin structure. Cell 34,75 -84.[CrossRef][Medline]
McLeod, M., Craft, S. and Broach, J. R. (1986).
Identification of the crossover site during FLP-mediated recombination in the
Saccharomyces cerevisiae plasmid 2 microns circle. Mol. Cell
Biol. 6,3357
-3367.
Metaxakis, A., Oehler, S., Klinakis, A. and Savakis, C.
(2005). Minos as a genetic and genomic tool in Drosophila
melanogaster. Genetics
171,571
-581.
Missirlis, P. I., Smailus, D. E. and Holt, R. A. (2006). A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination. BMC Genomics 7, 73.[CrossRef][Medline]
Misulovin, Z., Yang, X. W., Yu, W., Heintz, N. and Meffre, E. (2001). A rapid method for targeted modification and screening of recombinant bacterial artificial chromosome. J. Immunol. Methods 257,99 -105.[CrossRef][Medline]
Muyrers, J. P., Zhang, Y. and Stewart, A. F. (2001). Techniques: recombinogenic engineering - new options for cloning and manipulating DNA. Trends Biochem. Sci. 26,325 -331.[CrossRef][Medline]
Nimmo, D. D., Alphey, L., Meredith, J. M. and Eggleston, P. (2006). High efficiency site-specific genetic engineering of the mosquito genome. Insect Mol. Biol. 15,129 -136.[CrossRef][Medline]
Norga, K. K., Gurganus, M. C., Dilda, C. L., Yamamoto, A., Lyman, R. F., Patel, P. H., Rubin, G. M., Hoskins, R. A., Mackay, T. F. and Bellen, H. J. (2003). Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development. Curr. Biol. 13,1388 -1396.[CrossRef][Medline]
Oberstein, A., Pare, A., Kaplan, L. and Small, S. (2005). Site-specific transgenesis by Cre-mediated recombination in Drosophila. Nat. Methods 2, 583-585.[CrossRef][Medline]
O'Brochta, D. A. and Atkinson, P. W. (1996). Transposable elements and gene transformation in non-drosophilid insects. Insect Biochem. Mol. Biol. 26,739 -753.[CrossRef][Medline]
O'Brochta, D. A., Warren, W. D., Saville, K. J. and Atkinson, P. W. (1996). Hermes, a functional non-Drosophilid insect gene vector from Musca domestica. Genetics 142,907 -914.[Abstract]
O'Kane, C. J. and Gehring, W. J. (1987).
Detection in situ of genomic regulatory elements in Drosophila.
Proc. Natl. Acad. Sci. USA
84,9123
-9127.
O'Keefe, L. V., Smibert, P., Colella, A., Chataway, T. K., Saint, R. and Richards, R. I. (2007). Know thy fly. Trends Genet. 23,238 -242.[CrossRef][Medline]
Parks, A. L., Cook, K. R., Belvin, M., Dompe, N. A., Fawcett, R., Huppert, K., Tan, L. R., Winter, C. G., Bogart, K. P., Deal, J. E. et al. (2004). Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nat. Genet. 36,288 -292.[CrossRef][Medline]
Patton, J. S., Gomes, X. V. and Geyer, P. K.
(1992). Position-independent germline transformation in
Drosophila using a cuticle pigmentation gene as a selectable marker.
Nucleic Acids Res. 20,5859
-5860.
Pirrotta, V. (1988). Vectors for P-mediated transformation in Drosophila. Biotechnology 10,437 -456.[Medline]
Raymond, C. S. and Soriano, P. (2007).
High-efficiency FLP and C31 site-specific recombination in mammalian
cells. PLoS ONE 2,e162
.[CrossRef]
Rio, D. C. and Rubin, G. M. (1988).
Identification and purification of a Drosophila protein that binds to the
terminal 31-base-pair inverted repeats of the P transposable element.
Proc. Natl. Acad. Sci. USA
85,8929
-8933.
Rong, Y. S. and Golic, K. G. (2000). Gene
targeting by homologous recombination in Drosophila.
Science 288,2013
-2018.
Rong, Y. S., Titen, S. W., Xie, H. B., Golic, M. M., Bastiani,
M., Bandyopadhyay, P., Olivera, B. M., Brodsky, M., Rubin, G. M. and Golic, K.
G. (2002). Targeted mutagenesis by homologous recombination
in D. melanogaster. Genes Dev.
16,1568
-1581.
Roseman, R. R., Swan, J. M. and Geyer, P. K. (1995). A Drosophila insulator protein facilitates dosage compensation of the X chromosome min-white gene located at autosomal insertion sites. Development 121,3573 -3582.[Abstract]
Rubin, G. M. and Spradling, A. C. (1982).
Genetic transformation of Drosophila with transposable element vectors.
Science 218,348
-353.
Ryder, E. and Russell, S. (2003). Transposable
elements as tools for genomics and genetics in Drosophila. Brief.
Funct. Genomic. Proteomic. 2,57
-71.
Ryder, E., Blows, F., Ashburner, M., Bautista-Llacer, R.,
Coulson, D., Drummond, J., Webster, J., Gubb, D., Gunton, N., Johnson, G. et
al. (2004). The DrosDel collection: a set of P-element
insertions for generating custom chromosomal aberrations in Drosophila
melanogaster. Genetics
167,797
-813.
Sarkar, A., Atapattu, A., Belikoff, E. J., Heinrich, J. C., Li, X., Horn, C., Wimmer, E. A. and Scott, M. J. (2006). Insulated piggyBac vectors for insect transgenesis. BMC Biotechnol. 6,27 .[CrossRef][Medline]
Sarov, M., Schneider, S., Pozniakovski, A., Roguev, A., Ernst, S., Zhang, Y., Hyman, A. A. and Stewart, A. F. (2006). A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nat. Methods 3,839 -844.[CrossRef][Medline]
Sawitzke, J. A., Thomason, L. C., Costantino, N., Bubunenko, M., Datta, S. and Court, D. L. (2007). Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Meth. Enzymol. 421,171 -199.[Medline]
Schlake, T. and Bode, J. (1994). Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33,12746 -12751.[CrossRef][Medline]
Schmidt, E. E., Taylor, D. S., Prigge, J. R., Barnett, S. and
Capecchi, M. R. (2000). Illegitimate Cre-dependent chromosome
rearrangements in transgenic mouse spermatids. Proc. Natl. Acad.
Sci. USA 97,13702
-13707.
Seibler, J. and Bode, J. (1997). Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay. Biochemistry 36,1740 -1747.[CrossRef][Medline]
Seibler, J., Schubeler, D., Fiering, S., Groudine, M. and Bode, J. (1998). DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs. Biochemistry 37,6229 -6234.[CrossRef][Medline]
Senecoff, J. F., Rossmeissl, P. J. and Cox, M. M. (1988). DNA recognition by the FLP recombinase of the yeast 2 mu plasmid. A mutational analysis of the FLP binding site. J. Mol. Biol. 201,405 -421.[CrossRef][Medline]
Sepp, K. J. and Auld, V. J. (1999). Conversion
of lacZ enhancer trap lines to GAL4 lines using targeted transposition in
Drosophila melanogaster. Genetics
151,1093
-1101.
Shizuya, H., Birren, B., Kim, U. J., Mancino, V., Slepak, T.,
Tachiiri, Y. and Simon, M. (1992). Cloning and stable
maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli
using an F-factor-based vector. Proc. Natl. Acad. Sci.
USA 89,8794
-8797.
Siegal, M. L. and Hartl, D. L. (1996). Transgene Coplacement and high efficiency site-specific recombination with the Cre/loxP system in Drosophila. Genetics 144,715 -726.[Abstract]
Siegal, M. L. and Hartl, D. L. (2000). Application of Cre/loxP in Drosophila. Site-specific recombination and transgene coplacement. Methods Mol. Biol. 136,487 -495.[Medline]
Smith, D., Wohlgemuth, J., Calvi, B. R., Franklin, I. and Gelbart, W. M. (1993). hobo enhancer trapping mutagenesis in Drosophila reveals an insertion specificity different from P elements. Genetics 135,1063 -1076.[Abstract]
Sorrell, D. A. and Kolb, A. F. (2005). Targeted modification of mammalian genomes. Biotechnol. Adv. 23,431 -469.[CrossRef][Medline]
Spradling, A. C., Stern, D. M., Kiss, I., Roote, J., Laverty, T.
and Rubin, G. M. (1995). Gene disruptions using P
transposable elements: an integral component of the Drosophila genome project.
Proc. Natl. Acad. Sci. USA
92,10824
-10830.
Steller, H. and Pirrotta, V. (1985). A transposable P vector that confers selectable G418 resistance to Drosophila larvae. EMBO J. 4,167 -171.[Medline]
Sternberg, N. (1990). Bacteriophage P1 cloning
system for the isolation, amplification, and recovery of DNA fragments as
large as 100 kilobase pairs. Proc. Natl. Acad. Sci.
USA 87,103
-107.
Stilwell, G. E., Rocheleau, T. and Ffrench-Constant, R. H. (1995). GABA receptor minigene rescues insecticide resistance phenotypes in Drosophila. J. Mol. Biol. 253,223 -227.[CrossRef][Medline]
Sundararajan, P., Atkinson, P. W. and O'Brochta, D. A. (1999). Transposable element interactions in insects: crossmobilization of hobo and Hermes. Insect Mol. Biol. 8,359 -368.[CrossRef][Medline]
Takeuchi, H., Georgiev, O., Fetchko, M., Kappeler, M.,
Schaffner, W. and Egli, D. (2007). In vivo construction of
transgenes in Drosophila. Genetics
175,2019
-2028.
Thibault, S. T., Singer, M. A., Miyazaki, W. Y., Milash, B., Dompe, N. A., Singh, C. M., Buchholz, R., Demsky, M., Fawcett, R., Francis-Lang, H. L. et al. (2004). A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat. Genet. 36,283 -287.[CrossRef][Medline]
Thomason, L. C., Calendar, R. and Ow, D. W. (2001). Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system. Mol. Genet. Genomics 265,1031 -1038.[CrossRef][Medline]
Thorpe, H. M. and Smith, M. C. (1998). In vitro
site-specific integration of bacteriophage DNA catalyzed by a recombinase of
the resolvase/invertase family. Proc. Natl. Acad. Sci.
USA 95,5505
-5510.
Thummel, C. S. and Pirrotta, V. (1992). New pCaSpeR P element vectors. Drosoph. Inf. Serv. 71, 150.
Thyagarajan, B., Guimaraes, M. J., Groth, A. C. and Calos, M. P. (2000). Mammalian genomes contain active recombinase recognition sites. Gene 244, 47-54.[CrossRef][Medline]
Thyagarajan, B., Olivares, E. C., Hollis, R. P., Ginsburg, D. S.
and Calos, M. P. (2001). Site-specific genomic integration in
mammalian cells mediated by phage C31 integrase. Mol. Cell.
Biol. 21,3926
-3934.
Venken, K. J. and Bellen, H. J. (2005). Emerging technologies for gene manipulation in Drosophila melanogaster. Nat. Rev. Genet. 6,167 -178.[Medline]
Venken, K. J., He, Y., Hoskins, R. A. and Bellen, H. J.
(2006). P[acman]: a BAC transgenic platform for targeted
insertion of large DNA fragments in D. melanogaster.
Science 314,1747
-1751.
Vidal, M. and Cagan, R. L. (2006). Drosophila models for cancer research. Curr. Opin. Genet. Dev. 16, 10-16.[CrossRef][Medline]
Warming, S., Costantino, N., Court, D. L., Jenkins, N. A. and
Copeland, N. G. (2005). Simple and highly efficient BAC
recombineering using galK selection. Nucleic Acids
Res. 33,e36
.
Warren, W. D., Atkinson, P. W. and O'Brochta, D. A. (1994). The Hermes transposable element from the house fly, Musca domestica, is a short inverted repeat-type element of the hobo, Ac, and Tam3 (hAT) element family. Genet. Res. 64, 87-97.[Medline]
Wild, J., Hradecna, Z. and Szybalski, W.
(2002). Conditionally amplifiable BACs: switching from
single-copy to high-copy vectors and genomic clones. Genome
Res. 12,1434
-1444.
Wong, Q. N., Ng, V. C., Lin, M. C., Kung, H. F., Chan, D. and
Huang, J. D. (2005). Efficient and seamless DNA
recombineering using a thymidylate synthase A selection system in Escherichia
coli. Nucleic Acids Res.
33, e59.
Xu, T. and Rubin, G. M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117,1223 -1237.[Abstract]
Yang, X. W., Model, P. and Heintz, N. (1997). Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotechnol. 15,859 -865.[CrossRef][Medline]
Zappe, S., Fish, M., Scott, M. P. and Solgaard, O. (2006). Automated MEMS-based Drosophila embryo injection system for high-throughput RNAi screens. Lab Chip 6,1012 -1019.[CrossRef][Medline]
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