Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.106.065920

In Vivo Construction of Transgenes in Drosophila

Hajime Takeuchi,1 Oleg Georgiev, Michael Fetchko, Michael Kappeler,Walter Schaffner and Dieter Egli2

Institute of Molecular Biology, University of Zurich, Zurich CH-8057, Switzerland

Manuscript received September 17, 2006Accepted for publication December 14, 2006

ABSTRACT

Transgenic flies are generated by transposon-mediated transformation. A drawback of this approach isthe size limit of transposable elements. Here, we propose a novel method that allows the extension oftransgenes in vivo. This method is based on an incomplete transgene that has been constructed in vitroand integrated into the Drosophila genome by conventional transgenesis. The incomplete transgenecontains two short stretches of DNA homologous to the 59- and 39-ends of a larger DNA segment ofinterest. Between the short stretches of homology an I-SceI recognition site is located. Once activated,I-SceI endonuclease introduces a DNA double-strand break, which triggers ectopic recombination betweenthe stretches of homology and the endogenous locus. Through gap repair, the transgene obtains thecomplete region of interest in vivo. Our results show that this method is effective for copying up to 28 kbof genomic DNA into the transgene, thereby eliminating the technical difficulties associated with thein vitro construction of large transgenes and extending the size limits of current transgenesis protocols. Ingeneral, this method may be a useful technique for genetic engineering of eukaryotic model organisms.

IN Drosophila, transgenes can be integrated into thegenome by mobile elements, such as P elements

and piggyBacs (Rubin and Spradling 1982; Wimmer

2003). Transposon-mediated transgenesis is limited insize as the frequency of integration decreases dramat-ically with increasing size of the mobile element(Spradling 1986). It is inefficient to transform flieswith mobile elements that are .20–30 kb. The largestP elements that have been directly introduced into theDrosophila genome were based on cosmid vectors, set-ting an arbitrary upper limit of �40 kb (Haenlin et al.1985). A promising new approach for Drosophila trans-genesis is to generate transgenic flies by FC31-, Cre- orFlp-mediated site-specific recombination (Groth et al.2004; Horn and Handler 2005; Oberstein et al. 2005;Venken et al. 2006).

A remarkable ability of the cell is that its homologousrecombination (HR) machinery can find a templateDNA located anywhere in the whole genome. Areas ofhomology can be found and used as a template whetherthe homology resides on the sister chromatid, on thehomologous chromosome, at a nonallelic ectopic posi-tion in the genome, or even on an injected DNA (Banga

and Boyd 1992; Nassif et al. 1994; Keeler et al. 1996;Lankenau and Gloor 1998; Rong and Golic 2003).

Several techniques that take advantage of this abilityhave been developed to modify the genome in a tar-geted manner in model organisms from yeast to mouse(Capecchi 1989; Jasin 1996; Rong and Golic 2000;Egli et al. 2004). HR is also used to generate recombi-nant constructs in yeast and bacteria, thus alleviatingthe difficulties associated with cloning using restrictionenzymes (Zhang et al. 2000; Copeland et al. 2001;Muyrers et al. 2001).

Here, we propose a novel efficient method wherebywe can introduce long DNA segments into transgeneloci by HR. Furthermore, we show that HR may becoupled to nonhomologous end joining (NHEJ) evenafter extensive DNA repair synthesis at both ends of thebreak.

MATERIALS AND METHODS

DNA constructs: Constructs were made using the P-elementvector pTARG (GenBank accession no. DQ269206) andtransformed by micro-injection. The 59 part of the yellow gene(�2869–11608, with numbers referring to the transcriptionalinitiation site of yellow) was cloned into the SalI–SphI positionof pTARG. An I-SceI site was inserted at the SphI site and theresulting vector was termed pTARG-N. The following seg-ments downstream of the yellow gene were amplified byPCR: segment F (112,069–115,762), segment G (129,411–134,985), segment K (149,656–153,914), and segment J(187,554–192,103). PCR products were blunted with T4 DNApolymerase, digested with SphI, and cloned into the SphI–StuIsites of the pTARG-N plasmid. All constructs were namedaccording to their downstream segments of ‘‘F,’’ ‘‘G,’’ ‘‘K,’’ and‘‘J,’’ respectively. The construct ‘‘F–R’’ is derived from ‘‘F’’ by

1Present address: Faculty of Pharmaceutical Sciences, Kagawa Campus,Tokushima Bunri University, Kagawa 769-2193, Japan.

2Corresponding author: Department of Molecular and Cellular Biology,Harvard University, 437 Fairchild, 7 Divinity Ave., Cambridge, MA02138. E-mail: degli@mcb.harvard.edu

Genetics 175: 2019–2028 (April 2007)

the elimination of an EcoRI fragment and therefore contains ashorter segment from 112,069 to 112,627 from the transcrip-tional initiation site of yellow. The integration site of F–R2,F–R5, F1, G2, or J was determined by inverse PCR and found tobe at 33B1, 99E4, 7E6/7, 80A1, or 55C2, respectively. InversePCR was performed using primer pairs, Plac1–Plac4 and Pry1–Pry2, as described in http://www.fruitfly.org/ and PCR prod-ucts, were sequenced with primer P* as described in http://www.fruitfly.org/. The integration sites of other transgeneswere not identified. The two segments of the Cg25C constructcorrespond to the region from �17091 to �15063 and from14150 to 17103 from the transcription initiation site of theCg25C gene. The 59 segment was amplified and cloned into theSphI and NotI sites in pTARG. The 39 segment of this constructwas amplified and cloned into the MluI and SphI sites. An I-SceIsite was inserted at the SphI site. The red fluorescent protein(RFP) (dsRed), was cloned into the MluI site of the Cg25Cconstruct. The details of all primers are shown in supplemen-tal Table 1 at http://www.genetics.org/supplemental/.

Fly stocks and genetics: The stock y1 w1118; P[v1, 70I-SceI] isderived from y1 w1118;P[ry1, 70FLP]4 P[v1, 70I-SceI]2B Sco/S2CyO, kindly provided by Y. Rong and K. Golic (Rong et al.2002). Df(1)y3PLsc8R and Df(1)y3PLsc4R/D49 was provided by JuanModolell (Campuzano et al. 1985). Flies carrying a heat-inducible I-SceI gene and one of the constructs—F, F–R, G,K, J, or N—as transgenes were generated by crossing. Theywere kept in vials to lay eggs for 6 hr at 26�. A heat shock (at 38�for 1 hr) was given to offspring at the time points indicated.Subsequently, all heat-shocked flies were singly mated to y1

w1118 to analyze gap repair efficiency in germline cells.Molecular characterization of double-strand break repair

events: Using genomic DNA, gap repair events were analyzedby PCR using the primers shown in Figure 1C and supple-mental Table 1 at http://www.genetics.org/supplemental/.Gap repair events of the Cg25C construct were analyzed byPCR using a primer located in RFP (59-GTA CTG GAA CTGGGG GGA CAG-39) and a primer in the endogenous Cg25locus (59-CAG GGC GCT GTC GGA GTA CC-39) within the gapto be completed by HR. Events obtained by heat shockingat different developmental stages as well as with differentnumbers of heat shocks were included in the molecularanalysis.

RFP expression analysis and microscopy: For the analysis ofthe expression pattern of Cg25C-RFP, second instar Drosoph-ila larvae were photographed using a Leica DRB fluorescencestereomicroscope equipped with a Zeiss Axiocam.

RESULTS

In vivo assembly of transgenes by homologous recom-bination: The in vivo assembly of transgenes requires an

incomplete construct, made in vitro and containing twoDNA segments derived from two distant sequenceslocated in the chromosomal region of interest and anI-SceI recognition site between these segments (Figure1A). The entire assembly is inserted within a mobileelement vector for conventional transgenesis. In fliescarrying these constructs, the expression of I-SceI endo-nuclease leads to a double-strand break (DSB). Eachend at the breakpoint invades the homologous locusand initiates DNA synthesis. When synthesized DNAfrom each end reaches the complementary region, thetwo ends anneal to each other to restore a continuousstrand, a process referred to as gap repair (see alsoNassif et al. 1994). Thereby the sequence between thetwo pieces of DNA is copied from the endogenous locusinto the mobile element (Figure1A).

A genetic selection system for the in vivo construc-tion of transgenes in Drosophila: To test this system, weused the yellow–ASC region on the Drosophila X chro-mosome because of its extensive genetic characteriza-tion and the availability of mutations causing visiblephenotypes (Garcia-Bellido 1979; Ruiz-Gomez andModolell 1987). Several fly strains with w1-markedmobile elements carrying the wild-type 59 part of theyellow gene with one of several downstream segments—F, F–R, G, K, or J—were constructed (Figure 1B). Fliescarrying one of those constructs within a y1 mutant back-ground are y�w1 as the 59 part of the yellow gene in themobile element is truncated and therefore nonfunc-tional. The downstream segments of the construct are,when aligned with the X chromosome, separated fromthe 59 yellow segment by 10.5 kb for F and F–R, 27.8 kbfor G, 48.0 kb for K, and 85.9 kb for J. The differencebetween F and F–R is the size of the downstreamsegment (Figure 1C).

We induced DSBs by heat-shock-inducible I-SceI ex-pression. DSB repair using the endogenous y1 locus as atemplate restored a functional yellow gene within thetransgene, resulting in flies mosaic for the y1 gene. y1

patches indicate that the 39 part of the yellow gene hasbeen copied into the transgene (Figure 1D). The fre-quencies of y1 retrieval in the germline of these mosaicflies were determined by singly crossing them to y w flies

Figure 1.—(A) Principle of the construction of transgenes in vivo. To copy a desired region from the endogenous locus into atransgene, two segments (blue and orange boxes) at both ends of the target DNA are cloned into an insect transformation vector.An I-SceI recognition site connects the two segments. This element is integrated into the Drosophila genome at a position differentfrom the locus of interest. By crossing with flies expressing the endonuclease I-SceI, a DSB is introduced in the transgene betweenthe two segments. Subsequently, the transgene acquires the missing region from the endogenous locus by gap repair. (B–D) In vivoconstruction of transgenes of the yellow–ASC locus. (B) Segments used for cloning are indicated by bars and labeled with 59 yellow(blue), F, F–R, G, K, and J (orange). The size of each segment is indicated in kilobases. The w marker gene serves to identifytransgenic flies. (C) yellow–ASC genomic region. The positions of each segment and primers for the molecular characterizationof gap repair events are indicated. The y1 mutation is a point mutation in the yellow start codon; the mobile element constructs,however, contain a wild-type start codon. Primer numbers indicate the position relative to and downstream of the y transcriptionstart. (D) DSB repair produced a mosaic phenotype. This fly carries both the F construct and the heat-shock-inducible I-SceI trans-gene. A heat shock has been applied at larval stages to induce DSBs. Dark patches (y1) are gap repair events (green arrows). Thesymbols in the boxed legend are also used in Figures 2 and 3.

:

2020 H. Takeuchi et al.

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and screening their offspring for y1 (Tables 1 and 2).These results can be summarized as follows:

1. y1 rescue events occurred in the constructs with an11-, 28-, or a 48-kb gap but only rarely with an 86-kbgap.

2. The frequency of copying a y1 gene into the P-element locus decreases when the size of the gapincreases. This is particularly clear when the y1 rescuefrequencies obtained with insertions of F are com-pared with the ones obtained with insertions of Gor K.

3. The frequency of y1 rescue varies considerably withrespect to the insertion site of the mobile element inthe genome.

4. A segment of as little as 560 bp on one side of thebreak together with a longer segment on the otherside is able to support efficient copying of 11 kb ofDNA from the endogenous locus. Such a decrease in

homology is accompanied by a decrease in copyingefficiency (compare the average frequency of F withthe one for F–R).

5. The frequency of y1 rescue also depends on the de-velopmental stage at which the DSBs were induced,especially in the male germline where a heat shockat the first instar appears to result in the highestfrequency (Table 2). These variations may also reflectthe efficiency of the heat shock in inducing I-SceIexpression.

We also wanted to know whether a single arm ofhomology is sufficient for invading the homologoustemplate, initiating DNA synthesis, and copying anentire locus into the DSB. To address this question, weused a construct, termed ‘‘N,’’ with only the 59 segmentof the yellow gene but without a segment of downstreamhomology. In this case, a single arm would invade theyellow locus, start DNA synthesis, and possibly retrieve

TABLE 1

y1 reconstitution efficiency of different constructs and insertions

Transgene Fly line Sex% events/

crosses% y1 in total

offspring

% averageefficiency of

the constructs(events/crosses)

F1 (11-kb gap) F1 (X chromosome) M 100 (19/19) 15.7 (147/932) 60F 69 (24/35) 11.0 (438/3957)

F1–2 (second chromosome) M 28 (7/25) 0.79 (13/1633)F 25 (1/4) 0.37 (1/271)

F1–3 (third chromosome) M 52 (26/50) 1.95 (59/2958)F 86 (6/7) 3.66 (19/500)

F–R (11-kb gap) F–R2 (second chromosome) M 12 (4/33) 0.45 (9/1980)

17

F 24 (8/33) 0.50 (12/2395)F–R3 (second chromosome) M 23 (22/95) 0.58 (40/6870)

F 27 (21/79) 0.82 (54/6599)F–R4 (second chromosome) M 6 (1/18) 0.091 (1/1096)

F 13 (2/16) 0.50 (6/1173)F–R5 (third chromosome) M 11 (9/85) 0.16 (10/6218)

F 22 (19/86) 0.66 (40/6043)

G (28-kb gap) G1 (third chromosome) M 15 (12/80) 0.28 (21/7366)

13F 13.5 (10/74) 0.29 (16/5564)

G2 (third chromosome) M 0 (0/38) 0 (0/2119)F 23 (9/40) 0.29 (9/3018)

K (48-kb gap) K 3.8 (second chromosome) M 10 (12/118) 0.29 (30/10225)9.5

F 9 (9/102) 0.32 (28/8518)

J (87-kb gap) J1 (second chromosome)a ND 2.5 (11/450) 0.04 (11/30000) 2.5

N (one arm of homology) N2–3 ND 0.2 (1/500) 0.0022 (1/45000) 0.2

DSBs were generated by the heat-shock-inducible expression (1 hr at 38�) of the I-SceI endonuclease at the early third instarstage. F1, F1–2, F1–3, F–R2, F–R3, F–R4, F–R5, G1, G2, K3.8, J1, and N2–3 are transgenic fly lines for the corresponding constructsF, F–R, G, K, J, and N. M, male; F, female. The average efficiency for each construct was obtained by averaging the efficiency of eachinsertion of a particular construct. A single tube contains �80–120 flies of the relevant genotype. ND, not determined.

a For the J and the N construct, three instead of one heat shock (1 hr 38�) was applied on subsequent days. The differencebetween the sexes was not addressed for the insertions of the J or the N construct.

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the entire y locus. Indeed, among 45,000 chromosomesor 500 tubes screened, we recovered a single y1 event(0.0022% of all chromosomes or 0.2% of all tubes)(Table 1).

A DNA segment of up to 28 kb can be integrated intoa transgene in vivo: We further analyzed the y1 events bygenetic and molecular means (Figure 2, Table 3).Before inducing gap repair, none of the initial con-structs were able to rescue a mutation in the achaetelocus. Upon gap repair, the transgene obtained a part ofthe ASC locus whose size depends on the position ofthe downstream segment (Figure 1C). To test for pheno-typic rescue of achaete, we crossed the y1 events tothe deficiency Df(1)y3PLsc8R, which deletes the yellow andachaete loci. All the y1 events derived from the constructwith an 11-kb gap (F) that were tested rescued achaetemutants, whereas 22 of 29 (76%) independent y1 eventsfrom the construct with a 28-kb gap rescued achaete.However, none of 6 tested y1 events derived from theconstruct with a 48-kb gap rescued achaete nor did thesingle y1 event obtained with the N construct that lacks adownstream homology segment. Interestingly, 3 of the11 J-derived events were able to complement a mutationin the achaete locus. Events that did not rescue achaetewere considered as incomplete.

For the molecular analysis, we checked several y1

events by PCR using primer pairs indicated in Figure1C and supplemental Table 1 at http://www.genetics.org/supplemental/. PCR was performed in a Df(1)y3PLsc8R

background, which deletes the endogenous chromo-somal PCR template. Events obtained with the 48-kb gapand 86-kb gap constructs K and J were analyzed byquantitative PCR. Of 35 events that rescued both theyellow and the achaete gene derived from both the 11-kbgap construct (F) and the 28-kb gap construct (G), 34carried a completely copied yellow–ASC region in thetransgene (Figure 2A, Table 3). Sequencing of in vivo-cloned DNA did not show any evidence for smalldeletions or point mutations (data not shown). How-ever, none of the yellow or even the yellow–achaete rescueevents obtained with either the 48-kb gap or the 86-kbgap construct were complete. Accordingly, both genetic

and molecular data show that DNA sequences up to 28kb can be faithfully copied into transgenes by HR.

A two-side invasion can be resolved by NHEJ to yieldincomplete gap repair events: Gap repair events classi-fied as incomplete by genetic and molecular analysiscontained a single large deletion of several kilobases. Allof these events, however, had copied DNA from theendogenous locus at both ends of the break (examplesin Figure 2B). To examine the nature of partial gap re-pair events, junctions of two independent events de-rived from the construct with a 28-kb gap were amplifiedusing PCR and sequenced. Alignment with the wild-typegenomic sequence revealed a junction without ho-mology at the junction point (Figure 2C). Apparently,both ends invaded the homologous template and, afterextensive DNA synthesis, they ligated by NHEJ. Exten-sive DNA synthesis followed by NHEJ was also found forevents obtained with the 86-kb gap construct. Three ofthese were able to rescue the achaete locus, suggestingthat .10 kb was added to one end of the break beforeligation of the nonhomologous ends.

The endogenous template locus usually remains un-changed: Recombination between the endogenous lo-cus and the DNA sequence contained in the transgenecould potentially result in the modification of the endo-genous locus. While this could be useful for other ap-plications, it would complicate the selection system forthe construction of transgenes in vivo. We thereforewanted to know how frequent modifications of theendogenous locus occur in our system. To this end, wescreened for the correction of the CTG point mutationat the endogenous y1 locus by the correct ATG start co-don sequence contained in the transgene. In two inde-pendent cases among a total of 134,000 flies screened, awild-type y1 gene was restored at the endogenous locus,demonstrating that genetic information can flow fromthe broken strand to the intact DNA strand, albeit at avery low efficiency. This finding is in agreement withprevious observations in both mammals and flies thatthe template strand for DNA repair usually remainsunchanged (Engels et al. 1990; Richardson et al.1998). We conclude that the inefficiency of this process

TABLE 2

y1 reconstitution efficiency at different developmental stages

% events/crosses % y1 in total offspring (y1 flies/total no.)

Construct Sex EmbryoFirst

instarSecondinstar

Thirdinstar Embryo First instar Second instar Third instar

F–R3 M 62 (31/50) 74 (37/50) 59 (20/34) 23 (22/95) 4.6 (201/4390) 6.5 (282/4319) 4.7 (106/2269) 0.6 (40/6870)F 32 (15/47) 38 (13/34) 40 (14/50) 27 (21/79) 0.8 (38/4736) 1.3 (45/3437) 1.5 (40/2687) 0.8 (54/6599)

F–R5 M 10 (5/50) 48 (24/50) 28 (14/50) 11 (9/85) 0.8 (35/4421) 1.5 (66/4511) 0.7 (21/2824) 0.16 (10/6218)F 27 (14/51) 30 (15/50) 45 (20/44) 22 (19/86) 1.3 (54/4118) 1.1 (50/4739) 1.4 (47/3320) 0.65 (40/6043)

DSBs were induced at various developmental stages as indicated (1 hr 38�). F–R3 and F–R5 are transgenic fly lines for the cor-responding construct F–R.

Transgene Integration in Drosophila 2023

is unlikely to confound the use of HR for the completionof transgenes in vivo.

A complete transgene can be selected for by re-instating a fluorescent protein reporter: To test whetherthis method can be applied to a gene located elsewherein the genome, we generated transgenic flies carrying acopy of a partial Cg25C gene, the endogenous copy ofwhich is located on the second chromosome. Unlikewith the y locus, we used a wild-type copy of Cg25C as atemplate and therefore could not directly select for agenetic rescue by the transgene. Instead, in this partialconstruct, the segment corresponding to the 39-end ofthe coding region was fused in frame to an RFP (Figure3, A and B). Due to the incompleteness of the transgene,RFP was not expressed before the gap repair. Inductionof DSBs, however, gave rise to flies expressing RFP inthe larval fat body, as reported previously for theexpression of the endogenous Cg25C gene (Figure3C) (Yasothornsrikul et al. 1997). Using two differentP elements inserted on the second chromosome, RFP-expressing flies were recovered in 21.5% (9/42) of thecrosses or at a frequency of 2.5% (31/1249) of total fliesfor the first line and in 0.92% (3/325) of total flies forthe second line. Another line on the third chromosomedid not yield RFP-expressing flies among 600 fliesscreened. Molecular analysis of these gap repair eventsat the breakpoint revealed the integration of DNA fromthe endogenous Cg25C locus into the gap, therebyexcluding the possibility that integration of DNA from

Figure 2.—Examples of complete and partial gap repairevents derived from the construct with a 28-kb gap (G). (A)PCR of three (lanes 1–3) complete events in the Df(1)y3PLsc8R

background. The overlapping PCR products span a total of24.4 kb corresponding to the downstream region of y. Theprimer pairs used are indicated on the left (see also Figure1C). The size of the PCR product is indicated below theprimer numbers. M, size marker. Primer numbers indicatethe position relative to and downstream of the y transcriptionstart. (B) Examples of two incomplete gap repair events (‘‘#4’’and ‘‘#6’’). The in vivo-cloned DNA is in black with the sizeindicated above each segment. Orange and blue bars repre-sent segments of the starting construct. The chromosomal re-gion is shown below. Note that a considerable amount of DNAhas been integrated at both ends of the DSB, but a gap of .15kb remains. (C) Alignment of the DNA sequence at the junc-tion of the events 4 and 6 with wild-type genomic DNA. Thenumber indicates the length of the wild-type sequence notshown. Note the absence of (micro-)homology at the junctionpoint.

TABLE 3

Completeness of gap repair

Transgene Fly line

% offspringwith complete

gap repair

F (11-kb gap) F1 (X chromosome) 100 (12/12)F1–2 (second chromosome) 100 (1/1)F1–3 (third chromosome) 91 (10/11)

F–R (11-kb gap) F–R2 (second chromosome) 83 (5/6)F–R3 (second chromosome) 100 (9/9)F–R4 (second chromosome) 100 (1/1)F–R5 (third chromosome) 100 (5/5)

G (28-kb gap) G1 (third chromosome) 73 (11/15)G2 (third chromosome) 79 (11/14)

K (48-kb gap) K3.8 (second chromosome) 0 (0/6)

J (87-kb gap) J1 (X chromosome) 0 (0/11)

Numbers represent independent y1 events with a com-pletely copied region in percentage of all y1 events. Complete-ness of gap repair was analyzed by both genetic and molecularmeans. F1, F1–2, F1–3, F–R2, F–R3, F–R4, F–R5, G1, G2, K3.8,and J1 are transgenic fly lines for the corresponding con-structs F, F–R, G, K, and J. Incomplete in vivo cloning eventscarry a single large deletion (Figures 2 and 4).

2024 H. Takeuchi et al.

another nonhomologous locus by nonhomologous re-combination resulted in the expression of RFP (Figure3D). As the correct expression of a Cg25C–RFP fusionprotein requires the integration of the promoter as wellas the maintenance of the reading frame and the correctsplicing of six exons, it appears very likely that the entireCg25C gene was faithfully integrated into the transgene.

DISCUSSION

Several loci in the Drosophila genome are large,extending 40 kb in the case of Notch (Artavanis-Tsakonas et al. 1983) and 50 kb in the case of broad(DiBello et al. 1991), or very large, extending 90 kb inthe case of Cadherin-N (Iwai et al. 1997). We demon-strated that gap repair permits the efficient introduc-tion of an �30-kb DNA segment into a transgene thatalready contains part of the locus of interest. Togetherwith preexisting segments whose length is limited bythe limits of mobile-element-mediated transgenesis to�30 kb, a fly carrying a transgene of at least 60 kb may beconstructed. This would allow the genetic analysis oflarge genes and their regulatory regions. Possibly, thecurrent size limit of the in vivo extension of transgenescan be overcome by the use of three or more segmentsthat are linked by different meganuclease restrictionsites that can be used in successive rounds of extension.The availability of an increasing number of molecularlyand phenotypically characterized Drosophila mutantspermits a genetic selection strategy for complete trans-genes, similar to the one demonstrated in this studyfor y1, where the transgene contains part of the genespanning the region harboring a mutation in theendogenous gene. In other cases, the generation of afluorescent fusion protein may be desirable, requiring,

however, a strong promoter for detectable fluorescence.Our system therefore allows the generation of rescueand transcriptional reporter constructs and of fusionproteins. Also, the method presented could simplifycloning of long DNA segments by PCR and restrictionenzymes that may be associated with time-consumingdifficulties when DNA segments reach a size of severalkilobases. Rather than cloning a single large segmentinto the transgene vector, two short and incompletesegments can be cloned and the cloning process canthen be completed in vivo. A major challenge to thistechnique may be to conveniently sort out incompletefrom complete gap repair events especially when thesegment of interest is large. A clever choice of thesegments used for the construction of the partial trans-gene certainly can facilitate the secondary and necessaryselection step for full-length transgenes. Such an assaycan be genetic, PCR based, or a Southern blot.

In addition to introducing a new technique toDrosophila, this study also contributes to the under-standing of DNA repair mechanisms. It appears that inDrosophila, repair DNA synthesis tracts are limited to,50 kb. This finding is also reported by an indepen-dent study that shows efficient filling of an 11-kb gap,inefficient filling of a 43-kb gap, and no complete fillingof a 210-kb gap ( Johnson-Schlitz and Engels 2006).Such a limitation appears to be unlike that in yeast,where an entire chromosomal arm can be copied intoa double-strand break by break-induced replication(Morrow et al. 1997). Probably a low processivity ofDNA repair synthesis accounts for such an upper limit.It appears that repair synthesis proceeds throughmultiple cycles of strand invasion and dissociation(McVey et al. 2004). Each cycle may be an opportunityto complete the repair event by either a homologous or a

Figure 3.—A fluorescent-protein-basedselection system for the construction oftransgenes in vivo. (A) A partial P-elementconstruct. RFP is fused to a 39 segment(blue) of the target gene. (B) Cg25C geno-mic region. Black boxes indicate thecoding region and the left arrow indicatesthe transcription start of Cg25C. An orangeand a blue bar indicate the genomic posi-tion of each segment used for cloning.(C) Expression pattern of the in vivo-completed Cg25C–RFP fusion transgene.Shown are images of second instar larvaecarrying either a copy of the in vivo-completed Cg25C–RFP construct (left) or acopy of the original transgene showing onlyauto-fluorescence of the intestinal tract(right). (D) Molecular analysis of gap repairevents by PCR using the primers indicatedbyarrowheads inAandB. Lanes1and2 rep-resent independent gap repair events andlane 3 represents the transgenic fly beforegap repair. The product is of the expectedsize of 3.6 kb. M, size marker. From top tobottom: 5, 4, 3, and 2 kb.

Transgene Integration in Drosophila 2025

nonhomologous repair pathway. Such a coupling of HRto NHEJ has been reported previously (Richardson andJasin 2000). If this coupling were efficient, we wouldexpect similar frequencies of y1 recovery at differentgap sizes, assuming that strand invasion does not dependon the distance between the different targets. We find,however, a dramatic reduction in y1 rescue events with in-creasing gap size, possibly because NHEJ is not as efficientin sealing the break as when the entire gap is filled andsealing can occur by homologous end joining. In-terestingly, the incomplete gap repair events that weanalyzed have all invaded and initiated DNA synthesisat both ends of the break. Most likely, these incompletegap repair events reflect how complete repair eventsare initiated. We therefore suggest a two-end invasionfor both complete and incomplete events (Figure 4). Thistwo-end invasion model is also supported by the findingthat a single-end invasion by a single arm of homology

can only very rarely copy a larger DNA segment into thebreak. Remarkable is the length of the DNA segmentsand the sequence of the junction that we found in theincomplete gap repair events. At least in one case, a tractof .10 kb has been added to one side of the break. In thetwo cases where we sequenced the junction, a micro-homology was absent in one case and a possible micro-homology of a single C nucleotide was found in the othercase (see also Staveley et al. 1995; Adams et al. 2003). Itappears unlikely that two noncomplementary, very long,leading single strands are generated and then ligatedwithout the presence of homology, especially since theDrosophila genome appears to lack family X group DNApolymerases that could initiate DNA synthesis on non-homologous ends (Sekelsky et al. 2000; McElhinny et al.2005). Therefore, we speculate that lagging-strand syn-thesis occurs during gap repair of longer tracts. In thisscenario, lagging-strand synthesis restores two double

Figure 4.—Model of gap repair involving two-end invasion and lagging-strand synthesis. (A) Adouble-strand break is generated by I-SceI, fol-lowed by the processing of DNA ends, strand inva-sion, and DNA synthesis. Whether or not thebranches migrate following DNA synthesis hasnot been addressed in this study. Ligation of theends may occur by either homologous end joining(B) or nonhomologous end joining (NHEJ) afterresection of the single-stranded ends (C). Themodel in B is a modification of the SDSA modelproposed by Nassif et al. (1994). An alternativemodel involving Holliday junction formationand resolution without associated crossing overis also compatible with our data.

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strands that may be ligated by homologous end joining ornonhomologous end joining after resection of the single-stranded ends (Figure 4). The involvement of lagging-strand synthesis in DSB-induced gene conversions ofeven very short tracts has been suggested in yeast, but waslater again revised (Holmes and Haber 1999; Wang et al.2004). However, several models have been suggested forDSB repair events with long tracts of new DNA synthesis,such as a synthesis-dependent strand-annealing model(SDSA) with lagging-strand synthesis or a break-inducedreplication model that involves a true replication fork(for review see Paques and Haber 1999). Our data ap-pear to fit an SDSA model involving lagging-strand syn-thesis (Figure 4). The SDSA model is used frequently toexplain mitotic recombination events in Drosophila, as itcan explain the absence of crossover in most mitotic re-combination events and the unidirectional flow of in-formation from the template strand to the broken strand(Nassif et al. 1994). Also in this study, the flow of geneticinformation in almost all cases is from the intact to thebroken strand, with two remarkable exceptions where theendogenous y1 point mutation was corrected to wild typeupon DSB induction in the transgene. This reversiondepends on DSB formation in the transgene, as we havenever observed spontaneous reversion of the y1 mutationin any of our transgenic fly strains. These two cases can bereadily explained by a model involving Holliday junctionmigration with heteroduplex formation and with thecorrection of the mismatch.

Taken together, these results open promising newapplications for the modification of the Drosophilagenome. Transgenes can probably also be constructedin vivo in other model organisms with a relatively smallgenome and with efficient ectopic recombination, suchas Arabidopsis or Caenorhabiditis elegans.

We are grateful to Antonia Manova and Bruno Schmid for technicalassistance, to Fritz Ochsenbein for the preparation of figures, andto Primo Schar (University of Basel), Konrad Basler (University ofZurich), and Serge Gangloff (Commissariat a l’Energie Atomique deFontenay-aux-Roses) for stimulating discussions, and two anonymousreviewers for many helpful comments on the manuscript. This workwas supported by grants from the project Mechanisms of Gene Integra-tion (LSHG-CT-2003-503303) of the European Union, the SchweizerStaatssekretariat fur Bildung und Forschung, the Kanton Zurich, andthe Swiss National Science Foundation.

LITERATURE CITED

Adams, M. D., M. McVey and J. J. Sekelsky, 2003 Drosophila BLM indouble-strand break repair by synthesis-dependent strand an-nealing. Science 299: 265–267.

Artavanis-Tsakonas, S., M. A. Muskavitch and B. Yedvobnick,1983 Molecular cloning of Notch, a locus affecting neurogen-esis in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 80:1977–1981.

Banga, S. S., and J. B. Boyd, 1992 Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster. Proc. Natl. Acad.Sci. USA 89: 1735–1739.

Campuzano, S., L. Carramolino, C. V. Cabrera, M. Ruiz-Gomez,R. Villares et al., 1985 Molecular genetics of the achaete-scutegene complex of Drosophila melanogaster. Cell 40: 327–338.

Capecchi, M. R., 1989 Altering the genome by homologous re-combination. Science 244: 1288–1292.

Copeland, N. G., N. A. Jenkins and D. L. Court, 2001 Recom-bineering: a powerful new tool for mouse functional genomics.Nat. Rev. Genet. 2: 769–779.

DiBello, P. R., D. A. Withers, C. A. Bayer, J. W. Fristrom and G. M.Guild,1991 TheDrosophilaBroad-Complexencodesa familyofrelated proteins containing zinc fingers. Genetics 129: 385–397.

Egli, D., E. Hafen and W. Schaffner, 2004 An efficient method togenerate chromosomal rearrangements by targeted DNA double-strand breaks in Drosophila melanogaster. Genome Res. 14: 1382–1393.

Engels, W. R., D. M. Johnson-Schlitz, W. B. Eggleston and J. Sved,1990 High-frequency P element loss in Drosophila is homologdependent. Cell 62: 515–525.

Garcia-Bellido, A., 1979 Genetic analysis of the achaete-scute sys-tem of Drosophila melanogaster. Genetics 91: 491–520.

Groth, A. C., M. Fish, R. Nusse and M. P. Calos, 2004 Con-struction of transgenic Drosophila by using the site-specific inte-grase from phage FC31. Genetics 166: 1775–1782.

Haenlin, M., H. Steller, V. Pirrotta and E. Mohier, 1985 A 43kilobase cosmid P transposon rescues the fs(1)K10 morphoge-netic locus and three adjacent Drosophila developmental mutants.Cell 40: 827–837.

Holmes, A. M., and J. E. Haber, 1999 Double-strand break repair inyeast requires both leading and lagging strand DNA polymerases.Cell 96: 415–424.

Horn, C., and A. M. Handler, 2005 Site-specific genomic targetingin Drosophila. Proc. Natl. Acad. Sci. USA 102: 12483–12488.

Iwai, Y., T. Usui, S. Hirano, R. Steward, M. Takeichi et al.,1997 Axon patterning requires DN-cadherin, a novel neuronaladhesion receptor, in the Drosophila embryonic CNS. Neuron 19:77–89.

Jasin, M., 1996 Genetic manipulation of genomes with rare-cuttingendonucleases. Trends Genet. 12: 224–228.

Johnson-Schlitz, D. M., and W. R. Engels, 2006 The effect of gaplength on double-strand break repair in Drosophila. Genetics173: 2033–2038.

Keeler, K. J., T. Dray, J. E. Penney and G. B. Gloor, 1996 Genetargeting of a plasmid-borne sequence to a double-strandDNA break in Drosophila melanogaster. Mol. Cell. Biol. 16: 522–528.

Lankenau, D. H., and G. B. Gloor, 1998 In vivo gap repair in Dro-sophila: a one-way street with many destinations. BioEssays 20:317–327.

McElhinny, S. A. N., J. M. Havener, M. Garcia-Diaz, R. Juarez, K.Bevenek et al., 2005 A gradient of template dependence de-fines distinct biological roles for family X polymerases in non-homlogous end joining. Mol. Cell 19: 357–366.

McVey, M., D. Radut and J. J. Sekelsky, 2004 Evidence for multiplecycles of strand invasion during repair of double-strand gaps inDrosophila. Genetics 167: 699–705.

Morrow, D. M., C. Connelly and P. Hieter, 1997 ‘‘Break copy’’duplication: a model for chromosome fragment formation in Sac-charomyces cerevisiae. Genetics 147: 371–382.

Muyrers, J. P., Y. Zhang and A. F. Stewart, 2001 Techniques: re-combinogenic engineering—new options for cloning and ma-nipulating DNA. Trends Biochem. Sci. 26: 325–331.

Nassif, N., J. Penney, S. Pal, W. R. Engels and G. B. Gloor,1994 Efficient copying of nonhomologous sequences from ec-topic sites via P-element-induced gap repair. Mol. Cell. Biol. 14:1613–1625.

Oberstein, A., A. Pare, L. Kaplan and S. Small, 2005 Site-specifictransgenesis by Cre-mediated recombination in Drosophila. Nat.Methods 2: 583–585.

Paques, F., and J. E. Haber, 1999 Multiple pathways of recombina-tion induced by double-strand breaks in Saccharomyces cerevisiae.Microbiol. Mol. Biol. Rev. 63: 349–404.

Richardson, C., and M. Jasin, 2000 Coupled homologous and non-homologous repair of a double-strand break preserves genomeintegrity in mammalian cells. Mol. Cell. Biol. 20: 9068–9075.

Richardson, C., M. E. Moynahan and M. Jasin, 1998 Double-strandbreak repair by interchromosomal recombination: suppression ofchromosomal translocations. Genes Dev. 12: 3831–3842.

Rong, Y. S., and K. G. Golic, 2000 Gene targeting by homologousrecombination in Drosophila. Science 288: 2013–2018.

Transgene Integration in Drosophila 2027

Rong, Y. S., and K. G. Golic, 2003 The homologous chromosome isan effective template for the repair of mitotic DNA double-strandbreaks in Drosophila. Genetics 165: 1831–1842.

Rong, Y. S., S. W. Titen, H. B. Xie, M. M. Golic, M. Bastiani et al.,2002 Targeted mutagenesis by homologous recombination inDrosophila melanogaster. Genes Dev. 16: 1568–1581.

Rubin, G. M., and A. C. Spradling, 1982 Genetic transformationof Drosophila with transposable element vectors. Science 218:348–353.

Ruiz-Gomez, M., and J. Modolell, 1987 Deletion analysis of theachaete-scute locus of Drosophila melanogaster. Genes Dev. 1:1238–1246.

Sekelsky, J. J., M. H. Brodsky and K. C. Burtis, 2000 DNA repairin Drosophila: insights from the Drosophila genome sequence.J. Cell Biol. 150: F31–F36.

Spradling, A. C., 1986 Drosophila: A Practical Approach. IRL Press,Oxford.

Staveley, B. E., T. R. Heslip, R. B. Hodgetts and J. B. Bell,1995 Protected P-element termini suggest a role for inverted-

repeat-binding protein in transposase-induced gap repair inDrosophila melanogaster. Genetics 139: 1321–1329.

Venken, K. J., Y. He, R. A. Hoskins and H. J. Bellen, 2006 P[acman]:a BAC transgenic platform for targeted insertion of large DNAfragments in D. melanogaster. Science 314: 1747–1751.

Wang, X., G. Ira, J. A. Tercero, A. M. Holmes, J. F. X. Diffley et al.,2004 Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae. Mol. Cell. Biol.24: 6891–6899.

Wimmer, E. A., 2003 Innovations: applications of insect transgene-sis. Nat. Rev. Genet. 4: 225–232.

Yasothornsrikul, S., W. J. Davis, G. Cramer, D. A. Kimbrell andC. R. Dearolf, 1997 viking: identification and characterizationof a second type IV collagen in Drosophila. Gene 198: 17–25.

Zhang, Y., J. P. Muyrers, G. Testa and A. F. Stewart, 2000 DNAcloning by homologous recombination in Escherichia coli. Nat.Biotechnol. 18: 1314–1317.

Communicating editor: K. G. Golic

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