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P[Acman]: a BAC Transgenic Platform for Targeted Insertion of Large DNA

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P[Acman]: a BAC Transgenic Platform for Targeted Insertion of Large DNA RESEARCH ARTICLE specific integration using the integrase of bac- teriophage fC31 has been demonstrated in Drosophila (11). fC31-integrase mediates recom- P[acman]: A BAC Transgenic Platform bination between an engineered “docking” site, containing a phage attachment (attP) site, in the fly genome, and a bacterial attachment (attB) for Targeted Insertion of Large DNA site in the injected plasmid. Three pseudo-attP docking sites were identified within the Dro- Fragments in D. melanogaster sophila genome, potentially bypassing desired integration events in engineered attP sites. For- Koen J. T. Venken,1 Yuchun He,2,3 Roger A. Hoskins,4 Hugo J. Bellen1,2,3,5* tunately, they did not seem receptive to attB plasmids, because all integration events were at We describe a transgenesis platform for Drosophila melanogaster that integrates three recently the desired attP sites (11). Thus, recovery of large developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), DNA fragments by gap repair into a low-copy recombineering, and bacteriophage fC31–mediated transgenesis. The BAC is maintained at low plasmid containing an attB site, followed by copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy fC31-mediated transformation, might allow the number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap integration of any DNA fragment into any en- repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. fC31- gineered attP docking site dispersed throughout mediated transgenesis integrates these large DNA fragments at specific sites in the genome, the fly genome. allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform Here we describe new vectors that over- should greatly facilitate structure/function analyses of most Drosophila genes. come the limitations associated with P-element– mediated transgenesis. We developed P/fC31 ar- rosophila is an important model orga- experimentally induced to high copy number tificial chromosome for manipulation (P[acman]), nism for studying biology and disease, (7). Hence, the introduction of conditionally a conditionally amplifiable BAC vector that con- Dand new tools are continually being de- amplifiable BAC features into fly transformation tains recognition sites for both P-transposase– (3) veloped to facilitate this research (1, 2). A major vectors is a first key step to manipulating large and fC31-mediated integration (11). P[acman] advance was the development of P-element– DNA fragments in Drosophila. permits recombineering-mediated cloning of any mediated transformation after the injection of Cloning of large DNA fragments is limited by genomic DNA fragment from Drosophila P1 or plasmids into Drosophila embryos (3). Hence, conventional methods that rely on restriction BAC clones (12–15) and enables the transfer of P-element vectors have been engineered for enzymes and DNA ligases, hampering analyses large DNA fragments into the fly genome. The numerous applications (4). However, P-element­– of large genes and gene complexes. Recently, ability to easily manipulate these DNA frag- mediated transformation has a number of limi- efficient in vivo cloning technologies using en- ments through recombineering and to introduce tations: inability to clone large DNA fragments hanced and regulated recombination systems, them into specific sites in the fly genome will in available P-element vectors, difficulties in commonly known as recombineering, have been greatly facilitate and accelerate in vivo genetic manipulating large DNA fragments, inability developed (8). Recombineering greatly facilitates manipulations of Drosophila. to transfer large DNA fragments into the fly the retrieval of DNA fragments through gap genome, and failure to target DNA to specific repair and their subsequent site-directed muta- Results sites in the genome. genesis. Because recombineering is based on Construction of a BAC transgenesis vector Cloning large DNA fragments in high–copy- homologous recombination, restriction enzymes for Drosophila. The motivation to construct P number plasmids, such as typical P-element and DNA ligases are not required. Recom- [acman] grew from our inability to clone 29- and vectors, is inefficient because large fragments bineering is widely used by mouse geneticists 39-kb DNA fragments in existing P-element are unstable at high copy number in bacteria. to generate transgenic and knockout constructs. vectors for the transformation rescue of muta- Hence, low–copy-number vectors, including P1 Recombineering-mediated mutagenesis is much tions in the gene senseless2 (16). We were unable (5) and bacterial artificial chromosome (BAC) more efficient with low-copy plasmids (8). Hence, to identify restriction sites for cloning the entire (6) vectors, were developed to stably maintain using recombineering in a conditionally ampli- gene. Moreover, recombineering-mediated gap large cloned DNA fragments. Unfortunately, fiable BAC should greatly facilitate the gap repair repair in existing high-copy P-element vectors low–copy-number vectors hamper sequencing, of large DNA fragments and subsequent muta- was unsuccessful. This was consistent with pub- embryo injection, and other manipulations re- genesis at low copy number. lished data demonstrating that gap repair in high- quiring large amounts of plasmid DNA. An P-element–mediated transformation is lim- copy or medium-copy plasmids has an upper size elegant solution is a conditionally amplifiable ited by DNA size, precluding the study of large limit of 25 and 80 kb, respectively (17). Because plasmid that has two origins of replication genes (>40 kb) and gene complexes. In addi- high-copy P elements have a substantial size, gap (ori’s): oriS for low-copy propagation, typical tion, more than 75% of P elements insert in repair is limited to fragments of about 20 kb. We for P1 and BAC vectors; and oriV, which can be regulatory elements of genes (9), often disrupt- hypothesized that a low-copy plasmid might ing genes in subtle ways (10). Moreover, P ele- alleviate size limitations and improve the sta- 1 ments are subject to position effects: the effect bility of large DNA fragments. Hence, we added Program in Developmental Biology, Baylor College of of a local chromosomal environment on the lev- P-element components to a chloramphenicol- Medicine, Houston, TX 77030, USA. 2Department of Molecular and Human Genetics, Baylor College of Medicine, els or patterns of transgene expression. This resistant conditionally amplifiable BAC (7), R Houston, TX 77030, USA. 3Howard Hughes Medical Institute, necessitates the generation and characterization resulting in P[acman]-Cm (Fig. 1A). The P- Baylor College of Medicine, Houston, TX 77030, USA. of several transgenes for each DNA construct element components include the 5′P and 3′P 4 Department of Genome Biology, Lawrence Berkeley studied. Hence, site-specific integration would termini required for P-transposase–mediated National Laboratory, Berkeley, CA 94720–3200, USA. 5Department of Neuroscience, Baylor College of Medicine, greatly facilitate structure-function analysis of integration, a multiple cloning site (MCS), and + Houston, TX 77030, USA. transgenes, permitting direct comparison of dif- the white marker. The conditionally ampli- *To whom correspondence should be addressed. E-mail: ferently mutagenized DNA fragments integrated fiable BAC contains two origins of replication: [email protected] at the same site in the genome. Recently, site- oriS and oriV (7). oriS keeps P[acman] at low www.sciencemag.org SCIENCE VOL 314 15 DECEMBER 2006 1747 RESEARCH ARTICLE Table 1. Retrieval of genes in P[acman]. Genomic fragments containing genes of interest retrieved by gap repair into P[acman]-CmR or -ApR are shown. Donor plasmids, with clone coordinates, are P1, BACs, or ampicillin-modified BACs (BAC-ApR). Mutations in the corresponding genes were lethal or showed a phenotype. Rescue was obtained for most genes using P-element–mediated transformation. NA, not applicable. Gene Construct Size Donor Clone P[acman] Mutation Rescue sens2 sens2-22 22.3 kb P1 DS05421 CmR NA NA sens2 sens2-29 28.9 kb P1 DS05421 CmR NA NA sens2 sens2-39 38.9 kb P1 DS05421 CmR NA NA CG10805 CG10805-S 9.7 kb P1 DS05421 CmR Lethal Yes CG10805 CG10805-L 14.7 kb P1 DS05421 CmR Lethal Yes dap160 dap160 10.9 kb P1 DS02919 CmR Lethal Yes sens sens-S 12.1 kb BAC-ApR BACR17E13 CmR Lethal Yes sens sens-L 18.1 kb BAC-ApR BACR17E13 CmR Lethal Yes Dalpha7 Da7 29.4 kb BAC-ApR BACR02B03 CmR Phenotype Yes Drp Drp 9.4 kb BAC BACR30P05 ApR Lethal Yes* Sec8 Sec8-L 12 kb BAC BACR02L23 ApR Lethal No Eps15 Eps15-L 11.9 kb BAC BACR27P17 ApR Lethal Yes *From (25). fragments ranging from 9.4 to 39 kb, from To integrate attB-P[acman], we co-injected different donor plasmids (Table 1). Colony circular plasmid DNA and mRNA encoding polymerase chain reaction (PCR) screening fC31-integrase (11) into embryos carrying + Fig. 1. P[acman]: BAC transgenesis for Drosophila. identified correct recombination events at both piggyBac-yellow -attP (Fig. 2B). Integration junctions after gap repair. After plasmid copy- results in flies with a “yellow+” body color and (A)P[acman]containsP-element transposase sites + ′ ′ +, number induction, DNA fingerprinting and “white ” eye color phenotype. Because the (3 P and 5 P), white and an MCS. This P element is + inserted in the conditionally amplifiable BAC, sequencing demonstrated that the desired frag- attB site is upstream of the MCS and white is containing a low-copy origin of replication (oriS) ment was obtained. Hence, the methodology is downstream of the MCS, only transgenic flies + + and a copy-inducible origin of replication (oriV). (B) reliable and large DNA fragments can easily be that are “yellow white ” should have undergone P[acman] is linearized between both homology retrieved. an integration event containing most of the arms (LA and RA) and transformed into recom- To test the functionality of the gap-repaired injected DNA, including the cloned insert (Fig. bineering bacteria containing P1 or BAC clones. constructs, we used P-mediated transposition.
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