An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases

Abstract

Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a phiC31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the phiC31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the phiC31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, high-throughput screening of large cDNA sets and regulatory elements.

Fig. 1.

φC31-mediated integration into attP target sites. (a) Design of the pM{3xP3-RFPattP} landing site construct. This construct contains an attP docking site of 221 bp and a DsRFP marker gene attached to the tubulinα1 3′ UTR element. RFP expression is driven by the 3xP3 promoter, and the marker cassette is flanked by loxP sites. The landing site construct is bounded by mariner inverted repeats (inverted repeats, open triangles). (b) Diagram of the four major chromosomes indicating the cytological positions of the 25 ZH-attP landing sites that are located intergenically. Two lines exist at both positions 86D and 86F. (c) Integration mechanism of the pUASTattB vector into attP landing sites. The pUASTattB plasmid contains a 285-bp attB fragment, the white⁺ selectable marker, a UAS-MCS-SV40 cassette, and a single loxP site. The φC31 integrase mediates recombination between attB and attP sites, resulting in the integration of pUASTattB into the landing site, thereby creating the two hybrid sites attL and attR, which are refractory to the φC31 integrase. The final configuration at the landing site is directed by the orientation of the attB and attP elements. The loxP sites allow elimination of intervening sequences before or after integration of pUASTattB (indicated with flat arrowheads). In all of the injection experiments where we used the pUASTattB vector, it contained a lacZ reporter (not indicated). (The parallel diagonal lines indicate the presence of the plasmid backbone.) RFP, red fluorescence protein; UAS, upstream activating sequence; MCS, multiple cloning site.

Fig. 2.

Establishment of germ-line-specific φC31 integrase lines. (a) nanos- and vasa-φC31 constructs used to generate transgenic integrase lines. The φC31 ORF is flanked by either nanos (nos) or vasa (vas) regulatory elements, including promoters, 5′ UTR, and 3′ UTR. NLS-tagged φC31 integrase versions were also tested. All constructs contain a 3xP3-EGFP marker cassette, a loxP site, and an attB site for site-specific integration into attP sites. (b) Schematic of the two different setups used to compare the four different integrase versions depicted in a. Set-up I contains the integrase constructs in an attP site on the third chromosome (3R 86F) in combination with a free attP site on the fourth (102D); set-up II represents the reverse situation. The animals used were homozygous for the depicted situation. (c) Detection of β-galactosidase activity in third-instar wing discs of the transgenic ZH-attP-86Fb-lacZ line (here, the pUAS-lacZattB plasmid was introduced via coinjection of φC31 integrase mRNA). The observed patterns correspond to the apterous (Left) and omb (Right) expression domains. (d) Antibody staining against φC31 integrase. An embryo at stage 4–5 shows enhanced staining in the posterior pole cells, indicating accumulation of φC31 integrase protein in these cells. The depicted embryo is homozygous for vas-φC31.

Fig. 3.

Strategy for transforming an existing attP landing site into a split-white landing site. (a) Elimination of the 3xP3-RFP marker cassette. The attP line was crossed to a Cre-expressing line, leading to excision of the sequence between the loxP sites. (b) Placing of white (exons 3–6) into the modified attP site. Construct p3xP3-RFP/w^Ex3-6attP/B is introduced by φC31-mediated integration. To prevent intramolecular recombination between the two attachment sites, a shortened attP element of 54 bp was used and cloned immediately next to the attB element, which was trimmed at the 5′ region. (c) Germ-line transformation with the split-white vector pw^P-Ex2UASTattB via φC31-mediated integration. Correct attP targeting establishes the white transcriptional unit, allowing expression of a functional white gene.