Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster

Abstract

We constructed Drosophila melanogaster bacterial artificial chromosome libraries with 21-kilobase and 83-kilobase inserts in the P[acman] system. We mapped clones representing 12-fold coverage and encompassing more than 95% of annotated genes onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using UC31 integrase to rescue mutations. They can be modified through recombineering, for example, to incorporate protein tags and assess expression patterns.

Figure 1. The P(acman) BAC Vector and Mapped Clones in the eve Region

(a) Map of attB-P(acman)-Cm^R-BW with partitioning functions (parA parB and parC), low-copy replication functions (repE and oriS), selectable marker (chloramphenicol acetyl transferase, Cm^R), conditionally inducible origin of replication (oriV) and dominant eye color marker white ⁺. Genomic DNA was cloned into the BamHI site (Supplementary Fig. 2). (b) A 100-kb region surrounding the eve gene (yellow) on chromosome arm 2R. Mapped CHORI-321 and CHORI-322 clones are indicated below the FlyBase R5.9 gene annotation. CH322-103K22, selected for transformation (Supplementary Table 2) and protein tagging (Fig. 2), is indicated in red.

Figure 2. Expression of EGFP Fusion Proteins in Transgenic Embryos

Fusion proteins were detected using an anti-GFP antibody and peroxidase staining. (a-d) Expression of an even skipped fusion construct recapitulates the native pattern of Eve expression. (a) embryonic stage 5, (b) embryonic stage 9, (c) embryonic stage 11, (d) embryonic stage 15. (e) Dichaete, embryonic stage 5. (f) caudal, embryonic stage 9. (g) Deformed, embryonic stage 11. (h) tailless, embryonic stage 5. (i) sloppy paired 2, embryonic stage 17. (j) extradenticle, embryonic stage 15. (k) engrailed, embryonic stage 9. (l) hairy, embryonic stage 6. Scale bar (a) indicates 50 µm.