Cloning and Tagging Drosophila Centrosomal Proteins

The PACT domain of Drosophila DPLP55 (link) was cloned by PCR from pubiPCTGFP (a gift from J. Raff) using the primer pair 5′-gtacggtaccaacatgattgctctgcagaagaaatg-3′ and 5′-gtacctcgagatgatgccgcgcatgcgctctttttg-3′. The PCR product was subcloned into the pcDNA3 Flag1 EosFP tandem vector that contains the tandem dimer version of the photo-convertible EosFP56 (link) to construct the PACT–d2Eos fusion protein. Using PSI Blast57 (link), the Ensemble Genome Browser and InParanoid, we identified CG5690 as the putative Drosophila ortholog of mouse and human centrobin. We obtained the corresponding full-length cDNA, SD06673, from DGRC (http://dgrc.cgb.indiana.edu), and fused it to EYFP-C1 (Clontec). We subcloned asl47 (link) in the pTagFP635-C vector (mKATE, Evrogen). These three fusion constructs were subcloned into the Drosophila pUBq pWRpUbqu transformation vector, and transgenic lines expressing PACT–d2Eos, mKATE-ASL and YFP–CNB under the control of the poly-Ubiquitin promoter were obtained using standard techniques.