GENERATION OF A CASK‐GFP TRANSGENIC FLY LINE

Ca lcium/calmodulin‐dependent s erine protein k inase (CASK) belongs to the membrane‐associated guanylate kinases (MAGUK) protein family. The protein contains the classic MAGUK domains including a single PDZ, SH3 and guanylate kinase domain. CASK is unique among MAGUK family members in that it also contains an N‐terminal CamK‐like domain. Overall, the CASK protein has been most intensively studied in the central nervous system, where studies in Drosophila have shown that CASK plays an integral role in the formation of learning memory. Interestingly, Drosophila CASK is also highly expressed in ovaries and is implicated in the process of border cell migration in Drosophila oogenesis. There are two known classes of CASK isoforms in Drosophila , CASK‐alpha isoforms and CASK‐beta isoforms. CASK‐alpha isoforms encode transcripts that yield a version of the CASK protein lacking the N‐terminal CamK‐like domain. CASK‐beta isoforms encode transcripts that yield a CASK protein that possesses the full complement of canonical MAGUK domains and the CamK‐like domain. Currently, it is unknown how these two classes of isoforms contribute to border cell migration in Drosophila ovaries. In order to begin to delineate CASK's functional role in Drosophila ovaries the current study aims to clone the CASK‐RA isoform (a CASK‐alpha isoform) and CASK‐RB isoform (a CASK‐beta isoform) for generation of UAS‐CASK‐A‐GFP and UAS‐CASK‐B‐GFP transgenic fly lines. Primers were designed using MacVector software to clone the CASK‐A and CASK‐B transcripts. In order to clone the CASK‐A transcript RNA was isolated from Drosophila ovaries and subjected to cDNA synthesis with the Invitrogen Superscript IV cDNA synthesis kit. Attempts to amplify the CASK‐A open reading frame using the generated cDNA were unsuccessful. The CASK‐B open reading frame was available in a pFlc plasmid and PCR conducted on mini‐prepped pFlc‐CASK‐B plasmid with the CASK‐B primers yielded a product of the expected 2697 base pair size. Modified CASK‐B primers were designed such that the CASK‐B forward primer contained a CACC sequence for cloning into a pENTR‐TOPO cloning vector (CASK‐A TOPO forward primer). A modified CASK‐B reverse primer lacking the stop codon sequence was also designed (CASK‐B reverse no stop). Subsequent PCR conducted with CASK‐A TOPO forward primer and CASK‐A reverse or CASK‐A reverse no stop primers were successful and yielded the expected amplicons. The full length CASK‐B open reading frame was confirmed in both products via Sanger sequencing. Future work will focus on the cloning of the CASK‐B products into the pENTR‐TOPO vector and use of a Gateway cloning reaction to transfer the CASK‐B open reading frames into UAS‐GFP vectors that will produce transgenic constructs with either an N‐terminal or C‐terminal GFP tag. Work on CASK‐A cloning will continue via standard cloning attempts. The CASK‐GFP fly lines will serve to provide a platform for characterization of CASK protein localization and determination of CASK binding partners in vivo in Drosophila ovaries. Moreover, determination of CASK protein function in Drosophila ovaries will provide insight into CASK's mechanistic contribution to border cell migration, a model of collective cell migration that serves to provide biological insight into the molecular pathways that govern processes such as wound healing and tumor cell metastasis.