Cell Biology

PXGS: a Poly-Transgene Expression System based on Mutually Exclusive Splicing of Dscam

Renee Yin Yu
Alyeri Bucio-Méndez
Brian E Chen author has email address

Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montréal, Canada
Departments of Medicine and Neurology & Neurosurgery, McGill University, Montréal, Canada

https://doi.org/10.7554/eLife.111721.1

Open access
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Figures and data

PXGS takes advantage of the mutually exclusive splicing property of Dscam.
a, Endogenous Dscam1 mRNA is mutually exclusively spliced in four exons during transcription, exons 4, 6, 9, and 17. For each of the mutually exclusively spliced exons, with every round of transcription, one and only one of the possible exon variants is in the final mRNA product and ultimately the protein product. b, The entire Dscam exon 4 cluster is inserted downstream of a UAS sequence to create the PXGS vector. A 6× histidine tag is added at the end of exon 5 to distinguish PXGS expression from endogenous Dscam exon 4 expression. Any gene of interest (GOI) can then be inserted to replace any of the 12 exon 4 alternates for expression. With each round of transcription, only a single transgene is selected for incorporation into the final mRNA product. c, Exon 4 mutually exclusive splicing is maintained in PXGS. PXGS vectors with and without a 6× histidine tag were co-transfected with Actin5c-Gal4 in Drosophila S2 cells. As a positive control, reverse transcription (RT) of Exon 4 was performed on untransfected S2 cells to identify the endogenous Dscam Exon 4 mRNA. RT using PXGS-specific reverse primers on S2 cells was performed, with the no Actin5c-Gal4 co-transfection (PXGS only) as negative controls. PCR of Exon 4 in the PXGS vectors with and without a 6×His-tag showed DNA bands at the expected 500 bp to 600 bp sizes.

PXGS can express multiple fluorophores simultaneously in vivo.
Crossing the pan-neuronal nSyb-Gal4 driver line with the transgenic fly UAS-PXGS_GFP^4.1-BFP_nols^4.2-iRFP^4.11-RFP^4.12 demonstrates that nearly all neurons can splice and express multiple transgenes in vivo. However, the fluorescence intensity for any single color was low as its production was divided among 12 Exon 4 alternates.

PXGS can drive expression in non-neural cells.
Crossing the transgenic fly UAS-PXGS_iRFP_nols^4.1-COX8::RFP^4.2-BFP_nols^4.3-mCD8::GFP^4.4-iRFP_nols^4.5-COX8::RFP^4.6-mCD8::GFP^4.8-BFP_nols^4.9-iRFP_nols^4.10-BFP_nols^4.12 to the glial Repo-Gal4 driver (left column) or to the ubiquitous Tubulin-Gal4 driver (right column) showed expression in non-neural cells. The subcellular localization of the fluorophores was observed in the flight muscle (right column), but was less apparent in glial cells in the brain (left column). Scale bars are 50µm.

PXGS can express multiple cell surface receptors in vivo to manipulate neural wiring.
a, Representative example of the pSc axonal branching pattern in 455-Gal4; PXGS_fluorophores controls. A schematic of the three PXGS constructs expressing different combinations of cell surface receptors is shown on the right. b, Two representative examples of the pSc axonal branching in 455-Gal4/+; PXGS_dpr12^4.1-dpr8^4.2-Gal4^4.3/+ flies (left images), 455-Gal4/+; PXGS_kek1^4.7-kirre^4.8-tutl^4.9/+ flies (middle images), and 455-Gal4/+; PXGS_Toll-6^4.10-Bsg^4.11-sli^4.12/+ flies (right images). Yellow arrows point to the pSc axon exiting the thoracic ganglion anteriorly. White arrowheads denote missing branches. c, Overexpression of multiple cell surface receptors significantly increases axonal targeting errors. A frequency distribution shows the five error types found in the PXGS-cell surface receptor flies. A schematic for each error is shown below the x-axis, where blue branches indicate ectopic branches. Posterior shortening and contralateral anterior missing branches occurred significantly more frequently in 455-Gal4/+; PXGS_dpr12^4.1-dpr8^4.2-Gal4^4.3/+ flies and 455-Gal4/+; PXGS_Toll-6^4.10-Bsg^4.11-sli^4.12/+ flies. d, Both 455-Gal4/+; PXGS_dpr12^4.1-dpr8^4.2-Gal4^4.3/+ flies and 455-Gal4/+; PXGS_Toll-6^4.10-Bsg^4.11-sli^4.12/+ flies had significantly reduced total branch length and branch number compared to the control (left graphs). The 455-Gal4/PXGS_fluorophores and PXGS_fluorophores/+ both had significantly reduced total branch length compared to 455-Gal4/+, most likely due to genetic background. 455-Gal4/PXGS_fluorophores and PXGS_fluorophores/+ flies had no significant differences in branch length (right graphs). Dotted lines mark the midline. Scale bars are 50µm. Statistical significance comparisons to control are indicated directly above the bar representing each genotype. No asterisks indicate the comparison was not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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