Cellular labeling of endogenous virus replication (CLEVR) reveals de novo insertions of the gypsy endogenous retrovirus in cell culture and in both neurons and glial cells of aging fruit flies

Abstract

Evidence is rapidly mounting that transposable element expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, successful reporters of LINE element mobilization have been in use for some time, but similar strategies have not been developed to report Long Terminal Repeat (LTR) retrotransposons and endogenous retroviruses. We describe Cellular Labeling of Endogenous Virus Replication (CLEVR), which reports replication of the gypsy element in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR-retrotransposons, endogenous retroviruses and exogenous retroviruses.

Nuclear Transcriptomes of the Seven Neuronal Cell Types That Constitute theDrosophilaMushroom Bodies

“…Because of the change in genetic background, we re-characterized the expression pattern of each split-GAL4 combination to confirm that the expected cell subtype-specific pattern had not been altered. We used a novel membrane-GFP-P2A-nuclear-mCherry dual label reporter (Watermelon or WM for short; see Methods) (Chang et al 2018), which expresses membrane-tethered GFP and nuclear mCherry from a single transcript via a viral ribosome skip peptide (P2A; Daniels et al 2014). The GFP label revealed neuronal morphology, thereby confirming MB subtype specificity, and the nuclear-mCherry marked each nucleus, which permitted an accurate cell count.…”

“…For each split-GAL4 combination, we separately backcrossed the GAL4 activating domain and DNA-binding domain components, and then combined the two hemi-drivers as a split-GAL4 line in the MJ2 background thereafter using standard balancer chromosomes that had themselves been equilibrated to the MJ2 strain. The UAS-WM P{5XUAS-myr :: GFP-V5-p2A-His2B :: mCherry-HA} reporter strain was generated using standard approaches (Chang et al 2018). Flies were cultured on standard cornmeal food using the standard cornmeal recipe from Bloomington stock center.…”