Spontaneous excision and facilitated recovery as a control for phenotypes arising from RNA interference and other dominant transgenes

We present here such an approach, relying on facilitated recovery after spontaneous excision – or ‘popouts’ – of dominant transgenes stably inserted into the ribosomal RNA array, utilizing GFP as a marker and single cell sorting to recover regenerated WT controls. We validate its utility using RNA interference knockdowns of the paraflagellar rod gene PFR2 of L. (Viannia) braziliensis. The method yields stably modified lines suitable for long term studies of Leishmania virulence, relies solely on host rather than introduced genetic machinery, and is thus readily applied in many species and circumstances including functional genetic testing. Graphical abstract

Source: Molecular and Biochemical Parasitology - February 12, 2018 Category: Parasitology Source Type: research