FIG 1.

Gene-silencing system targeting an HS. The system is based on a single-marker BF cell line that has been stably transfected with a stem-loop construct (smHS cell line) containing a doxycycline (dox)-inducible promoter for the expression of a heterologous dsRNA. In the absence of further genetic manipulations, induction of this dsRNA expression has no consequence for trypanosome proliferation. To enable a specific knockdown of any single-copy gene X, two further consecutive transfections of smHS cells are necessary: one to eliminate an X allele by the hygromycin resistance marker (HYG) and one to integrate a plasmid into the remaining X allele, which will fuse the HS to the X mRNA sequence. Doxycycline-induced (+ dox) expression of HS dsRNA then targets and destroys the X-HS mRNA hybrid.