Gene knockdown in Homalodisca vitripennis cell cultures

The glass-winged sharpshooter, Homalodisca vitripennis, is an invasive Hemipteran pest of economically important plans in the United States.  H. vitripennis is the vector of the bacterium Xylella fastidiosa, the causal agent of Pierce’s disease in grape vines.  There are currently several chemical treatment options for the control of H. vitripennis but they can be costly and may have off-target effects.  A more specific control strategy is needed. RNA interference is a post transcriptional regulation mechanism that targets specific messenger RNAs by binding and enzymatic cleavage.   This naturally occurring process can be utilized for the determination of ideal gene targets for biological control.  In order to streamline the process an H. vitripennis cell culture was established.  19,530 EST sequences were batch-downloaded from NCBI (www.ncbi.nlm.nih.gov) and compiled into 1758 contigs using Geneious (www.geneious.com) and the Primer3 algorithm was used to design primer sets against many nuclear and mitochondrial genes.  The primer sets that successfully amplified the expected band were then used to make double-stranded RNA (dsRNA), using the HiScribe T7 in vitro transcription kit.  H. vitripennis cells were then grown in media containing the dsRNA and control cells were grown in dsRNA (-) media.  Changes in growth or proliferation patterns were documented to determine the best candidate gene targets for the control of this agricultural pest.