An Anopheles stephensi Promoter-Trap: Augmenting Genome Annotation and Functional Genomics

Abstract

The piggyBac transposon was modified to generate gene trap constructs, which were then incorporated into the genome of the Asian malaria vector, Anopheles stephensi and remobilized through genetic crosses using a piggyBac transposase expressing line. A total of 620 remobilization events were documented, and 73 were further characterized at the DNA level to identify patterns in insertion site preferences, remobilization frequencies, and remobilization patterns. Overall, the use of the tetameric AmCyan reporter as the fusion peptide displayed a preference for insertion into the 5′-end of transcripts. Notably 183 – 44882 bp upstream of the An. stephensi v1.0 ab initio gene models, which demonstrated that the promoter regions for the genes of An. stephensi are further upstream of the 5′-proximal regions of the genes in the ab inito models than may be otherwise predicted. RNA-Seq transcript coverage supported the insertion of the splice acceptor gene trap element into 5′-UTR introns for nearly half of all insertions identified. The use of a gene trap element that prefers insertion into the 5′-end of genes supports the use of this technology for the random generation of knock-out mutants, as well as the experimental confirmation of 5′-UTR introns in An. stephensi.

Keywords

• insect genomics
• malaria
• transposon gene vectors
• Asian mosquito