Transgenic Mosquitoes Expressing a Phospholipase A2 Gene Have a Fitness Advantage When Fed Plasmodium falciparum-Infected Blood
Figure 5
Identification of transgene integration sites.
Genomic positions of transgene integration were identified for the AP (-) (A) and AP (+) (B) transgenic mosquito lines using splinkerette PCR. Identification was based on multiple PCR products for each end of the transgene, suggesting that each transgene integrated at a single, canonical piggyBac integration site. Using the An. stephensi genome database, 20 kb of sequence flanking each end of the insertion was identified. No annotated transcripts are present around the AP (-) integration site, while for the AP (+) line integration occurred at a highly transcribed locus (B). To determine whether the transgene influences surrounding gene function, the most proximal gene to the AP (+) integration site, a predicted AP4 transcription factor (ASTM 012538), was analyzed by qRT-PCR in (C). For each sample, the relative AP4 transcript abundance was measured in whole mosquitoes using rpS7 as an internal reference, and the abundance in wild type An. stephensi (WT) was used to calculate fold change. The abundance of the AP4 transcript was significantly higher in AP (+) transgenic mosquitoes using a one-way ANOVA with a Tukey post-test. pBac LE: left end of the piggyBac inverted repeat; pBac RE: right end of the piggyBac inverted repeat; W: DNA from wild type mosquitoes; (-): DNA from AP(-) mosquitoes; (+): DNA from AP(+) mosquitoes; TTAA: piggyBac canonical insertion site.
