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. 2021 Apr 13;10:e58791. doi: 10.7554/eLife.58791

Figure 3. Splicing of the artificial intron and expression analysis.

(A) Schematic showing mRNA expression of the modified host genes of the three markerless strains assuming correct splicing of the artificial intron. Black arrows indicate RT-PCR primers used in B. (B) RT-PCR (left) and sequencing of the amplicons (right) indicate precise splicing of the three artificial introns. Midguts of homozygous strains Sco-CP, ScoG-AP2, and Aper1-Sco were dissected 3 hr after blood feeding and RNA was extracted for RT-PCR. (C) Relative mRNA expression of Scorpine in the transgenic strains with and without the marker cassette. (D) Expression of the host genes CP, AP2, and Aper1 in transgenic strains with and without the marker cassette relative to the wild type. RNA was extracted from 10 to 15 midguts 3 hr after the blood meal in the case of CP and Aper1, and non-blood-fed for AP2. qPCR with the primer pairs indicated as coloured half arrows in the schematic below was conducted on cDNA and expression was normalized to the S7 house-keeping reference gene. Data derive from three biological replicates with three technical replicates each. p-values were calculated on ΔCt values using the unpaired Student’s t-test. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, and ns: not significant.

Figure 3.

Figure 3—figure supplement 1. Scorpine expression levels and mass spectrometry of co-opted host genes.

Figure 3—figure supplement 1.

(A) RT-PCR with primers spanning the splice site (indicated as half arrows in the schematic below) on 3 hr blood-fed guts. (B) qPCR for the integrated Scorpine sequence was performed on cDNA from both non-blood-fed guts and guts dissected 3 hr after blood-feed of all three markerless transgenic strains. The primer-pair binding to Scorpine is indicated as yellow half arrows in the inset. Error bars indicate standard deviation from three technical replicates normalized to the S7 reference gene. G3 wild-type controls (blood-fed and non-blood-fed) run in parallel were negative (Ct mean values above 35). (C) Peptides identified in mass-spectrometry that map to the endogenous host genes. Proteins were extracted from non-blood-fed guts of the homozygous transgenic lines Sco-CP and Aper1-Sco. Note that Sco-CP (but not Aper1-Sco) is expected to generate two protein products due to the presence of the 2A peptide. Green rectangles indicate high confidence peptides with amino acid sequences shown below.