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The RRM and linker region are the most conserved components of TRA-2 proteins among dipteran and non-dipteran insects [TA transactivator gene, but homozygous females Tet yielded progeny with a 1:1 sex ratio and normal hatching and survival rates, whereas homozygous females –Tet yielded progeny showed a significant bias towards male without intersex adult phenotypes, indicating that the abnormal sex ratio should be attributed to the construct was also designed to kill the carrier’s offspring from the third larval stage, hence also reducing the survival in the absence of Tet.
Mass crossing between heterozygous males and females allowed the selection of second-instar larvae and pupae in the next generation showing the brightest Ds Red fluorescence, corresponding to homozygous insertions.The performance of transgenic males was not significantly different to wild-type males in narrow-cage competitive mating experiments.gene expression causes segregation distortion acting at the level of gametic function, which is reinforced by sex-specific zygotic lethality.The resulting plasmid was cloned in competent cells then isolated and digested with Sac II and Bam HI to remove the intron fragment, which was amplified using primers 5 and 6. The posterior poles of 2500 Rock embryos were microinjected with a cocktail of the Zoo-2 and transposase constructs (0.6 and 0.3 μg/μl, respectively) using a Femtotip II with the Ni Kon Eclipse TS100 microinjector system.The RNAi core structures were then digested with Sac II and Bam HI and mixed with the linear plasmid described above in a 1:3 molar ratio. The transposase construct was a helper plasmid carrying the 5′ sequence was used to design two pairs of nested primers which amplify the same insertion and flanking regions but generate products differing in size by 214 bp (TaqαI-PB5-1, 5′-GAT GAG GTA CAT GAA GTG CAG CCA-3′ and TaqαI-PB5-2, 5′-CAT GCG TCA TTT TGA CTC ACG C-3′; TaqαI-PB5-3, 5′-TAG CCG AGT CTC TGC ACT GAA C-3′ and TaqαI-PB5-4, 5′-CAG TGA CAC TTA CCG CAT TGA C-3′).