Generation of 12 Drosophila Transgenic Negative Control Lines Based on Site-specific ΦC31 Integrase and pUASTattB Vector

Abstract

Objective Construction of a negative control line for the Drosophila transgenic system based on ΦC31 integrase and vector plasmid pUASTattB to provide a more scientific negative control for transgenic Drosophila research experiments.Methods The vector plasmid pUASTattB was microinjected into four different genetic backgrounds Drosophila lines attP-25C6, attP-68A4, attP-75B1 and attP-86F8 embryos carrying ΦC31 integrase. All of the injected embryos were incubuated to get G0 adults, and each of them was crossed with balancer stock ywR13S separately in a single vial (1 adult of the G0 generation and 3 of the ywR13S in each vial). The probability of successful insertion was calculated by observing the colour of the compound eyes of the G1 generation of Drosophila to determine whether there was a mini-White insertion. The G1 generation Drosophila adults successfully inserted into mini-White were then selected to make single-vial crosses (one G1 generation male Drosophila crossed with three virgins of balancer Drosophila line) with each of the three balancer Drosophila strains DB, ywR13S and yw122, respectively, for balanced seed preservation. The genomic DNA of the conserved Drosophila lines was extracted and the vector plasmid pUASTattB was identified for transfer by PCR.Results 12 Drosophila strains were obtained, all of which were red-eyedDrosophila melanogaster carrying the mini-White marker, and were identified by PCR as having the pUASTattB sequence insertion.Conclusion The 12 transgenic Drosophila strains can meet the negative control requirements for the transgenic fly research experiments that constructed with pUASTattB as the vector basically, enriching the Drosophila resources in the National Drosophila Resource Center of China.

Keywords

• drosophila melanogaster
• transgenic
• negative control
• microinjection
• balancers