Abstract
Transgenic sexing strains (TSS) that carry conditional female lethal genes are advantageous for genetic control programs based on the sterile insect technique (SIT). It is desirable if females die early in development as larval diet is a major cost for mass production facilities. This can be achieved by using a gene promoter that is only active in embryos to drive expression of the tetracycline transactivator (tTA), the transcription factor commonly used in two-component TSS. While an embryo-specific promoter is ideal it may not be essential for assembling an effective TSS as tTA can be repressed by addition of tetracycline to the diet at larval and/or adult stages. Here we have investigated this idea by isolating and employing the promoters from the Lucilia spitting image and actin 5C genes to drive tTA expression in embryos and later stages. L. cuprina TSS with the tTA drivers and tTA-regulated tetO-Lshid effectors produced only females when raised on a limited tetracycline diet. The Lshid transgene contains a sex-specific intron and as a consequence only females produce LsHID protein. TSS females died at early larval stages, which makes the lines advantageous for an SIT program.
Highlights
The ability to make transgenic insects has been invaluable for genetic analysis and offers the potential for developing strains that have practical applications[1]
The initial L. cuprina Transgenic sexing strains (TSS) we developed carried a single transgene that consisted of a tetO21-hsp[70] enhancer-promoter driving expression of tetracycline transactivator transcripts that were sex- spliced as they contained the regulated intron from the Cochliomyia hominivorax transformer (Chtra) gene[5]
In this study we have developed L. cuprina early-larval TSS using gene promoters that are active throughout development to control expression of tTA
Summary
The ability to make transgenic insects has been invaluable for genetic analysis and offers the potential for developing strains that have practical applications[1]. We previously developed efficient methods for germline transformation of the Australian sheep blow fly Lucilia cuprina[2, 3], a major pest of sheep[4] This technology was used to create transgenic sexing strains (TSS) of L. cuprina for potential use in a genetic control program[5, 6]. The initial L. cuprina TSSs we developed carried a single transgene that consisted of a tetO21-hsp[70] enhancer-promoter driving expression of tetracycline transactivator (tTA) transcripts that were sex- spliced as they contained the regulated intron from the Cochliomyia hominivorax transformer (Chtra) gene[5]. It should be possible to rear larvae and adults on diet without tetracycline This would be important as females fed tetracycline would be expected to pass on the antibiotic to their offspring, which would inhibit tTA binding to DNA in the developing embryo. These results suggested that it might not be necessary to find an embryo-specific promoter to build a TSS with early stage female lethality, if a suitable tetracycline-feeding schedule can be established
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