NovoFly™ contains a single insertion of the pAll-in-one construct integrated into the genome through piggyBac-mediated germline transformation system. The construct contains three functional coding sequences that together constitute a tetracycline-repressible (Tet-Off) genetic sexing system with female-specific lethality and a fluorescent marker.

1. Transcription of the optimized tetracycline-controlled transactivator (tTAo) is driven by the embryonic Lucilia sericata bottleneck (Lsbnk) promoter and terminates at the P10 polyadenylation signal. Expression of tTAo during early embryonic development provides the driver component of the Tet-Off system. In the absence of tetracycline-class antibiotics, tTAo binds to the 21 tandem tetracycline operator sequences (tetO21) associated with the Lucilia cuprina heat shock protein 70 (Lchsp70) promoter and activates transcription of the downstream female-lethal effector.

2. The effector cassette encodes the modified Lucilia sericata head involution defective protein (LshidAla2) and terminates at the SV40 polyadenylation signal. A sex-specific intron derived from the Cochliomyia hominivorax transformer gene (Chtra intron) is incorporated into the cassette, producing a functional transcript only in females. Male-specific splicing generates a non-functional isoform containing premature stop codons. In the presence of sufficient concentrations of tetracycline or doxycycline, the antibiotic binds tTAo and reduces its affinity for the tetracycline operator sequences, thereby suppressing activation of LshidAla2 expression and allowing survival of both sexes and maintenance of mixed-sex colonies under laboratory rearing conditions.

3. Transcription of the DsRed-Express2 (DsRedex2) fluorescent marker is driven by the L. cuprina heat shock protein 83 (Lchsp83) promoter and terminates at the L. cuprina tubulin (Lctub) polyadenylation signal.

The construct additionally contains a φC31 attP attachment site, which provides a recognition site for φC31 integrase-mediated site-specific recombination, and piggyBac terminal sequence(s) associated with the transposon-mediated germline transformation system.

Notes:
-The TD1#12 genetic construct is present as a single, stably integrated copy in a homozygous state and is inherited in a Mendelian manner.
-Whole-genome sequencing confirmed a single insertion event and the absence of plasmid backbone sequences.
-The insertion is located within an intronic region and is not predicted to disrupt endogenous genes or open reading frames.
-DsRedex2 is constitutively expressed in all cell types and throughout the insect life cycle and permits identification of transgenic individuals. Therefore fluorescence is detectable in all tissues and life stages of both sexes.
-DsRedex2 is expressed at substantially higher levels than tTAo and LshidAla2. Mean DsRedex2 protein levels were approximately 113 ng per adult male and 0.2 ng per egg.
-Expression of tTAo and LshidAla2 was low, close to the lower detection limit of the assay (5–10 pg/mL), and occurred primarily during early embryonic development.