Gene block

Mammalian expression constructs used include: Retroviral vectors encoding shRNA for luciferase-RNAi and EWS::FLI-RNAi, as well as cDNA-containing vectors encoding 3xFLAG-EF, 3xFLAG-EF DBD, 3xFLAG-EF DBD+ (Boone et al., 2021 (link)). The EWS::FLI DBD, and EWS::FLI DBD+ were ordered as gene blocks (Integrated DNA Technologies) and cloned into the pMSCV-hygro plasmid between BamHI and AgeI restriction sites.

Chimeric 5′UTR-luciferase constructs were cloned by FastCloning using Phusion HF polymerase according to manufacturer’s protocol (NEB) (Li et al, 2011 (link)). pGEM-luc and pSP64 Poly(A) vectors were purchased from Promega. NotI and BglII sites were first cloned into pSP64 Poly(A) vector, and luciferase was cloned upstream of the poly(A) sequence. 5′UTR from intestine and kidney/liver isoforms were cloned from GeneBlocks (Integrated DNA Technologies) directly 5′ to the luciferase sequence. Plasmid sequences were all verified by Sanger sequencing across inserts.

The cDNA sequence encoding the ectodomain of desired HAs, or the variable domains from the heavy and light chains of the antibodies, were codon optimised for Chinese hamster (Cricetulus griseus) expression before synthesis as Geneblocks (Integrated DNA Technologies) together with the necessary 5′ and 3′ overlaps for subsequent InFusion cloning (ClonTech) into in-house generated mammalian expression vectors. These vectors contained either no trimerisation domain, the foldon trimerisation domain or the molecular clamp domain (derived from HIV-1 glycoprotein 41 heptad repeat (HR) regions, HR1 (aa540–576) and HR2 (aa619–656)) for the HA constructs, or the constant regions of human or mouse IgG1 heavy or light chain for the antibodies. The HA stem constructs, which were based on previously designed antigens³⁷ were cloned into a vector containing a monomeric Fc domain^{82 (link)}. After successful InFusion cloning, plasmid DNA was prepared by Midi- or Maxi-prep (Promega) and sequence confirmed at the Australian Genome Research Facility (AGRF).

We used the following stocks in these experiments: yw (wild type), ±/Cyo bcd⁺;bcd^E1/bcd^E1, Cyo bcd⁺/Sco;bcd^E1/bcd^E1, and ΦC31 (y+);38F1 (w+). We cloned an injection plasmid (piattB40-Bcd) containing two inverted ΦC31-specific recombination sequences (AgeI/HindIII) for Recombination Mediated Cassette Exchange (RMCE), Gmr-GFP (HindIII/AscI), and a polylinker flanked by 1.9 kb bcd promoter and 0.8 kb 3’UTR (AgeI/AscI). The bcd and zen coding regions were amplified by PCR from pBS-SK + cDNA clones, digested with RsrII and AscI and ligated into piattB40-Bcd. We used standard cloning techniques to generate homeodomain swaps and residue changes. Gene Blocks coding for the ancestral HD and variant sequences together with the flanking Bcd coding sequence were obtained from Integrated DNA Technologies (IDT). They were digested with AscI and BspEI and ligated to the piattB40-Bcd vector digested with the same restriction enzymes. All transgenic lines were generated using the ΦC31 integration system, and constructs were integrated into the 38F1 landing site on the second chromosome (Bateman et al., 2006 (link)). Each transgene was crossed to Cyo bcd⁺/Sco; bcd^E1/bcd^E1 to generate Cyo bcd⁺/[transgene]; bcd^E1/bcd^E1 stocks. Embryos and larvae from homozygous transgenic females were assayed for gene expression and cuticle phenotype.