Plasmid midi kit

Chromosome preparations were carried out as described by Rodriguez et al. [2 (link)], using larvae (1–3 days after hatching) of S. senegalensis. The DNA-BAC was purified using Plasmid Midi Kit (Quiagen, Hilden, Germany) following manufacturer’s instructions, and then labelled using Biotin or Digoxigenin Nick Translation Mix (Roche Molecular Biochemical), as described by manufacturer’s instructions. Pre-treatment of chromosome preparations and hybridization were carried out following the protocol described by García-Cegarra et al. [30 (link)]. For the immunocytochemistry detection, the antibodies described in Rodriguez et al. [2 (link)] were used. The antibodies were prepared in Tween Non-Fat Milk (TNFM, 4x SSC, 0.05% Tween 20.5% skim milk). Chromosome staining was carried out with 0.4 mg/mL of 4’,6-diamidino-2-phenylindole DAPI-Vectashield (Antifade Mounting Medium) (Vector), and the images were examined with a Zeiss Palm MicroBeam microdissector and fluorescence microscopes equipped with an AxioCam MRm digital camera.

Plasmid DNAs were isolated using the Quiagen plasmid Midi Kit, and germline transformation was performed as described in refs (Berghammer et al., 1999 (link); Berghammer et al., 2009 (link)). In one experimental series, 408 vermilion white embryos were injected of which 22% hatched. 44 crosses were set up, from which 10 transgenic strains could be generated. In another experimental series, 210 embryos were injected with a hatch rate of 56%. 59 crosses were set up, from which five transgenic strains could be generated. Ten of these hs-hb lines were tested for heat-shock phenotypes. Phenotype strength was measured by determining the proportion of larvae which (i) developed homeotic transformations, and (ii) which displayed, in addition to the homeotic transformations, additional trunk segments (see Appendix 1 for basic description of the cuticle phenotype of heat-shocked hs-hb embryos). Two out of those ten lines (hs-hb one and hs-hb 2) seemed most effective in generating heat-shock phenotypes and were further studied. The strain hs-hb two was used to generate the data in this paper.

Purified BSP amplification products were ligated to the pGEM-T Easy Vector and transformed into competent JM109 cells by using the pGEM-T Easy Vector system I (Promega, Madison, WI, USA). White colonies were selected from the ampicillin/X-Gal/IPTG plates and colony PCR was performed using vector-directed primers to confirm the presence of inserts based on their expected fragment size. Ten positive colonies from each plate were inoculated into LB medium with ampicillin for plasmid isolation. Plasmids containing the target DNA were extracted and purified by using the Plasmid Midi Kit (QIAGEN) and sequenced by using the ABI Prism BioDye Terminator version 3.1 sequencing system. Cytosine methylation results were analyzed using the CyMATE online methylation sequence analysis software48 (link). Average methylation level expressed as percentage (%) per site for each of the three types of cytosines, CG, CHG and CHH, were calculated by dividing the number of non-converted (methylated) cytosines by the total number of cytosines of each type within the sequenced region.

To establish a purely tick promoter reporter plasmid, the H. longicornis actin (HlActin) promoter was used to replace the SV40 promoter of the Renilla luciferase (hrLuc) of the pmirGLO plasmid vector (Promega, Madison, WI, USA). First, the previously identified HlActin promoter region was amplified from the previously constructed pmirGLO-HlActin pro [12 (link)] using a primer pair of HlActin-Renilla F and HlActin-Renilla R (Additional file 1: Table S1) by PCR with KOD-Plus-Neo (Toyobo, Osaka, Japan). After amplification, the PCR products were electrophorosed in 1.0% agarose gel. The PCR product was purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The pmirGLO vector was then double-digested using KpnI and PfmI to remove the SV40 promoter. After double digestion, the plasmid vector was electrophoresed in 1.0% agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen). The digested vector and purified product were mixed with an In-Fusion HD cloning kit (Takara, Shiga, Japan). After the ligation of pmirGLO/HlActin-hrLuc, the plasmid was transformed into the Escherichia coli Stellar strain and purified using a Plasmid Midi Kit (Qiagen).

Yellow fluorescent protein (Venus) expression vectors with an HlFer1(F2) promoter region were constructed. To produce a pmirGLO/Fer1 (F2)-Venus plasmid, the Fer1 (F2) promoter was amplified by PCR using KOD-Plus-Neo (Toyobo) with pmirGLO-HlFer2-Venus F and pmirGLO-HlFer2-Venus R primers, while the Venus gene was also amplified using pmirGLO-Venus F and pmirGLO Venus-XhoI R. These PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen). The pmirGLO plasmid was double digested with BglII and XhoI to remove the PGK promoter and luciferase gene from the vector. The obtained vector and two purified products were mixed with an In-Fusion HD Cloning Kit (Takara) and incubated. After ligation, the plasmid was transformed into the E. coli Stellar cells and purified using a Plasmid Midi Kit (Qiagen). The pmirGLO/no promoter-Venus was constructed by blunt ligation of the purified Venus gene with the digested vector using DNA Ligation Kit Vector 2.1 (Takara).

Protein expression and purification was performed following standard protocols as previously described by Stadlbauer et al. (21 (link)) and Byrum et al. (41 (link)) with some modifications. The viral proteins RBD, S, and N are based on the genomic sequence of the Wuhan-Hu-1 isolate (2 (link)). Plasmids pCAGGS encoding SARS-CoV-2 Spike (with a C-terminal hexa-histidine tag), and the RBD genes (with a C-terminal hexa-histidine tag) were obtained from a donation of Dr. Florian Krammer (Department of Microbiology, Icahn School of Medicine at Mount Sinai, NY, United States) (21 (link)). Plasmid pET-28 vector (41 (link)) encoding SARS-CoV-2 N gene was donated by Dr. Eva Harris and Dr. Scott Biering (Division of Infectious Diseases and Vaccinology University of California, Berkeley, CA, United States).
All SARS-CoV-2 protein-encoding plasmids were initially amplified by transforming chemically competent Escherichia coli DH5α cells using approximately 100 ng of purified plasmid (QIAGEN Plasmid Midi Kit, United States) grown in LB medium supplemented with Ampicillin (1 μg/mL). PCR positive colonies were grown for mass production, and plasmids were recovered and purified following manufacturer’s protocols for standard DNA plasmid purification (Zippy™ Plasmid Midiprep Kit, ZYMO Research).