Transit insect transfection reagent

The genes of candidate marker antigens were amplified by polymerase chain reaction (PCR) using the plasmid DNA containing the candidate marker antigen gene as templates. Six histidine residues (His) were added at the N- and C-terminus of genes during PCR amplification. The target genes were cloned into the pFastBac1 vector (Invitrogen) and confirmed by sequencing. The recombinant bacmid was generated using the Bac-to-Bac System (Invitrogen). The recombinant baculovirus was generated by transfecting the recombinant bacmid to Sf9 cells using TransIT-Insect Transfection Reagent (Mirus Bio). The recombinant proteins that were extracted from baculovirus-infected cells were then analyzed by SDS-PAGE and western blotting (WB). Finally, the proteins were purified using a His-tagged protein purification kit (CoWin Biosciences).

Mouse and human NELL1, NELL2, Robo1, and Robo3 proteins were expressed using baculoviruses. The mature domains or domain truncations of these genes were cloned into pAcGP67A (BD Biosciences) with C-terminal hexahistidine tags and co-transfected into Sf9 cells (Spodoptera frugiperda) with linearized baculovirus BestBac 2.0 (Expression Systems) using the TransIT-Insect transfection reagent (Mirus) according to manufacturer’s instructions. Sf9 cells were cultured in SF900 SFM III (Fisher, 12658-019) with 2 mM l-glutamine (HyClone SH30034.02), 20 µg/ml gentamicin sulfate and 10% fetal bovine serum. For infection of High Five cells with baculoviruses, 2–5 ml of virus-containing conditioned media from Sf9 cultures were added per 1 l of High Five culture.
The produced viruses were used to infect cultures of High Five cells (Trichoplusia ni, BTI-Tn-5B1-4) grown in suspension (120 r.p.m. on shakers) at 27–28 °C in Insect-XPRESS media (Lonza, BE12-730Q) with 10 µg/ml gentamicin sulfate (Lonza, 17-518 L). Proteins were expressed for 66 h at 27–28 °C post infection. For producing biotinylated proteins, a C-terminal Avi-tag (GLNDIFEAQKIEWHE) followed by a hexahistidine tag was added to expression constructs. Avi-tagged proteins were biotinylated with the Escherichiacoli biotin ligase BirA.
Primer sequences used for cloning are provided in Supplementary Table 4.

Mosquito C6/36 cells were plated at a density of 4 × 10⁵ cells per well in 24-well plates. The next day, various doses of recombinant plasmid DNA (1 µg, 2 µg and 3 µg in Supplementary Fig. 2 and 0.5 µg in Fig. 3) were mixed with respective amounts of transfection reagent (TransIT-Insect Transfection Reagent; Mirus) as per manufacturer recommendations and incubated for 30 min at RT, before the DNA + transfection reagent mixture was added to the cells. After 5 h in a 28 °C incubator, the cells were removed and washed with 1X DPBS, before 10%-FBS complete medium was added. The cells were then incubated at 28 °C for 2 days before being collected for flow cytometry analyses.

For transfection of 3 × 10⁶ Drosophila S2 cells in six-well plate tissue culture format, 1 µg DNA plasmid was complexed with 4 µL Lipofectamine^TM3000 (ThermoFisher Scientific, Waltham, MA, USA) and 4 µL P3000 transfection reagent in 100 µL Opti-MEM, incubated for 15 min at room temperature prior to addition to 5 × 10⁶ cells. S2 transfections were incubated at 28 °C and harvested at 40–44 h post-transfection. RNA transfections in BSRT7/5 cells were mediated using 4 µL Trans-IT Insect transfection reagent (MIRUS-Bio, Madison, WI, USA) per 1 µg in vitro transcribed/capped RNA per six-well plate well containing 3 × 10⁶ cells. BSRT7/5 transfections were incubated for 3 h at 37 °C, transferred to 32 °C, and harvested at 40–44 h post-transfection. Transfections were scaled as needed according to tissue culture vessel size.

Baculovirus stocks were stored as baculovirus-infected insect cells (BIICs). BIIC stocks were generated by transfection of Sf9 cells with bacmid DNA using TransIT-Insect Transfection Reagent (# MIR 6100, Mirus Bio, Madison, WI) as follows: A 30 mL culture of Sf9 cells (2 × 10⁶ cells/mL) was prepared via the dilution of seed train into an ESF medium. Bacmid-transfection complexes were prepared by combining 200 ng of bacmid DNA, 5 µL of TransIT Reagent, and 200 µL of non-supplemented Grace’s Salts (#G8142, Sigma-Aldrich, St. Louis, MO, USA), followed by incubation at room temperature for 20 min. The entire mixture was then added directly to the Sf9 culture to initiate the reconstitution of infectious baculovirus.
At 3 days post-transfection, the cells were pelleted via centrifugation (5 min, 300× g), and virus-containing supernatant was transferred to a separate tube and stored in the dark at 4 °C.

Competent DH10Bac™ E. coli cells were transformed with the donor plasmid pFBD-3BT-GS, following the manufacturer’s recommendations. Each recombinant bacmid DNA was purified by in-house minipreps (62 (link)) for transfection into Sf9 cells, using TransIT^®-Insect Transfection Reagent (Mirus Bio LLC). Passage 0 (P0) of BacDual-3BT was harvested and titrated as previously described (63 (link)). To obtain high titer P2 and P3 of Bac-GFP and BacDual-3BT, Sf9 cells at 1.2 x 10⁶ cells/mL were infected with a multiplicity of infection (MOI) of 0.1 PFU/cell. At 80 h post-infection, baculoviral supernatants were harvested (64 (link)). To reach Bac-GFP and BacDual-3BT titers of 1 x 10⁹ PFU/mL, utilized for immunization, supernatants were centrifugated at 14,000 x g 90 min at 4°C. Supernatants were removed, and viral pellets were soaked with sterile/endotoxin-free PBS overnight at 4°C. Baculoviral pellets were gently resuspended, titrated, aliquoted, and stored protected from light at 4°C (64 (link)).