Nanoinject 2

To prepare the gene-specific dsRNA, 200–600bp long gene fragments were amplified from I. ricinus cDNA and cloned into the pll10 vector with two T7 promoters in reverse orientations (29 (link)), using primers listed in Supplemental Table 2 and containing additional restriction sites ApaI and XbaI. The dsRNA was synthesized as described previously (30 (link)). The dsRNA (3 μg/μl) was injected through the coxa of the third pair of legs into the hemocoel of nymphs (32 nl) using Nanoinject II (Drummond). After 3 days of rest in a humid chamber at room temperature, the nymphs (20 nymphs per mouse, 3 mice per group) were fed on BALB/c mice (Velaz, CR). The level of gene silencing was checked by qRT-PCR in a mix of five fully fed nymphs and compared to the dsGFP control group. For each group, we recorded feeding success, length of feeding, the weight of individual nymphs after feeding, and molting into adults (took approximately 2 months; recorded every 2 weeks until molting in the dsGFP control group reached 80%).

Wasps were anesthetized by chilling on ice for 3 min. Wings were ablated to limit the mobility of the wasp upon recovery. Venom obtained by a milking procedure described previously (Kaiser and Libersat, 2015 (link)) was collected using a nanovolumetric injector (Drummond Nanoinject II). The needle of the nanoinjector was kept close to ice throughout the process. The milking procedure did not take more than 25 min.

Ten days after EAE induction, rats received bilateral ICV injections of 1 × 10⁶ PMSCs, EMSC or phosphate-buffered saline (PBS) in a volume of 2 μL using a Hamilton 10 μL syringe with a 26-gauge needle. The coordinates of the injections were as follows: AP + 0.6 mm, ML ± 0.7 mm, and V −3 mm, from Bregma based on the mouse stereotaxic atlas (Paxinos and Watson). Two hours before transplantation, the GFP+ labeled cells were detached from the dishes by using a cell lifter, collected by centrifugation at 1000 × g for 4 min, and resuspended in 1 mL culture medium. After cell counting and viability assessment with Trypan blue in a hemacytometer, the cell suspension was centrifuged a second time and resuspended in a smaller volume to give a density of 1 × 10⁶ viable cells/μL.
For ITH transplantation, the vertebrae were carefully separated using two fine tweezers in order to reveal the lumbar spinal cord (L4–L5). EMSCs, PMECs (1 × 10⁶ cells) or saline were injected intrathecally with a glass micropipette connected to the nanoinjector (Nanoinject II; Drummond Scientific Company, Broomall, PA, USA) at the rate of 1 μL/minute for a total volume of 4 μL.

Stage V-VI de-folliculated Xenopus laevis oocytes were obtained from Ecocyte Bioscience US LLC (USA). On the day of delivery, the oocytes were selected and injected (Nanoinject II, Drummond Scientific, USA) with 50 nl of either mRNA or with H₂O (control). All experiments were done on the third day after injection. Oocytes after injection were cultured in a modified L-15 media (OR-3) and kept at 15–20 °C. Oocyte batches from three different shipments were used in the western blot and transport experiments.

The Nanoinject II (Drummond, 3–000204) was used to conduct RNAi inoculation on 1-day-old female An. arabiensis mosquitoes [22 ]. Cold anesthetised mosquitoes were inoculated with 69nl (3mg/ml) of Akirin dsRNA, Akirin siRNA or Mouse-ß2m siRNA. A subset of cold anesthetised mosquitoes injected with PBS was used as a handling control, while a subset of mosquitoes that were not subjected to inoculation, but had undergone cold anesthetisation, were used as an untreated control. Fatalities as a result of the injection procedure were minimal. However, if a fatality occurred immediately after injection, the mosquito was discarded from the analysis.
Inoculations were conducted using a total of 300 female mosquitoes per each treatment. Fifty mosquitoes were randomly selected from each treatment for quantitative-PCR (qPCR) analysis (10 mosquitoes per replicate, 5 replicates), while 150 female mosquitoes were randomly selected for longevity analysis (30 mosquitoes per replicate, 5 replicates). The remaining 100 female mosquitoes from each treatment were used to analyse vector fertility and fecundity (20 mosquitoes per replicate, 5 replicates). All treatments were carried out in 32.5cm³ insect rearing cages (BugDorm®, 211476).