The transcriptome associated to P. vivax infection revealed a variety of transcripts that play a key role in autophagy. In order to evaluate the effect of the autophagy process in the outcome of infection, we inoculated mosquitoes with wortmannin (an inhibitor of phosphatidylinositol 3-kinase DPI3K) and spermidine (an autophagy activator) [36 (link), 37 (link)]. Three- to four-day-old female mosquitoes were cold-anesthetized and inoculated intrathoraxically with 69 nl of a 5 μM and 0.05 μM solution of wortmannin (Merck, Darmstadt, Germany) or with the same volume of H2O Ultra Pure and with 69 nl of a 100 μM solution of spermidine (Sigma) or DMSO (0.05%) using a Nanoject micro-injector (Drummond Scientific, Pennsylvania, USA). Twenty-four hours after injection with the solutions, the mosquitoes were fed with a P. vivax-infected blood meal as described above. Three independent biological replicates were performed for each experiment. Mosquitoes were dissected 18–24 h after feeding; batches of 20–30 midguts were dissected in cold DEPC-treated phosphate-buffered saline (PBS) and processed for RNA preparation and cDNA synthesis using the same protocols mentioned above. Mosquito midguts were also collected on the 8th day post-infection to determine the prevalence and intensity of infection.
Nanoject microinjector
Manufactured by Drummond
Sourced in United States
The Nanoject microinjector is a laboratory instrument used for the precise injection of nanoliter volumes of liquids, such as DNA, RNA, or other biological samples, into cells or embryos. The device is designed to deliver consistent and reproducible injection volumes with high accuracy and precision.
Automatically generated - may contain errors
Lab products found in correlation
Mmessage mmachine t7 kit, by Thermo Fisher Scientific (2 mentions)
Matrigel, by BD (2 mentions)
Spermidine, by Merck Group (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Megascript sp6 kit, by Thermo Fisher Scientific (1 mentions)
H3k4me2 3, by Abcam (1 mentions)
P entry cloning system, by Thermo Fisher Scientific (1 mentions)
H3k9me2 3, by Cell Signaling Technology (1 mentions)
H3k27me3, by Cell Signaling Technology (1 mentions)
Transcriptaid t7 high yield transcription kit, by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
Dna template prep kit 2, by Pacific Biosciences (1 mentions)
Megaclear kit, by Thermo Fisher Scientific (1 mentions)
High pure pcr purification kit, by Roche (1 mentions)
13 protocols using nanoject microinjector
1
Modulating Autophagy in Mosquitoes to Understand P. vivax Infection
2
Overexpression of Chromatin Modifiers
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Mouse KDM6B (aa1025–1642) or KDM5B (aa1–770) were cloned using the p-Entry cloning system (Invitrogen, #K2400-20 and 11791-020) into a pCS2+ plasmid with a C-terminal HA-tag and NLS-tag. mRNA was synthesized in vitro using a MEGAscript SP6 Kit (Ambion, AM1330M) following the manufacturer's instructions. Eggs were in vitro–fertilized and de-jellied using a 2% cysteine solution in 0.1 × MMR. Injections into one-cell stage embryos were performed in injection solution (Smith et al. 2006 (link)) using a Drummond Nanoject microinjector, delivering 9.2 ng of mRNA per injection (mRNA at 1 mg/mL in DEPC H2O). Embryos were cultured at 18°C and collected for Western blot analysis at stage 21 (Nieuwkoop and Faber 1994 ). Western blot analyses were performed on 12% polyacrylamide gels using antibodies against H3K27me3 (Cell Signalling, #9733), H3K9me2/3 (Cell Signalling, #5327), H3K4me2/3 (Abcam, #8580), H4 (Abcam, #31830), and against H3 (Abcam, #18521)
3
Mosquito Malaria Infection Analysis
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A. gambiae s.s. mosquitoes (Yaoundé strain) were reared and maintained as described previously [17 (link)]. Three to four day old female mosquitoes were cold-anaesthetized and inoculated intrathoracically with 69 nl of a 50, 100 or 200 μg/ml solution of sHz or with the same volume of endotoxin-free PBS, using a Nanoject micro-injector (Drummond Scientific). Mosquitoes were left to rest for 24 h. Female CD1 mice were intraperitoneally inoculated with 107Plasmodium berghei (ANKA) GFPcon 259 cl2 parasitized red blood cells/ml and mosquitoes were fed as previously optimized in our Lab [17 (link)]. Four independent biological replicates were performed for each experiment. Between 8 and 10 days post-infection, mosquito midguts were collected to determine infection rate (prevalence) and intensity.
4
Xenopus Oocyte Expression of D2 Receptors
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Oocytes from the African clawed
toad, Xenopus laevis, were isolated surgically as
described previously.35 (link) The surgical procedures
were approved by the Swedish National Board for Laboratory Animals
and the Stockholm Ethical Committee. Following 24-h incubation at
12 °C, oocytes were injected (using a Nanoject microinjector;
Drummond Scientific) with 0.2 ng of D2L receptor cRNA,
40 ng of RGS4 cRNA, and 1 ng of each GIRK1 and GIRK4 cRNA in a volume
of 50 nL per oocyte. RGS4 is a GTPase activating protein and was included
in order to speed up the kinetics of G protein turnover, such that
GIRK channel opening more closely follows D2R occupancy
by DA.
toad, Xenopus laevis, were isolated surgically as
described previously.35 (link) The surgical procedures
were approved by the Swedish National Board for Laboratory Animals
and the Stockholm Ethical Committee. Following 24-h incubation at
12 °C, oocytes were injected (using a Nanoject microinjector;
Drummond Scientific) with 0.2 ng of D2L receptor cRNA,
40 ng of RGS4 cRNA, and 1 ng of each GIRK1 and GIRK4 cRNA in a volume
of 50 nL per oocyte. RGS4 is a GTPase activating protein and was included
in order to speed up the kinetics of G protein turnover, such that
GIRK channel opening more closely follows D2R occupancy
by DA.
5
RNAi-Mediated Gene Silencing in Planarians
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RNAi experiments were performed as described in [18 (link)]. Briefly, DjMap1B double-stranded RNA (dsRNA) was in vitro synthesized by using TranscriptAid T7 High Yield Transcription kit (Thermo Scientific) using the following primers:
Animals were injected with 3 shots of 32 nl each of dsRNA or water (control) once a week for 8 consecutive weeks using a Nanoject Microinjector (Drummond). For the experiment with low-dose irradiation, we also used as control animals injected with DjOps dsRNA prepared as previously described [18 (link)].
Animals were injected with 3 shots of 32 nl each of dsRNA or water (control) once a week for 8 consecutive weeks using a Nanoject Microinjector (Drummond). For the experiment with low-dose irradiation, we also used as control animals injected with DjOps dsRNA prepared as previously described [18 (link)].
6
Expression and Electrophysiology of UNC-49 Receptors
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Expression of unc-49 receptor cRNA was according to methods outlined in Abdelmassih et al. (2018 ) and adhered to the guidelines of the Canadian Council of Animal Care. Xenopus laevis were anesthetized using 0.15% 3-aminobenzoic acid ethyl ester, methane sulphate salt buffered to pH 7 with sodium bicarbonate. Oocytes were surgically removed and cut into small clumps of approximately 20 eggs per clump. These were incubated in OR-2 (82 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.5) with type-II collagenase (Sigma Aldrich, Canada) for 2 h while shaking. These were then placed in ND96 supplemented with pyruvate and gentamycin. Oocytes were injected with 50 nL of either wild-type (hco-unc-49b/c) or mutant hco-unc-49b/ wildtype hco-unc-49c cRNA, using the Drummond Nanoject microinjector. These were stored at 20ᵒC for 48 h, replacing the supplemented ND96 twice before electrophysiology.
7
Kv Channel Expression and Regulation in Xenopus Oocytes
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mRNA was generated from linearized pXOOM plasmids using the Ambion T7 mMessage mMachine kit (Ambion, Austin, TX, USA) according to manufacturer's instructions. The mRNA concentrations were determined using a ND-1000 NanoDrop UV spectrophotometer, and mRNA integrity was confirmed by gel electrophoresis. mRNA was stored at −80°C until injection.
Xenopus laevis oocytes were purchased from EcoCyte Bioscience, Germany. Upon delivery, they were kept at 19°C for 24 hours in Kulori solution (4 mM KCl, 90 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, pH 7.4). Subsequently, they were injected with 50 nl of Kv1.4 mRNA (50 pg) using a Nanoject microinjector (Drummond Scientific, Broomall, PA, USA). For co-expression of Kv1.4 and Nedd4-1 or Nedd4-2, the mRNAs were mixed in molar ratios of 1:1, 1:2, 1:5 and 1:10 prior to oocyte injection. For measurements on Kv7.1, 10 ng of Kv7.1 mRNA was injected alone or in combination with Nedd4-1/Nedd4-2 in a 20:1 molar ratio. For optimal expression conditions, the oocytes were kept in Kulori medium at 19°C for 48–72 hours before functional measurements were performed.
Xenopus laevis oocytes were purchased from EcoCyte Bioscience, Germany. Upon delivery, they were kept at 19°C for 24 hours in Kulori solution (4 mM KCl, 90 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, pH 7.4). Subsequently, they were injected with 50 nl of Kv1.4 mRNA (50 pg) using a Nanoject microinjector (Drummond Scientific, Broomall, PA, USA). For co-expression of Kv1.4 and Nedd4-1 or Nedd4-2, the mRNAs were mixed in molar ratios of 1:1, 1:2, 1:5 and 1:10 prior to oocyte injection. For measurements on Kv7.1, 10 ng of Kv7.1 mRNA was injected alone or in combination with Nedd4-1/Nedd4-2 in a 20:1 molar ratio. For optimal expression conditions, the oocytes were kept in Kulori medium at 19°C for 48–72 hours before functional measurements were performed.
8
Extracting Spiroplasma Genomic DNA from Fly Hemolymph
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Cell-free cultivation of spiroplasmas is not currently possible. To obtain the required amount of genomic DNA to generate a genomic library (~3 µg), we extracted ~50 µl of pure fly hemolymph with a Nanoject microinjector (Drummond Scientific) from 1-month-old infected flies. The hemolymph extracts were immediately mixed with 500 µl of phosphate-buffered saline (PBS) on ice to avoid melanization. Bacteria were pelleted, washed twice with 500 µl of PBS, and then filtered through a 0.45-µm filter to eliminate other potential bacterial contaminants or Drosophila hemolymph cells (36 (link)). Bacteria were subsequently pelleted. For optical mapping, the bacterial pellet was sent to OpGen. For sequencing, DNA was extracted by phenol-chloroform extraction. The DNA library was prepared with the Pacific Biosciences DNA Template prep kit 2.0 (3 to 10 kb) with 3 µg of S. poulsonii genomic DNA with an expected insert length of 25 kb.
9
Generating Mouse Ileal Organoids
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Mouse organoids were generated as previously described (Fernando et al., 2017 ). Briefly, the ileum was removed from 8 to 12‐week‐old male BALB/c mice and washed thoroughly in ice‐cold Ca2+/Mg2+‐free DPBS. After washing and cutting into small sections, the tissue was incubated in 3 mM EDTA, DTT, and sucrose at 4 ºC for 30 min. Crypts were collected in chelation buffer (sucrose, sorbitol, and bovine serum albumin), centrifuged at 300 × g for 10 minutes, and embedded in Matrigel (BD Biosciences). After Matrigel polymerization, Matrigel domes were covered with CMGF+media (purchased from the BCM GEMS Core) without Wnt containing 10 µM Y‐27632 rock inhibitor (Chang‐Graham et al., 2019 ). Organoids were used at passage 3 to ensure the remaining debris was removed. After 5 days of growth, organoids were microinjected with 17.6 nl of solution (uninoculated LDM4 media control, 100 ng/mL of LPS or L. reuteri LDM4 conditioned media or L. reuteri UV‐irradiated bacteria) using a Nanoject microinjector (Drummond Scientific Company) as previously described (Engevik et al., 2013 ). Organoids were incubated overnight and the supernatant was analyzed using Luminex Multiplex Assay.
10
Fluorescent RNA Localization in Xenopus Oocytes
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In vitro transcribed fluorescently labeled RNAs (fluorescein-U2 snRNA, fluorescein-U7 snRNA, Cy5-DmscaRNA:MeU2-C28) were injected into X. laevis oocytes using a Nanoject microinjector (Drummond). The injection procedure and amount of injected RNAs were exactly the same as in the in vitro modification assay with unlabeled U2 snRNA and its guide scaRNA:MeU2-C28 (Deryusheva and Gall 2009 (link)). After overnight incubation, injected and control Xenopus oocyte nuclei were isolated under mineral oil. While still in oil, nuclei were transferred onto slides, gently covered with a coverslip, and analyzed by confocal microscopy. All manipulations with Xenopus were performed according to approved IACUC protocols.
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