Journal: iScience

Article Title: Genetic manipulation for the non-model protozoan Eimeria : Advancements, challenges, and future perspective

doi: 10.1016/j.isci.2025.112060

Figure Lengend Snippet: Schematic for the development of genetically modified (GM) Eimeria oocysts The process of gene editing in Eimeria species and the subsequent evaluation of GM oocysts. Transfection and cultivation: The gene of interest (GOI), driven by a specific promoter (as detailed in Table 1 ), is delivered into the Eimeria genome via electroporation using a shuttle plasmid. This process involves the use of sporozoites, merozoites, or sporocysts as recipient cells, and may employ systems like the U-033 electroporation system. After the electroporation, the GM parasites are cultured in vitro for 24 h or more using cell lines such as Vero, MDBK, or PCKCs. The presence and expression of the reporter gene are then observed under a microscope. In vivo application : The transfected sporozoites, merozoites, or sporocysts are administered to chickens via oral, cloacal, or intravenous routes depending on the species and parasitic site of Eimeria . When drug resistance genes are present, the corresponding drug should be included in the first-generation application. It’s crucial to note that to ensure the expression of drug resistance genes in the transfected parasites, the drug is typically added 18 to 24 h after inoculation. Selection and propagation : After obtaining genetically modified oocysts, it is essential to perform reporter gene detection. Depending on the type of fluorescent reporter gene used, select the appropriate excitation wavelength for the flow cytometry channel to sort the oocysts or sporocysts. These genetically modified oocysts or sporocysts can then be enriched and subjected to further passage and amplification. When utilizing drug resistance genes, the addition of the corresponding drug can significantly enhance selection efficiency. It has been demonstrated that drug resistance genes for pyrimethamine, halofuginone, and diclazuril (unpublished data) are effective in screening GM Eimeria oocysts. Evaluation of GM oocysts : After stabilizing a population of GM oocysts, various identification methods can be employed to verify the modifications. At the genetic level, PCR can confirm the stable presence of the gene of interest, while techniques such as genome walking or plasmid rescue can analyze the genomic insertion site. Due to potential challenges in RNA extraction quality, identifying transcripts of the gene of interest using reverse transcription PCR (RT-PCR) may be technically demanding. At the protein level, GOI specific antibodies or tagged antibodies can be utilized in Western blotting (WB) and indirect immunofluorescence assays (IFA) verify the expression of GOI. In addition, depending on the purpose of genetic modification, biological analyses of GM oocysts—such as morphology, oocyst shedding patterns, pathogenicity, and immunogenicity—can be systematically compared with those of the parental strain.

Article Snippet: Lonza’s patented Nucleofector nuclear transfection technology, which integrates optimized electroporation instruments, buffers, and programs, can deliver foreign nucleic acids into the cytoplasm and directly into the nucleus, significantly improving transfection efficiency.

Techniques: Genetically Modified, Transfection, Electroporation, Plasmid Preparation, Cell Culture, In Vitro, Expressing, Microscopy, In Vivo, Selection, Flow Cytometry, Amplification, RNA Extraction, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Modification, Immunopeptidomics

Journal: Frontiers in Cellular Neuroscience

Article Title: Functional Reintegration of Sensory Neurons and Transitional Dendritic Reduction of Mitral/Tufted Cells during Injury-Induced Recovery of the Larval Xenopus Olfactory Circuit

doi: 10.3389/fncel.2017.00380

Figure Lengend Snippet: Olfactory nerve transection induces transitional OB volume reduction due to axonal degradation of olfactory receptor neurons and subsequent reinnervation by new neurons. (A) Graph shows relative changes in OB volume recovering after ON transection (filled black circles, black line connects mean values for each time-point analyzed) and of animals subjected to weekly ON transection to hinder reconnection of ORN axons to the OB (empty circles, dotted line connects mean values for each time-point analyzed). Animals were transected unilaterally, and changes in OB volume are shown as the percentage of decrease in volume of the transected side in relation to the non-transected side. (B) Non-transected OB with ORN axons (white) stained by nasal electroporation of fluorescent dextrans. Typical ventral glomerular clusters are outlined with a dotted white line: lateral (LC), intermediate (IC), small cluster (SC) and medial cluster (MC). The ORN axons of the accessory olfactory bulb (AOB) are also visible on the lateral side of the OB. (C) ON transection induces gradual axonal degradation in the OB. Axons (cyan) were labeled by microRuby via the ON, which is anterogradely transported along the axons. Two days post-transection degeneration of axonal fibers became apparent and fluorescent dye began to accumulate in aggregates that gradually dispersed through the OB over time (posterior agglomerates highlighted by open arrowheads, glomerular clusters are outlined with a dotted white line). (D) Representative images of the OB showing reconnecting ORN axons (magenta) stained by nasal electroporation at different time-points after ON transection (1, 2, 3 and 7 weeks). Examples of individual axons are highlighted by filled arrowheads and glomerular clusters are outlined with a dotted white line. A, anterior; AOB, accessory olfactory bulb; a.t., after transection; IC, intermediate cluster; L, lateral; LC, lateral cluster; M, medial; MC, medial cluster; MOB, main olfactory bulb; n.t., non-transected; OB, olfactory bulb; ON, olfactory nerve; ORN, olfactory receptor neuron; P, posterior; SC, small cluster. Statistical significance was tested using Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test with Holm-Bonferroni correction (* p < 0.05, *** p < 0.001).

Article Snippet: Electroporation micropipettes (Warner instruments, resistance 10–15 MΩ) were filled with Alexa 488 or Alexa 594 dextran solution (1–5 μl, 3 mM dissolved in bath solution) and mounted on a pipette holder containing a silver wire electrode covered with silver chloride.

Techniques: Staining, Electroporation, Labeling, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Functional Reintegration of Sensory Neurons and Transitional Dendritic Reduction of Mitral/Tufted Cells during Injury-Induced Recovery of the Larval Xenopus Olfactory Circuit

doi: 10.3389/fncel.2017.00380

Figure Lengend Snippet: Dynamic changes of mitral/tufted cell dendritic tuft complexity in the OB after olfactory nerve transection. (A) Top row shows individual MTCs stained via sparse cell electroporation. Maximum intensity projections of image stacks of representative MTCs are shown for each time-point after ON transection. Animals were transected unilaterally and MTCs were stained and analyzed on both the non-transected side of the OB, used as control, and on the transected side, 1, 3 and 7 weeks a.t. Bottom row shows a magnification of the tufted regions (boxed outline). (B) Top row illustrates quantification of complexity of the tufts shown in (A) using Sholl analysis. The number of intersections on the three-dimensional tuft is represented as a color gradient on the tuft morphology—blue areas indicate very few intersections and magenta indicates many intersections. Bottom row shows linear Sholl plots for each of the presented tufts with number of intersections indicated as dots and best fit polynomial function as line. (C) The average linear tuft-complexity curves (of all curves as shown in B ) for tufts of each respective group are shown. A distance of ±10 μm around the maximum is shown. The shaded areas around the curves indicate the SEM within each group. (D) Scatter plot showing the maximum number of intersections for each tuft analyzed in the control group and at each respective time point a.t. Lines show the mean of all analyzed tufts for each time-point. a.t., after transection; MTC, mitral/tufted cell; n.t., non-transected; OB, olfactory bulb; ON, olfactory nerve. Statistical significance was tested using Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test with Holm-Bonferroni correction (* p < 0.05).

Article Snippet: Electroporation micropipettes (Warner instruments, resistance 10–15 MΩ) were filled with Alexa 488 or Alexa 594 dextran solution (1–5 μl, 3 mM dissolved in bath solution) and mounted on a pipette holder containing a silver wire electrode covered with silver chloride.

Techniques: Staining, Electroporation, Control, Comparison