<i>COWP8</i> is not essential for infection or transmission.
<p>A) Strategy to replace the full open reading frame of <i><i>cowp8</i></i> (cgd6_200) with a repair cassette where mScarlet-I and NanoLuciferase-Neomycin resistance fusion protein (NLuc-Neo<sup>R</sup>) is expressed by the constitutive <i><i>En...
محفوظ في:
| المؤلف الرئيسي: | |
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| مؤلفون آخرون: | , , , , , , , , , , , |
| منشور في: |
2025
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| الموضوعات: | |
| الوسوم: |
إضافة وسم
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| الملخص: | <p>A) Strategy to replace the full open reading frame of <i><i>cowp8</i></i> (cgd6_200) with a repair cassette where mScarlet-I and NanoLuciferase-Neomycin resistance fusion protein (NLuc-Neo<sup>R</sup>) is expressed by the constitutive <i><i>Enolase</i></i> promoter. The same gRNA (black arrow) that was used to target the C-terminus for fluorescent fusion and the same downstream region of 50 bp of homology (grey) was used to target the gene for deletion (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1013561#ppat.1013561.s004" target="_blank">S4A Fig</a>). B) PCR with primer pairs indicated in (A) was performed using genomic DNA extracted from wild type and <i>∆cowp8.</i> C) Whole genome sequencing of wild type (yellow) and <i>∆cowp8</i> (blue) confirms gene deletion. Histogram illustrates sequencing coverage mapped at the <i><i>cowp8</i></i> locus. Confocal microscopy of live wild type (D and E) and <i>∆cowp8</i> (F and G) confirms mScarlet-I cytoplasmic expression of <i>∆cowp8</i> strain (magenta); scale bar 5 µm. H) Super resolution microscopy of tissue collected from interferon-gamma knockout mouse infected with <i>∆cowp8</i> culled at peak infection and processed for histology. Brush border (F-actin stained with phalloidin, white) of ileal villi (DNA stained with Hoechst, cyan) are highly infected with <i>∆cowp8</i> (mScarlet-I expressed in the parasite cytoplasm, purple). Images collected on a Zeiss LSM880 Airyscan microscope, (Airyscan mode for P). Representative images shown. J-L) Experimental design for passage of <i>∆cowp8</i> strain in interferon-gamma knockout mice. J) Initial passage where inoculum is wild type sporozoites transfected with CRISPR machinery to target <i>co<i>wp8</i></i> for deletion, K) second passage infected with slurry made of faecal material from first passage, L) and third passage infected with oocysts purified from the second passage. Infection level of COWP8-mNeon (white circles) and <i>∆cowp8</i> strains (black circles) is similar and follows typical acute infection pattern in M) first, N) second, and P) third passages. Infection level of mice as determined by faecal NLuc, limit of detection at 500 RLU/mg, dotted line. Average and SD of three technical replicates of one biological replicate.</p> |
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