qtl-mapping-crisprcas9-editing-to-identify-drug-resistance-gene

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in <em>Toxoplasma gondii</em>

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

Louisiana State University

In many mammalian species, surface markers have been used to obtain enriched populations of spermatogonial stem cells (SSCs) for assisted reproduction and other applications; however, little is known about the expression patterns of feline SSCs. In this study, we assessed expression of the SSC surface markers commonly used in other species, KIT, ITGA6, CD9, GFRalpha1, ADGRA3, and THY1, in addition to the less frequently used pluripotent markers TRA-1-60, TRA-1-81, SSEA-1, and SSEA-4 in SSCs of both prepubertal and adult domestic cats (Felis catus). To further characterize cat SSCs, we sorted cells using SSC-specific markers and evaluated the expression of the pluripotent transcription factors NANOG, POU5F1, and SOX2 and the proto-oncogene MYC within these populations. We concluded that SSC surface markers used in other mammalian species were not specific for identifying cat SSCs. However, the pluripotent markers we evaluated were more specific to cat spermatogonia, and the presence of SSEA-1 and SSEA-4 in fewer and primarily individual cells suggests that these two markers may be used for enrichment of cat SSCs. The expression of pluripotent transcription factors at mRNA level by single-stained cells positive for SSEA-4 and by dual-stained cells positive for both GFRalpha1 and SSEA-4 reflects the undifferentiated stage of cat SSCs. The absence of transcription factors in double-stained cells positive for only one marker implies the loss of the stem cell-like identity with the loss of either GFRalpha1 or SSEA-4. Further investigation is warranted to elucidate the biological characteristics of these spermatogonial subpopulations.

Phenotypic and Molecular Characterization of Domestic Cat (Felis catus) Spermatogonial Stem Cells.

Recent years have seen significant developments in the ability to continuously propagate organoids derived from intestinal crypts. These advancements have been applied to mouse and human samples providing models for gastrointestinal tissue development and disease. We adapt these methods for the propagation of intestinal organoids (enteroids) from various large farm and small companion (LF/SC) animals, including cat, dog, cow, horse, pig, sheep and chicken. We show that LF/SC enteroids propagate and expand in L-WRN conditioned media containing signaling factors Wnt3a, R-spondin-3, and Noggin (WRN). Multiple successful isolations were achieved for each species, and the growth of LF/SC enteroids was maintained to high passage number. LF/SC enteroids expressed crypt stem cell marker LGR5 and low levels of mesenchymal marker VIM. Labeling with EdU also showed distinct regions of cell proliferation within the enteroids marking crypt-like regions. The ability to grow and maintain LF/SC enteroid cell lines provides additional models for the study of gastrointestinal developmental biology as well as platforms for the study of host-pathogen interactions between intestinal cells and zoonotic enteric pathogens of medical importance.

Robin H. Powell

Pathobiological Sciences,
School of Veterinary Medicine,
Pathobiological Sciences, School of Veterinary Medicine

Louisiana State University

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Robin H. Powell

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Louisiana State University

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Pathobiological Sciences,
School of Veterinary Medicine,
Pathobiological Sciences, School of Veterinary Medicine