RESUMO
Extinction rates are rising, and current conservation technologies may not be adequate for reducing species losses. Future conservation efforts may be aided by the generation of induced pluripotent stem cells (iPSCs) from highly endangered species. Generation of a set of iPSCs from multiple members of a species can capture some of the dwindling genetic diversity of a disappearing species. We generated iPSCs from fibroblasts cryopreserved in the Frozen Zoo®: nine genetically diverse individuals of the functionally extinct northern white rhinoceros (Ceratotherium simum cottoni) and two from the closely related southern white rhinoceros (Ceratotherium simum simum). We used a nonintegrating Sendai virus reprogramming method and developed analyses to confirm the cells' pluripotency and differentiation potential. This work is the first step of a long-term interdisciplinary plan to apply assisted reproduction techniques to the conservation of this highly endangered species. Advances in iPSC differentiation may enable generation of gametes in vitro from deceased and nonreproductive individuals that could be used to repopulate the species.
Assuntos
Bancos de Espécimes Biológicos , Espécies em Perigo de Extinção , Extinção Biológica , Variação Genética , Células-Tronco Pluripotentes Induzidas/citologia , Perissodáctilos/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Criopreservação/métodos , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariotipagem , Proteína Homeobox Nanog/genética , Perissodáctilos/classificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Especificidade da EspécieRESUMO
Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.