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INTACT method for gene expression and chromatin profiling of individual cell types within a tissue

ABSTRACT: We used in vivo biotin labeling of a nuclear envelope protein in individual cell types followed by affinity isolation of labeled nuclei to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. Keywords: Chromatin affinity-purification on microarray

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE19654 | GEO | 2010/06/14

SECONDARY ACCESSION(S): PRJNA122553

REPOSITORIES: GEO

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Project description:Abstract: Controlled release of immunomodulatory cytokines like interleukin-4 (IL-4) from biomaterials can modulate host immune cell behavior to improve tissue integration, however options for modifying complex biomaterials remain limited. Bioconjugation strategies using biotin-avidin interactions offer a promising approach since biotin can be conjugated to proteins, biomaterials, and even cells, without compromising their function. Although it is known that conjugation of biotin to large biomolecules reduces its binding affinity for avidin and avidin variants, the potential to control release of biotinylated molecules via leveraging affinity interactions has not been thoroughly investigated. Moreover, the effects of biotin and avidin variants on innate immunity are poorly understood. Therefore, the goal of this study was to determine if biotin-avidin interactions could be manipulated to control the release of IL-4 from biomaterials and to investigate the subsequent effects on primary human macrophage phenotype. First, we characterized the effects of soluble biotin, avidin, and avidin variants, streptavidin and CaptAvidin, on the phenotype of primary human macrophages using RNA sequencing and found that CaptAvidin significantly influenced macrophage gene expression while other variants did not. Then, after evaluating how bioconjugation parameters influenced biotin density and avidin variant binding to porous gelatin scaffolds, we found that biotin-avidin affinity interactions influenced the spontaneous release of biotinylated IL-4 (bIL-4) from biotinylated and desthiobiotinylated scaffolds bound with either avidin or streptavidin for up to ten days in vitro. Finally, we measured the response of primary human macrophages (8 donors) cultured on modified scaffolds, finding an increase in IL-4 driven genes when bIL-4 was released via biotin-streptavidin affinity interactions compared to desorption-based release. These results highlight how biotin-avidin interactions can be leveraged for spontaneous release to achieve an immunomodulatory drug delivery system.

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INTACT method for gene expression and chromatin profiling of individual cell types within a tissue

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