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BMC Biotechnol. 2014 Oct 11;14:86. doi: 10.1186/1472-6750-14-86.

Simultaneous silencing of multiple RB and p53 pathway members induces cell cycle reentry in intact human pancreatic islets.

Author information

1
Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Craniofacial and Mesenchymal Biology Program, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, CA 94143, USA. jason.pomerantz@ucsfmedctr.org.

Abstract

BACKGROUND:

Human pancreatic islet structure poses challenges to investigations that require specific modulation of gene expression. Yet dissociation of islets into individual cells destroys cellular interactions important to islet physiology. Approaches that improve transient targeting of gene expression in intact human islets are needed in order to effectively perturb intracellular pathways to achieve biological effects in the most relevant tissue contexts.

RESULTS:

Electroporation of intact human cadaveric islets resulted in robust and specific suppression of gene expression. Two genes were simultaneously suppressed by 80% from baseline levels. When multiple (up to 5) genes were simultaneously targeted, effective suppression of 3 of 5 genes occurred. Enzymatic pretreatment of islets was not required. Simultaneous targeting of RB and p53 pathway members resulted in cell cycle reentry as measured by EDU incorporation in 10% of islet nuclei.

CONCLUSIONS:

At least three genes can be effectively suppressed simultaneously in cultured intact human pancreatic islets without disruption of islet architecture or overt alterations in function. This enabled the effective modulation of two central growth control pathways resulting in the phenotypic outcome of cell cycle reentry in postmitotic islet cells. Transient exposure to multiple siRNAs is an effective approach to modify islets for study with the potential to aid clinical applications.

PMID:
25305068
PMCID:
PMC4287515
DOI:
10.1186/1472-6750-14-86
[Indexed for MEDLINE]
Free PMC Article

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