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A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors.

Current gene therapy (2015-07-01)
Carmen Maria Jimenez-Moreno, Irene de Gracia Herrera-Gomez, Livia Lopez-Noriega, Petra Isabel Lorenzo, Nadia Cobo-Vuilleumier, Esther Fuente-Martin, Jose Manuel Mellado-Gil, Geraldine Parnaud, Domenico Bosco, Benoit Raymond Gauthier, Alejandro Martin-Montalvo
ABSTRACT

Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional β-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
HEPES, ≥99.5% (titration)
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Formaldehyde solution, ACS reagent, 37 wt. % in H2O, contains 10-15% Methanol as stabilizer (to prevent polymerization)
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Sodium chloride, BioXtra, ≥99.5% (AT)
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DAPI, for nucleic acid staining
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Formaldehyde solution, for molecular biology, 36.5-38% in H2O
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HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
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Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
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Sodium chloride solution, 0.9% in water, BioXtra, suitable for cell culture
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Donkey serum
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Sodium chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
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Sodium pyruvate, ReagentPlus®, ≥99%
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Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
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HEPES buffer solution, 1 M in H2O
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Sodium chloride solution, 5 M
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Formaldehyde solution, for molecular biology, BioReagent, ≥36.0% in H2O (T)
SAFC
Sodium chloride solution, 5 M
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Sodium pyruvate, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99%
SAFC
HEPES
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Sodium chloride, meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%
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Magnesium sulfate, BioReagent, suitable for cell culture, suitable for insect cell culture
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HEPES, BioUltra, for molecular biology, ≥99.5% (T)
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Sodium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
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Sodium chloride solution, 0.85%
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Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
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Magnesium sulfate solution, for molecular biology, 1.00 M±0.04 M
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Sodium chloride, 99.999% trace metals basis
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Formaldehyde solution, meets analytical specification of USP, ≥34.5 wt. %
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Sodium pyruvate, powder, BioXtra, suitable for mouse embryo cell culture
SAFC
HEPES
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Sodium pyruvate, BioXtra, ≥99%