Reprogramming of Pancreatic Acinar Cells to Functional Beta Cells by In Vivo Transduction of a Polycistronic Construct Containing Pdx1, Ngn3, MafA in Mice

C. Cavelti‐Weder1, A. Zumsteg2, W. Li3, Q. Zhou4

1 University Hospital of Basel, Department of Endocrinology, Diabetes, and Metabolism, Basel, 2 Covagen AG, Schlieren, 3 Translational Medical Center for Stem Cell Therapy and Institute for Regenerative Medicine, Shanghai East Hospital, Tongji University School of Life Sciences and Technology, Shanghai, 4 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.10
DOI:  10.1002/cpsc.21
Online Posting Date:  February, 2017
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To generate new beta cells after birth is a key focus of regenerative medicine, which could greatly aid the major health burden of diabetes. Beta‐cell regeneration has been described using four different approaches: (1) the development of beta cells from putative precursor cells of the adult pancreas, which is termed neogenesis, (2) replication of existing beta cells, (3) differentiation from embryonic or induced pluripotent stem cells, and (4) reprogramming of non‐beta cells to beta cells. Studies from the authors’ laboratory have shown that beta‐cell reprogramming can be achieved by transduction of adult pancreatic tissues with viral constructs containing the three developmentally important transcription factors Pdx1, Ngn3, and MafA. This protocol outlines the generation of a polycistronic construct containing the three transcription factors, the expansion and purification of the polycistronic virus, and in vivo transduction for acinar to beta‐cell reprogramming in adult mice. The ultimate goal is to generate beta‐like cells that resemble as closely as possible endogenous beta cells in phenotype and function for potential translational applications. © 2017 by John Wiley & Sons, Inc.

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Table of Contents

  • Significance Statement
  • Introduction
  • Basic Protocol 1: Making Polycistronic Constructs
  • Basic Protocol 2: Viral Production and Purification
  • Basic Protocol 3: Surgical Procedure
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Making Polycistronic Constructs

  • Gateway pENTR2B vector (Invitrogen)
  • Restriction enzymes and corresponding buffers
  • pAd/CMV/V5‐DEST Gateway Vector Kit (Invitrogen)
  • QIAEX II Gel Extraction Kit
  • KAPA High‐Fidelity DNA polymerase
  • T4 DNA ligase
  • Mouse cDNA
  • Agarose gel electrophoresis station
  • Heating block or incubator
  • Access to DNA sequencing service
Follow basic molecular biology protocols. As a template for the murine transcription factors, cDNA from reverse‐transcribed newborn mouse pancreas RNA is a good source. Primer sequences are available upon request.

Basic Protocol 2: Viral Production and Purification

  • M3C fragment (see protocol 1)
  • ViraPower Adenoviral Expression System (Invitrogen), containing pAd/CMV/V5‐DEST adenoviral vector and an optimized 293A cell line
  • Pac I enzyme and buffer
  • Phenol/chloroform or QIAEX II Gel Extraction Kit (Qiagen)
  • Sterile water
  • 293A cell line complete growth medium (see recipe)
  • Opti‐MEM I medium (Gibco) with and without serum
  • Lipofectamine 2000 (Invitrogen)
  • Trypsin
  • Dry ice/ethanol bath
  • Vivapure Adenopack 100 (Sartorius)
  • Storage buffer (see recipe)
  • 6‐ and 24‐well cell culture plates
  • Fluorescence microscope
  • 10‐ and 15‐cm tissue culture plates
  • 10‐ml tissue culture pipettes
  • 15‐ml capped tubes
  • 37°C water bath
  • Tabletop centrifuge

Basic Protocol 3: Surgical Procedure

  • Alcohol preps (Kendall)
  • Rag1−/− mice (B6.129S7‐Rag1<tm1Mom>/J)
  • Anesthesia (see recipe)
  • Betadine solution (Santa Cruz)
  • Adenovirus (see protocol 2)
  • Storage buffer (see recipe)
  • Banamine (Merck)
  • Animal balance
  • Shaver
  • 18‐ and 27‐G needles (BD)
  • 1‐ and 3/10‐ml syringes
  • Surgical gloves (sterile) and facemasks
  • Sterile surgical tools (stapler, staples, small scissors, and forceps)
  • Warming pads and Delta Phase operating board (Braintree Scientific)
  • Dissecting microscope (Leica stereo zoom 7)
  • Blue sterile tissues (IMCO)
  • Suture (5‐0 Chromic gut) (Butler Schein)
  • Sterile drape (IMCO)
  • Bead sterilizer (Fine Science Tool)
  • Heating lamp
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Literature Cited

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