Excision of a Viral Reprogramming Cassette by Delivery of Synthetic Cre mRNA

Yuin‐Han Loh1, Jimmy Chen Yang1, Alejandro De Los Angeles1, Chunguang Guo2, Anne Cherry3, Derrick J. Rossi2, In‐Hyun Park4, George Q. Daley4

1 These authors contributed equally to this work and are co‐first authors., 2 Immune Disease Institute, Program in Cellular and Molecular Medicine, Children's Hospital Boston, Harvard Stem Cell Institute, and the Department of Pathology, Harvard Medical School, Boston, Massachusetts, 3 Harvard Stem Cell Institute, Cambridge, Massachusetts, 4 These authors are co‐corresponding authors.
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.5
DOI:  10.1002/9780470151808.sc04a05s21
Online Posting Date:  May, 2012
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The generation of patient‐specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro‐ and lenti‐viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene‐free methods using adenovirus and protein transduction are limited by low efficiency. This unit describes a protocol for the generation of transgene‐free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene‐free human iPS cells. Curr. Protoc. Stem Cell Biol. 21:4A.5.1‐4A.5.16. © 2012 by John Wiley & Sons, Inc.

Keywords: reprogramming; induced pluripotent stem cells; synthetic mRNA; Cre‐excision; transgene‐free iPSCs

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Table of Contents

  • Introduction
  • Basic Protocol 1: Direct Reprogramming of Human Fibroblasts using a Cre‐Excisable Retroviral Reprogramming Monovector
  • Basic Protocol 2: Synthesis of Modified Cre mRNA
  • Basic Protocol 3: Efficient Excision of Reprogramming Factors using mRNA Transfection of Cre Recombinase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Direct Reprogramming of Human Fibroblasts using a Cre‐Excisable Retroviral Reprogramming Monovector

  • 293T cells for generating retrovirus (ATCC #CRL11268) growing in a 100‐mm tissue culture dish
  • 293T medium (see recipe)
  • Fugene 6 (Roche Applied Science, cat no. 1181509001)
  • DMEM
  • DMEM/F12 (Invitrogen, cat. no. 10565‐018)
  • pE4‐OSKM vector (available from author upon request)
  • Gag‐Pol (Addgene, clone 8455)
  • VSV‐G (Addgene, clone 8454)
  • 10% FBS medium
  • Human fibroblasts
  • Protamine sulfate (Sigma Aldrich, cat no. P4380)
  • PBS
  • Human fibroblast medium (see recipe)
  • Irradiated CF‐1 mouse embryonic fibroblast (MEFs) (GlobalStem, cat. no. GSC‐6001G)
  • CF1 MEF feeder medium (see 10% FBS medium recipe)
  • 0.05% trypsin (Invitrogen, cat no. 2530054)
  • hESC growth medium (see recipe)
  • Y‐27632 (Sigma‐Aldrich, cat no. Y0503)
  • Platform rocker
  • 37°C, 5% CO 2 humidified incubator
  • 6‐well plates
  • 10‐ml disposable syringe (BD, cat no. 301030)
  • Low protein–binding, 0.45‐µm pore size cellulose acetate filter (Pall Life Sciences, cat no. PN4184)
  • 1 × 3‐1/2 polyallomer centrifuge tube (Beckman, cat no. 326823)
  • Beckman XL‐90 ultracentrifuge
  • 100‐mm plates coated with 0.1% gelatin (for gelatin, Sigma‐Aldrich, cat no. G1890)
  • Hemacytometer
  • 12‐well plates coated with 0.1% gelatin (for gelatin, Sigma‐Aldrich, cat no. G1890)
  • Stereomicroscope
  • Cell lifter (Corning, cat no. 3008)

Basic Protocol 2: Synthesis of Modified Cre mRNA

  • 5′ ORF oligonucleotide: 5′ TCCAATTTACTGACCGTACACC3′
  • T4 polynucleotide kinase (PNK) with 10× buffer (New England Biolabs, cat. no. M0201)
  • Adenosine‐5′‐triphosphate (ATP) (100 mM, USB; Affymetrix, cat. no. 77241)
  • TE buffer, RNase‐free (10 mM Tris⋅Cl, pH 8/1 mM EDTA)
  • KAPA HiFi Hotstart ReadyMix (Harvard Biopolymers, cat. no. KK2601)
  • 3′ ORF oligonucleotide: 5′CTAATCGCCATCTTCCAGCAGG3′
  • pBABE‐puro‐Cre (kindly provided by Dr. Zhe Li, Harvard Medical School)
  • 1.2% agarose gel
  • 100‐bp DNA ladder (NEB, cat. no. N3231S)
  • QIAquick PCR purification kit (Qiagen) containing:
    • Buffer EB
    • Buffer PE
    • Buffer
    • PB Loading dye
    • QIAquick spin columns
  • Nuclease‐free water
  • 5′ UTR oligonucleotide:
  • 3′ UTR oligonucleotide (phosphorylated):
  • 5′ Splint oligonucleotide:
  • 3′ Splint oligonucleotide:
  • Ampligase enzyme with 10× buffer (Epicentre Biotechnologies, cat. no. A32750)
  • KAPA HiFi Hotstart Readymix (Harvard Biopolymers, cat. no. KK2601)
  • Tail forward primer (generic): 5′TTGGACCCTCGTACAGAAGCTAATACG3′
  • Tail reverse primer (generic; PAGE purified):
  • NTP mix (see recipe)
  • MEGAscript T7 kit (Ambion, cat. no. AM1334M) containing:
    • 10× reaction buffer
    • Enzyme mix
    • ATP solution
    • GTP solution
    • CTP solution
    • UTP solution
  • TURBO DNase (Invitrogen, cat. no. AM‐2238)
  • MEGAclear kit (Ambion, cat. no. AM1908M) containing:
    • Binding solution concentrate
    • Wash solution
    • 5 M ammonium acetate
    • Elution solution
    • Filter cartridges
    • Collection and elution tubes
  • 100% ethanol
  • Antarctic phosphatase (New England Biolabs, cat. no. M0289)
  • 37°C and 65°C heating blocks
  • Thermal cycler
  • 1.7‐ml microcentrifuge tubes, RNase‐free
  • Spectrophotometer (NanoDrop)
  • PCR strip

Basic Protocol 3: Efficient Excision of Reprogramming Factors using mRNA Transfection of Cre Recombinase

  • Matrigel hESC‐qualified matrix (BD Biosciences, cat. no. 354277)
  • DMEM/F12 (Invitrogen, cat. no. 10565‐018)
  • Growing hiPSCs generated with pE4‐OSKM retrovirus (see protocol 1) at 80% to 90% confluency in 60‐mm dish
  • Accutase (Invitrogen, cat. no. A11105‐01)
  • Y‐27632 (Sigma‐Aldrich, cat. no. Y0503)
  • mTeSR1 medium (Stem Cell Technologies, cat. no. 05850)
  • Cre mRNA (see protocol 2)
  • Opti‐MEM reduced serum medium (Invitrogen, cat. no. 31985‐070)
  • RNAiMax (Invitrogen, cat. no. 13778‐100)
  • B18R interferon inhibitor (Invitrogen, cat. no. 34‐8185)
  • CF1 MEF feeder cells
  • MEF medium (10% FBS/DMEM)
  • hESC medium (see recipe)
  • Qiagen DNeasy blood and tissue kit
  • pE4 retroviral vector primers:
    • Forward 5′ CATCGCAGCTTGGATACA 3′
    • Reverse 5′ CCGATGATTAATTGTCAA 3′
  • EcoRI and HindIII enzymes
  • 15‐ml conical tubes, sterile
  • 6‐well plates
  • 37°C incubator
  • Stereo microscope
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Literature Cited

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