Identifying Adult Stem Cells Using Cre‐Mediated Lineage Tracing

Diana L. Carlone1

1 Boston Children's Hospital, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5A.2
DOI:  10.1002/9780470151808.sc05a02s36
Online Posting Date:  February, 2016
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Lineage‐tracing has been used for decades to establish cell fate maps during development. Recently, with the advent of genetic lineage‐tracing techniques (employing Cre‐lox recombination), it has been possible to permanently mark progenitor/stem cell populations within somatic tissues. In addition, pulse‐chase studies have shown that only stem cells are capable of producing labeled progeny after an extensive period of chase. This unit focuses on the protocols used to target putative adult stem cells in vivo. Using these techniques, one should be able to functionally confirm or deny the stem cell capacity of a given cell population. © 2016 by John Wiley & Sons, Inc.

Keywords: tamoxifen‐inducible Cre recombination; lineage contribution; reporter activity; whole‐mount analysis

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Table of Contents

  • Introduction
  • Basic Protocol 1: Whole‐Mount Analysis of β‐Galactosidase Activity
  • Alternate Protocol 1: Whole‐Mount Analysis of Alkaline Phosphatase Activity
  • Alternate Protocol 2: Sectional Analysis for β‐Galactosidase or Alkaline Phosphatase Activity
  • Alternate Protocol 3: Analysis Using Fluorescent Reporter Mouse Models
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Whole‐Mount Analysis of β‐Galactosidase Activity

  • Tamoxifen‐inducible Cre :: Rosa26R or Z/AP bigenic mice
  • Tamoxifen or 4‐hydroxytamoxifen (see recipe)
  • Negative control mice (oil‐treated bigenic or treated monogenic mice)
  • Positive control mice (Rosa26; Jackson Laboratories cat. no. 002292)
  • Phosphate‐buffered saline, Ca++‐ and Mg++‐free (CMF‐PBS)
  • LacZ fixative, wash, and staining buffers (see reciperecipes)
  • 32% (w/v) paraformaldehyde solution (EMS, cat. no. 15714‐S)
  • 35% 70%, 80%, 90%, 95%, and 100% ethanol
  • Xylene
  • Paraffin
  • 10 mM sodium citrate, pH 6.0
  • 3% H 2O 2, optional
  • Avidin and biotin blocking solutions (Vector Laboratories, cat. no. SP‐2001)
  • Normal serum (species selection should match that of the secondary antibody; Sigma)
  • Differentiation‐specific antibodies
  • Vectastain ABC Elite kit (species‐specific kits are available dependent upon the primary antibody; Vector Laboratories)
  • DAB substrate kit (Vector Laboratories, cat. no. SK‐4100)
  • Nuclear Fast Red (Sigma, cat. no. N3020)
  • Cytoseal XYL mounting medium (Richard‐Allan Scientific, cat. no. 8312‐4)
  • 1.5‐ml microcentrifuge tubes or 6‐well tissue culture plates
  • Platform shaker
  • 37°C incubator
  • Microtome
  • Microscope slides
  • Coplin jars
  • Pressure cooker, microwave, or water bath
  • Coverslips
  • NOTE: Unless indicated, all steps in this protocol are performed at room temperature.

Alternate Protocol 1: Whole‐Mount Analysis of Alkaline Phosphatase Activity

  Additional Materials (also see protocol 1Basic Protocol)
  • Alkaline phosphatase fixative solution (see recipe)
  • AP buffer (see recipe)
  • BM Purple AP substrate (Roche Diagnostics, cat. no. 11 442 074 001)
  • PTM buffer (see recipe)
  • 70° to 75°C incubator

Alternate Protocol 2: Sectional Analysis for β‐Galactosidase or Alkaline Phosphatase Activity

  Additional Materials (also see protocol 1Basic Protocol)
  • Tissue‐Tek OCT (Sakura, cat. no. 4583)
  • 0.2% glutaraldehyde/2 mM MgCl 2 in CMF‐PBS
  • 0.2% glutaraldehyde in CMF‐PBS
  • AP buffer (see recipe)
  • 5‐bromo‐4‐chloro‐3‐indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) solution (see recipe or Vector Laboratories, cat. no. SK‐5400)
  • Cryomolds
  • Cryostat
  • 70° to 75°C incubator

Alternate Protocol 3: Analysis Using Fluorescent Reporter Mouse Models

  Additional Materials (also see protocol 1Basic Protocol)
  • Tamoxifen‐inducible Cre :: Rosa26 TdTomato or Rosa26 mT/mG bigenic mice
  • 0.6 M sucrose in CMF‐PBS
  • Dry ice
  • 4′6′‐diamidino‐2‐phenylindole (Dapi; Sigma, cat. no. D9542)
  • Prolong Gold Antifade Mountant (Molecular Probes, cat. no. P36930)
  • Cryomolds
  • Cryostat
  • Pap pen (Vector Labs, cat. no. H‐4000)
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Literature Cited

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