Mapping Lineage Using BAC‐Cre Reporter Lines

Qing Xu1, Stewart A. Anderson1

1 Weill Medical College of Cornell University, New York, New York
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.19
DOI:  10.1002/0471142301.ns0119s50
Online Posting Date:  January, 2010
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As the brain develops, progenitor cells acquire the features of specific neuronal or glial subtypes through dynamic expression of the fate‐determining signaling molecules and their targeting transcription factors. An effective and versatile approach for tracing lineage of progenitors into adult cell types is to target the promoter of an interested gene with Cre (a phage DNA recombinase) to achieve simultaneous activation during neurogenesis. The bacterial artificial chromosome (BAC) is an efficient Cre carrier. Not only the targeted gene remains diploidy in BAC‐Cre transgenic mice, but also the large portions of the gene's regulatory elements to be incorporated in the BAC allow Cre to sufficiently and reliably reproduce the endogenous gene expression pattern. When the BAC‐Cre mouse is crossed to a Cre reporter mouse, even Cre is transiently expressed. Cre‐loxP mediated recombination can permanently activate a reporter gene, such as green fluorescent protein (GFP) in all lineage cells of the gene. Experimental designs and procedures for RecA‐based BAC DNA modification and preparation for pronuclear injection are highlighted. Suggestions for the use of BAC‐Cre transgenic mice in fate‐mapping analyses are also provided. Curr. Protoc. Neurosci. 50:1.19.1‐1.19.29. © 2010 by John Wiley & Sons, Inc.

Keywords: bacterial artificial chromosome (BAC); Cre recombinase; homologous recombination; lineage; development

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Table of Contents

  • Introduction
  • Basic Protocol 1: Generating Cre‐PolyA Modified BAC with RecA System
  • Basic Protocol 2: Preparation of Embryonic and Neonatal Tissue for Immunohistochemical Staining
  • Basic Protocol 3: Lineage Identification by Immunodetection with Fluorescence or Bright‐Field Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Generating Cre‐PolyA Modified BAC with RecA System

  • Cre‐polyA‐containing expression vector
  • E. coli strains: BAC clones (DH10B), Pir2 cell, and DH5α or Top10 (Invitrogen)
  • LB medium ( appendix 2A) with and without 17 µg/ml chloramphenicol in ethyl alcohol
  • Chloramphenicol
  • Dimethyl sulfoxide (DMSO)
  • Autoclaved distilled water
  • DNA preparation miniprep kit (Invitrogen, cat. no. K2100‐11)
  • Isopropanol
  • 70% (v/v) ethanol
  • TE buffer ( appendix 2A)
  • 5× PCR buffer
  • Deoxynucleotide solution mix (dNTP, 10 mM each deoxynucleotide; New England Biolabs, cat. no. N0447S)
  • Phusion DNA polymerase (New England Biolabs, cat. no. F‐530S)
  • DNA gel extraction kit (e.g., Qiagen, cat. no. 28704 or Invitrogen, cat. no. K2100‐12)
  • DNA restriction enzymes (including Sal I, BamH I, EcoR I, and Hind III)
  • Building vector (any common cloning vector, e.g., pBluescript)
  • Calf intestinal alkaline phosphatase (CIAP)
  • T4 DNA ligase
  • Shuttle vector, e.g., pLD53 (unit 5.21) or equivalent
  • 10% (v/v) glycerol, sterile and ice‐cold
  • SOC medium (see recipe)
  • Ampicillin
  • LB agar plates with and without antibiotic(s) and 6% sucrose (see recipe)
  • Hispeed Plasmid Midi kit (Qiagen, cat. no. 12643) containing:
    • P3 buffer
    • P1 buffer
    • P2 buffer
    • QBT buffer
    • QC buffer
    • QF buffer
    • Qiagen Hispeed columns
  • Spermidine (Sigma‐Aldrich, cat. no. S0266)
  • Standard DNA size markers (e.g., 1‐kb marker; Promega) or midrange PFGE Marker I (Biolabs, cat. no. N3551S)
  • NEBlot Phototope Kit (New England Biolabs)
  • Bio‐Rad ChEF gel size standards or MidRange PFG MarkerI (Biolabs, N3551S) or lambda‐Hind III marker for PFGE
  • 0.5 µg/ml ethidium bromide ( appendix 2A)
  • 0.5× TBE or 1× TAE buffer (see reciperecipes)
  • Sepharose CL‐4B (Amersham Biosiences)
  • BAC purification buffer (see recipe)
  • 10× DNA loading buffer: 0.1% (w/v) bromphenol blue and 50% glycerol (store at 4°C)
  • Injection buffer (see recipe)
  • 14‐ml test tubes with loosely fitting snap‐caps
  • 37°C shaking incubator
  • 1‐ml, 200‐µl, 20‐µl, and 2‐µl pipets and tips
  • Microcentrifuge tubes
  • Platform rocking shaker (VWR)
  • Autoclave
  • Refrigerator (4°C) and Freezers (−20°C and −80°C)
  • Microcentrifuge
  • Benchtop centrifuge
  • 200‐µl PCR tubes
  • Thermal Cycler (e.g., Veriti Thermal Cycler from Applied Biosystems)
  • Plastic box with sealing lid for Southern blot hybridization
  • 1‐liter flasks
  • 250‐ml centrifuge bottles
  • Chilled electroporation cuvettes with 1‐mm gap
  • Electroporator, e.g., ECM 830 (BTX)
  • Spectrophotometer
  • Parafilm
  • UV light illuminator
  • Aluminum foil
  • 250‐ml and 500‐ml flasks
  • Vortex
  • 50‐ml scaled Falcon tubes
  • 50‐ml tubes
  • 30‐ml centrifuge tubes with caps
  • GeneScreen Plus hybridization transfer membrane (Perkin‐Elmer Life Sciences)
  • Two glass trays for capillary transfer of DNA from agarose gel
  • 65°C incubator with rocking platform for Southern blot hybridization
  • Spectroline autoradiography cassettes (e.g., Krackeler Scientific)
  • Kodak X‐Omat LS film
  • Pulse field‐gel electrophoresis system (PFGE), e.g., Bio‐Rad CHEF‐DR III System with Chiller
  • Sepharose CL‐4B columns
  • Additional reagents and equipment for synthesis and purification of oligonucleotides (Ellington and Pollard, 1998), agarose gel electrophoresis ( appendix 1N), digestion of DNA with restriction endonucleases ( appendix 1M), preparation of bacterial plasmid DNA (Seidman et al., ), miniprep DNA ( appendix 1J), enzymatic amplification of DNA by PCR (Kramer and Coen, ), Southern blot analysis (Brown, ), sequencing the cassette (Ausubel et al., Chapter 7), quantifying DNA using a spectrophotometer ( appendix 1K), performing molecular biology techniques ( appendix 1A), and molecular cloning (Sambrook and Russell, )
NOTE: All materials and reagents coming into contact with bacteria must be sterile.NOTE: DNA primers are dissolved in autoclaved distilled water (e.g., add 1 ml of water to 100 nmole of primer to make a 500× primary stock (0.1 mM) and make a 50× secondary stock of 200 nM working concentration in most cases). Store at −20°C.

Basic Protocol 2: Preparation of Embryonic and Neonatal Tissue for Immunohistochemical Staining

  • BAC‐Cre transgenic mice mated with Cre reporters, such as R26R‐YFP
  • Phosphate‐buffered saline (PBS; see recipe)
  • 30% sucrose in PB (see recipe)
  • OCT Compound (Tissue‐Tek)
  • Sodium pentobarbital
  • 10% (w/v) paraformaldehyde (PFA; see recipe)
  • 25% glutaraldehyde (Sigma, cat. no. G5882)
  • PB buffer (see recipe)
  • Low‐melting‐point agarose
  • Cyanoacrylate glue (Instant Krazy glue)
  • Antifreeze solution (see recipe)
  • 5‐ and 10‐cm petri dishes
  • Microcentrifuge tubes
  • Blades: industrial razor blades for trimming tissue (VWR); low‐profile blades for cryostat (Feather Safety Razor); double‐edged blades for vibratome (Wilkinson Seord)
  • Plastic embedding mold
  • Shallow dish
  • Cryostat (e.g., Leica CM 3050 S)
  • Superfrost slides (VWR)
  • Coverslips (VWR)
  • Slide box
  • Syringes and needles: 1‐ml syringes with 26‐G, 3/ needles for delivering anesthetic, 23‐G to 27‐G, 1/2 in. needles for delivering PBS and fixative to the heart ventricle (convenient with the Terumo Winged Infusion Set)
  • Styrofoam
  • Needles (to pin down the limbs of the mouse)
  • Small thin dissecting scissors
  • Peristaltic pump (1∼10 ml/min)
  • Small thin dissecting forceps
  • Small thin dissecting tweezers
  • Small rongeur
  • Microwave oven
  • 42°C water bath
  • Acrylic mouse brain matrix (e.g., Zivic, cat. no. BSM001.1)
  • Vibratome (e.g., Leica VT 1000S)
  • Fine eyebrow brush
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Basic Protocol 3: Lineage Identification by Immunodetection with Fluorescence or Bright‐Field Microscopy

  • Cryostat sections ( protocol 2)
  • PBT (see recipe)
  • 10% paraformaldehyde (PFA; see recipe)
  • 0.5 M EDTA, pH 8.0
  • Blocking solution (see recipe)
  • Primary antibodies against Cre, GFP (or β‐galactosidase), and cell specific neurochemical markers
  • Secondary antibody conjugated with various fluorophores (Alexa Fluor series from Invitrogen, and Cy5 from Jackson Immuno Research Laboratories)
  • 4′,6‐Diamidino‐2‐phenylindole (DAPI)
  • Mounting medium for fluorescence: Prolong Gold antifade reagent (Invitrogen, cat. no. P36930)
  • Nail varnish
  • β‐galactosidase staining solution (see recipe)
  • Biotin‐conjugated secondary antibody (Vector Laboratories)
  • Vectastain ABC peroxidase kit (Vector Laboratories)
  • DAB (diaminobezidine; Sigma)
  • M Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 30% hydrogen peroxide
  • 0.1% gelatin in PBS (see recipe)
  • 100% (v/v) ethanol
  • Xylene
  • Mounting medium for bright field: DPX (VWR) or Krystalon (EMD)
  • Slide folder
  • Coplin jars
  • 65° and 37°C water baths
  • Pap pen (Liquid‐repellent slide marker pen)
  • Humidity box with slides holders for incubation with antibodies
  • 2‐ml microcentrifuge tubes
  • 14‐ml tubes
  • Platform rocking shaker (VWR)
  • 1000‐µl pipet
  • Vortexer (VWR)
  • 10‐cm petri dishes
  • Superfrost slides
  • Fine spatula or forceps
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