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Mapping Lineage Using BAC‐Cre Reporter Lines

Qing Xu1,  Stewart A. Anderson1

1Weill 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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Abstract

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
  • Topics
    • Neuroscience
    • Molecular Biology
     
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Materials

Basic Protocol 1: Generating Cre-PolyA Modified BAC with RecA System

 Materials
  • Cre-polyA-containing expression vector
  • E. coli strains: BAC clones (DH10B), Pir2 cell, and DH5 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 recipes)
  • 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., 1997), miniprep DNA (appendix 1J), enzymatic amplification of DNA by PCR (Kramer and Coen, 2001), Southern blot analysis (Brown, 1999), 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, 2001)

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

 Materials
  • 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, 3/8.in. 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

 Materials
  • Cryostat sections (Basic 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 -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
  • -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
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 1.19.1
    Schema for the RecA-mediated BAC DNA modification. This BAC modification method was initially created in the N. Heintz laboratory using the pLD53 series shuttle vector (Yang et al., 1997; Gong et al., 2002). The two-step modification strategy leads to a clean insertion of the Cre-polyA in the transcription initiation site, while the target gene is pushed behind the Cre-polyA with its ATG start codon removed, so it is unlikely to be transcribed or translated. The modification can be illustrated: (A) The A and B arms of the BAC modification cassette are obtained by PCR, and ligated into the building vector that contains the Cre-polyA. (B) The cassette is then inserted into the shuttle vector. The RecA recombinase transiently expressed from the pLD53-basic shuttle vector in the BAC host E. coli DH10B cells will mediate a homological recombination between the BAC and the shuttle vector within either of the two recombination arms to generate the co-integrated BAC (C). The pLD53-basic shuttle vector will not persist alone, as R6K origin limits its replication in DH10B cells. Inclusion of chloramphenicol and ampicillin in the LB medium ensures that only cells carrying the co-integrated BAC will survive. The RecA recombinase can mediate a second round of recombination within either of the two recombination arms, resulting in excision of the shuttle vector from the BAC. The Cre-polyA will remain in the BAC when the two rounds of recombination occur in different arms (D). When co-integrated BACs cells are inoculated into LB medium containing sucrose and chloramphenicol, the cells that do not undergo this second round of recombination and therefore still carry the shuttle vector in the BAC will be eliminated as a result of their expression of the sacB gene in the shuttle vector. SacB encodes levansucrase, which converts sucrose into toxic levan.

    View Image
  • Figure 1.19.2
    Example of immunohistochemical staining for characterization of the cortical Nkx2.1 lineage with BAC-Nkx2.1-Cre;Z/EG mice. The Nkx2.1 lineage covers most of the cortical interneuron subgroups that express parvalbumin (PV) and somatostatin (Fogarty et al., 2007; Xu et al., 2008). This example shows that at P100, GFP+ cells partially represent the Nkx2.1 lineage (A; boxed regions are shown in B and C at higher magnification). They are not double stained with Tbr1, the cortical projection neuron marker (B), and calretinin (Calr, C) in the bipolar cortical interneurons whose origin is independent of Nkx2.1 (Xu et al., 2004). In contrast, more than half of the GFP+ cells express PV in the cortex and hippocampus (E and F, respectively). E and F are higher magnification views of the regions boxed in D. DAPI stains all the nuclei, which can delineate the laminar structures. An ectopic GFP-expressing example of BAC-Nkx2.1-Cre;Z/EG mouse, F2 generation, at P50 in G shows the GFP+ cells are all in pyramidal morphology. None of them expresses PV in cortex and hippocampus as shown in H and I, respectively, from the boxed regions of G at higher magnification. Scale bars = 100 µm in A; = 50 µm in B and applies to C, E, F, H, and I; = 250 µm in D and applies to G. For color version of this figure go to http://www.currentprotocols.com/protocol/ns0119.

    View Image

Literature Cited

Literature Cited
    Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A.,and Struhl, K. (eds.) 2009. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
    Branda, C.S. and Dymecki, S.M. 2004. Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6:7-28.
    Brown, T. 1999. Southern blotting. Curr. Protoc. Mol. Biol. 68:2.9.1-2.9.20.
    Ellington, A. and Pollard, J.D. Synthesis and purification of oligonucleotides. Curr. Protoc. Mol. Biol. 42:2.11.1-2.11.25.
    Feil, R., Wagner, J., Metzger, D., and Chambon, P. 1997. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237:752-757.
    Fogarty, M., Grist, M., Gelman, D., Marin, O., Pachnis, V., and Kessaris, N. 2007. Spatial genetic patterning of the embryonic neuroepithelium generates GABAergic interneuron diversity in the adult cortex. J. Neurosci. 27:10935-10946.
    García-Otín, A. and Guillou, F. 2006. Mammalian genome targeting using site-specific recombinases. Front. Biosci. 11:1108-1136.
    Gong, S. and Yang, X.W. 2005. Modification of bacterial artificial chromosomes (BACs) and preparation of intact BAC DNA for generation of transgenic mice. Curr. Protoc. Neurosci. 31:5 21.1-5.21.13.
    Gong, S., Yang, X.W., Li, C., and Heintz, N. 2002. Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication. Genome Res. 12:1992-1998.
    Gong, S., Zheng, C., Doughty, M.L., Losos, K., Didkovsky, N., Schambra, U.B., Nowak, N.J., Joyner, A., Leblanc, G., Hatten, M.E., and Heintz, N. 2003. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917-925.
    Gong, S., Doughty, M., Harbaugh, C.R., Cummins, A., Hatten, M.E., Heintz, N., and Gerfen, C.R. 2007. Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs. J. Neurosci. 27:9817-9823.
    Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1-15.1.14.
    Lee, E.C., Yu, D., Martinez de Velasco, J., Tessarollo, L., Swing, D.A., Court, D.L., Jenkins, N.A., and Copeland, N.G. 2001. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56-65.
    Mao, X., Fujiwara, Y., Chapdelaine, A., Yang, H., and Orkin, S.H. 2001. Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain. Blood 97:324-326.
    Matsuda, T. and Cepko, C.L. 2007. Controlled expression of transgenes introduced by in vivo electroporation. Proc. Natl. Acad. Sci. U.S.A. 104:1027-1032.
    Miyoshi, G., Butt, S.J., Takebayashi, H., and Fishell, G. 2007. Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors. J. Neurosci. 27:7786-7798.
    Miyoshi, G. and Fishell, G. 2006. Directing neuron-specific transgene expression in the mouse CNS. Curr. Opin. Neurobiol. 16:577-584.
    Muyrers, J.P., Zhang, Y., Testa, G., and Stewart, A.F. 1999. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27:1555-1557.
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    Novak, A., Guo, C., Yang, W., Nagy, A., and Lobe, C.G. 2000. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28:147-155.
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    Yang, X.W., Model, P., and Heintz, N. 1997. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotechnol. 15:859-865.
    Zhang, Y., Buchholz, F., Muyrers, J.P., and Stewart, A.F. 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20:123-128.
 Internet Resources

The Web pages may have been updated time after time. The routes here may not be exactly as shown on the pages when you open them; nevertheless, the new pages should be designed for easier use.

 Finding BAC clones:
    http://www.ncbi.nlm.nih.gov/mapview

The NCBI MapViewer search by gene's name open the Maps & Options use ADD and REMOVE to display the Clone Apply choose and click to show the BAC.

    http://genome.ucsc.edu/cgi-bin/hgGateway

The UCSC Genome Browser search by gene's name click the result open the Mapping and Sequencing Tracks in BAC End Pairs choose full choose and click to show the BAC.

    http://www.ensembl.org/Mus_musculus/index.html

The Ensembl search by gene's name open the ContigView open the DAS sources choose BAC map choose and click to show the BAC.

 Order BAC clones:
    http://bacpac.chori.org/

Children's Hospital Oakland Research Institute

    http://www.atcc.org/

American Type Culture Collection (ATCC)

 Find or order BAC-Cre mice:
    http://www.gensat.org/index.html

Gensat

    http://www.mmrrc.org/index.html

The Mutant Mouse Regional Resource Centers (MMRRC

    http://jaxmice.jax.org/

The Jackson Laboratory

     
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