Recombineering‐Based Procedure for Creating BAC Transgene Constructs for Animals and Cell Lines

Steven M. Hollenback1, Suzanne Lyman1, JrGang Cheng1

1 Neuroscience Center, UNC‐Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.14
DOI:  10.1002/0471142727.mb2314s95
Online Posting Date:  July, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The use of BAC/P1 as a vector for the generation of a transgene has gained popularity after the genomic annotation of many organisms was completed (often based on the respective BAC library). Large‐scale generation of BAC transgenic mice has proven that BAC transgene approaches have less integration position effects and dosage artifacts when compared with traditional transgenic approaches. Also, a BAC can achieve the same tissue‐specific expression as a knock‐in of the same gene with less effort and shorter time of establishment. The λ‐RED recombinogenic system has been used to manipulate DNA constructs with site‐directed mutagenesis, truncation, and tagging with an epitope tag or as a fusion protein by homologous recombination, as well as used here to modify many BACs with various transgenes. The recombineering plasmid, pKD46, is used to fabricate BAC transgenic constructs that can be used in generating transgenic organisms as well as used in mammalian cell culture. Curr. Protoc. Mol. Biol. 95:23.14.1‐23.14.28. © 2011 by John Wiley & Sons, Inc.

Keywords: BAC; recombineering; transgene

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Fabrication of BAC Transgene Construct Through Recombineering
  • Support Protocol 1: Purify Targeting Cassette with a Safeblue Gel
  • Support Protocol 2: Prepare Electrocompetent Cells
  • Support Protocol 3: Electroporation of a Plasmid into DH10B and its Derivatives
  • Support Protocol 4: Storing and Handling Recombinogenic Bacteria Strains
  • Support Protocol 5: Acquisition and Confirmation of BACs
  • Support Protocol 6: Obtain BAC DNA for Transformation
  • Alternate Protocol 1: Assemble a Targeting Cassette by PCR with Long Primers
  • Alternate Protocol 2: Overlapping PCR
  • Alternate Protocol 3: Arm Extension on the Targeting Cassette
  • Alternate Protocol 4: Sequential Targeting for Transgene Swapping
  • Alternate Protocol 5: Selection Marker Removal with Titration of Recombinase
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Fabrication of BAC Transgene Construct Through Recombineering

  Materials
  • Bacteria containing BAC
  • Low‐salt LB medium (unit 1.1)
  • 20 µg/ml chloramphenicol (34 mg/ml stock in ethanol; Sigma)
  • pKD46 and its derivatives (Dr. B. Wanner)
  • Electrocompetent cells (see protocol 3)
  • SOC medium (NEB)
  • Agar plates (unit 1.1)
  • 100 µg/ml ampicillin (50 mg/ml stock in water; Sigma)
  • Phusion high‐fidelity DNA polymerase (Finnzymes) or similar high‐fidelity enzyme
  • pGEM‐T‐Easy (Promega)
  • E. coli competent cells for cloning: DH10B (Invitrogen), NEB 10‐beta (NEB), Stable 2 or 4 (stb2/4, Invitrogen)
  • Isopropylthio‐β‐D‐galactoside (IPTG; 1 M, 20 µl/plate)
  • 5‐Bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside (X‐gal, 50 mg/ml in dimethylformamide, 50 µl/plate)
  • Restriction endonucleases (NEB)
  • Antibiotics:
    • 25 µg/ml kanamycin (50 mg/ml stock in water; Sigma)
    • 25 µg/ml zeocin (100 mg/ml stock in water; Invitrogen)
    • 50 µg/ml hygromycin (100 mg/ml stock in water; Invivogen)
  • Gel extraction kit (Qiaquick, Qiagen)
  • 0.1% L‐Arabinose (10% stock in water; Sigma)
  • 32°C orbital shaker incubators
  • Spectrophotometer with 1‐ml disposable plastic cuvettes
  • 1.5‐ and 2‐ml microcentrifuge tubes
  • Electroporator (Eppendorf Electroporator 2510) with 1‐mm electroporation cuvettes
  • 2‐ml centrifuge tubes
  • Bench‐top centrifuge with temperature control (Eppendorf 5810R)
  • 50‐ml culture tubes
  • Spectrophotometer (Nanodrop ND1000) with 1‐ml disposable plastic cuvettes (ThermoSpectronic‐Biomate 3)
  • 96‐well assay plates
  • 14‐ml centrifuge tubes
  • Additional reagents and equipment for PCR (unit 15.1), agarose gel electrophoresis (unit 2.5), DNA preparation and restriction endonuclease digestion (unit 3.1), and pulsed‐field gel electrophoresis (unit 2.5)

Support Protocol 1: Purify Targeting Cassette with a Safeblue Gel

  Materials
  • Safeblue dye (SYBR safe, Invitrogen)
  • DNA sample
  • Gel purification kit (Qiagen)
  • Safe Imager blue‐light transilluminator (Invitrogen)
  • Scalpel or razor blade
  • NanoDrop
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)

Support Protocol 2: Prepare Electrocompetent Cells

  Materials
  • High‐density culture (culture grown to saturation) of appropriate cells, e.g., DH10B (Invitrogen) or Stable 2 or 4 (stb2/4, Invitrogen)
  • 10% glycerol
  • Spectrophotometer
  • 2‐ml microcentrifuge tubes
  • 50‐ml centrifuge tubes
  • Refrigerated centrifuge

Support Protocol 3: Electroporation of a Plasmid into DH10B and its Derivatives

  Materials
  • Resuspended electrocompetent cells (DH10B or its derived cells, DY380, EL250, EL350; see protocol 3)
  • Freshly prepared DNA: BAC (1/10 miniprep), plasmid (50 ng) or targeting cassette (100∼200 ng)
  • SOC medium (NEB)
  • LB agar plate containing appropriate antibiotics for selection
  • Electroporator (Eppendorf electroporator 2510) with 1‐mm electroporation cuvettes
  • 1.5‐ml microcentrifuge tubes
  • 32°C (37°C for non‐recombinogenic bacteria) orbital shaking incubator

Support Protocol 4: Storing and Handling Recombinogenic Bacteria Strains

  Materials
  • Recombinogenic bacteria
  • LB medium
  • Glycerol (or DMSO) to make final 25% (or 7%)
  • 5‐ml transparent centrifuge tubes
  • 32° and 42°C incubator

Support Protocol 5: Acquisition and Confirmation of BACs

  Materials
  • LB medium with 20 µg/ml chloramphenicol
  • BAC‐containing cells in glycerol stock or grown on agar
  • 50 mM Tris⋅Cl (pH 8.0)/10 mM EDTA/100 µg/ml RNase A
  • Miniprep kit (Qiagen), optional
  • Isopropanol
  • 70% ethanol
  • 10 mM Tris⋅Cl buffer, pH 8.5 ( appendix 22)
  • 14‐ml conical tubes
  • 32°C orbital shaking incubator
  • 2‐ml centrifuge tubes
  • Tabletop centrifuge
  • 1.5‐ml microcentrifuge
  • 1‐ml pipets

Support Protocol 6: Obtain BAC DNA for Transformation

  Materials
  • Plasmid DNA
  • DpnI enzyme
  • HFPCR‐amplified transgene with selection marker
  • Phusion DNA polymerase (Finnzymes) or similar high‐fidelity enzyme
  • PCR primers
  • PCR nucleotide mix
  • Thermal cycler

Alternate Protocol 1: Assemble a Targeting Cassette by PCR with Long Primers

  Materials
  • Targeting cassette with the 50‐mer homologous arms (see protocol 8) generated using two sets of primers: transgene primers set or 50 mer+transgene primer set
  • Primer L/R (can be same as overlapping PCR primer pairs or ID primers)
  • Taq polymerase (NEB)
  • pGEM‐T‐Easy (Promega)
  • Agar plate with 100 µg/ml ampicillin as well as X‐Gal and IPTG
  • LB medium
  • Miniprep kit (Qiagen)
  • pKD46 and its derivatives
  • Recombinogenic bacteria (DY380/SW102, EL250/SW105. EL350/SW106) (Dr. N. Copeland)
  • Thermal cycler
  • Electroporator and cuvettes

Alternate Protocol 2: Overlapping PCR

  Materials
  • Targeting cassette with 50‐mer homologous arms (see protocol 8) that is generated by two sets of primers: transgene primer set and 50 mer+transgene primer set or targeted BAC with shared components (such as eGFP‐pA BAC)
  • Larger transgene with shared components (second TC), preferably with a different selection marker then the first targeting cassette
  • Recombinogenic plasmid: pKD46
  • Genotyping primer‐sets that can distinguish first targeting and second targeting product

Alternate Protocol 3: Arm Extension on the Targeting Cassette

  Materials
  • BAC‐tg
  • EL250 or EL350 cells
  • Agar plates with proper selection
  • LB medium
  • Arabinose
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Copeland, N.G., Jenkins, N.A., and Court, D.L. 2001. Recombineering: A powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2:769‐779.
   Datsenko, K.A. and Wanner, B.L. 2000. One‐step inactivation of chromosomal genes in Escherichia coli K‐12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97:6640‐6645.
   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.
   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.
   Poser, I., Sarov, M., Hutchins, J.R., Hériché, J.K., Toyoda, Y., Pozniakovsky, A., Weigl, D., Nitzsche, A., Hegemann, B., Bird, A.W., Pelletier, L., Kittler, R., Hua, S., Naumann, R., Augsburg, M., Sykora, M.M., Hofemeister, H., Zhang, Y., Nasmyth, K., White, K.P., Dietzel, S., Mechtler, K., Durbin, R., Stewart, A.F., Peters, J.M., Buchholz, F., Hyman, A.A. and 2008. BAC TransgeneOmics: A high‐throughput method for exploration of protein function in mammals. Nat. Methods 5:409‐415.
   Wagner, M. and Koszinowski, U.H. 2004. Mutagenesis of viral BACs with linear PCR fragments (ET recombination). Methods Mol. Biol. 256:257‐268.
   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
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library