Molecular Biology > Cloning and Recombination Methods

User Ratings

Your rating: None (1 vote)
Your rating: None (1 vote)
Your rating: None (1 vote)
 Add your comments

Human Artificial Chromosome Assembly by Transposon‐Based Retrofitting of Genomic BACs with Synthetic Alpha‐Satellite Arrays

Joydeep Basu1,  Huntington F. Willard1,  Gregory Stromberg2

1Duke Institute for Genome Sciences and Policy, Durham, North Carolina
2Athersys, Inc., Cleveland, Ohio


Publication Name: 
Current Protocols in Human Genetics
Unit Number: 
Unit 5.18
DOI: 
10.1002/0471142905.hg0518s52
Online Posting Date: 
January, 2007
GO TO THE FULL TEXT:
PDF or HTML at Wiley Online Library
Are you the author of this protocol? Login or register and return to this page.

Abstract

The development of methodologies for the rapid assembly of synthetic alpha-satellite arrays recapitulating the higher-order periodic organization of native human centromeres permits the systematic investigation of the significance of primary sequence and sequence organization in centromere function. Synthetic arrays with defined mutations affecting sequence and/or organization may be evaluated in a de novo human artificial chromosome assay. This unit describes strategies for the assembly of custom built alpha-satellite arrays containing any desired mutation as well as strategies for the construction and manipulation of alpha satellite–based transposons. Transposons permit the rapid and reliable retrofitting of any genomic bacterial artificial chromosome (BAC) with synthetic alpha-satellite arrays and other functional components, thereby facilitating conversion into BAC-based human artificial chromosome vectors. These techniques permit identification and optimization of the critical parameters underlying the unique ability of alpha-satellite DNA to facilitate de novo centromere assembly, and they will establish the foundation for the next generation of human artificial chromosome vectors.

Keywords: Transposon; alpha satellite; centromere; BAC; human artificial chromosome

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

Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Construction of a Transposon Vector Containing Higher-Order Alpha-Satellite Array
  • Support Protocol 1: Design and Assembly of a Synthetic, Higher-Order Alpha-Satellite Repeat Unit
  • Support Protocol 2: Assembly of a Higher-Order Alpha-Satellite Array
  • Basic Protocol 2: Creation of BAC-Based Human Artificial Chromosome Vectors by Transposon-Mediated Modification of a Genomic BAC Target with a Higher-Order Alpha-Satellite Array
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Construction of a Transposon Vector Containing Higher-Order Alpha-Satellite Array

 Materials
  • 50 mM MgSO4
  • Plasmid EGFP-c1 or similar vector containing dual neomycin/kanamycin resistance cassette (neo/kan; Clontech)
  • Primers containing the appropriate restriction enzyme overhangs (custom designed and synthesized) to permit directional cloning of the neo/kan cassette into the pMOD transposon vector
  • 5 U/µl High Fidelity Platinum Taq DNA polymerase and High Fidelity polymerase buffer (Invitrogen)
  • 10 MM dNTPs plasmid pMod transposon construction vector (Epicentre)
  • Primers containing the NotI restriction site and 4 to 6 base pairs 5¢ to the site to allow cloning into the
  • BAC-vector: pBeloBAC (NEB)
  • Restriction enzymes: BamHI and BglII (NEB)
  • Phosphatase: calf intestinal phosphatase (CIP) or shrimp alkaline phosphatase (SAP); NEB
  • 0.7% agarose gel (unit 2.7)
  • Alpha-satellite array (Support Protocol 2)
  • Qiagen large-construct DNA preparation kit (or equivalent)
  • Buffer 2 (NEB)
  • Low-melting point agarose (SeaKem)
  • GELase and GELase buffer (Epicentre)
  • T4 DNA ligase and 10× T4 ligase buffer (NEB)
  • Electrocompetent DH10B E. coli (Epicentre)
  • LB agar plates (appendix 2D) supplemented with 12.5 µg/ml chloramphenicol
  • LB medium (appendix 2D) supplemented with 12.5 µg/ml chloramphenicol
  • Buffer set P1, P2, P3 (Qiagen)
  • Isopropanol
  • 70% (v/v) ethanol
  • Thermal cycler
  • Pulsed-field gel electrophoresis (PFGE) apparatus (CHEF-mapper, Bio-Rad; or comparable)
  • UV light source
  • 70°C and 45°C water baths
  • Electroporator (Gene-pulser II, Bio-Rad; or equivalent)
  • Additional equipment and reagents for DNA cloning (unit 3.16) restriction endonuclease digestion of DNA (cpmb unit 3.1), agarose gel electrophoresis (unit 2.7), pulsed-field electrophoresis (unit 5.1), and transformation by electroporation (cpmb unit 1.8)

Support Protocol 1: Design and Assembly of a Synthetic, Higher-Order Alpha-Satellite Repeat Unit

 Materials
  • Custom-designed (see below), PAGE- or HPLC-purified oligonucleotides (e.g., IDT or Operon Technologies)
  • 25 mM ATP (Epicentre)
  • 10× kinase buffer (NEB)
  • 10 U/µl polynucleotide kinase (NEB)
  • NEB buffer 3, optional
  • 400 U/µl T4 DNA ligase (NEB) and 10× T4 ligation buffer (NEB)
  • 2% agarose gel (see unit 2.7)
  • Qiaex resin or Qiaquick gel purification system spin columns (Qiagen)
  • 5 U/µl High Fidelity Platinum Taq DNA polymerase (Invitrogen)
  • Type IIS restriction enzymes (e.g., SapI and/or BsaI; NEB)
  • pGEMTeasy T/A subcloning system (Promega)
  • BAC vector: pBeloBAC (NEB; contains a chloramphenicol resistance gene for selection)
  • Thermal cycler
  • Additional equipment and reagents for agarose gel electrophoresis (unit 2.7) and restriction endonuclease digestion of DNA (cpmb unit 3.1)

Support Protocol 2: Assembly of a Higher-Order Alpha-Satellite Array

 Materials
  • HOR/pBeloBAC subclone DNA (Support Protocol 1)
  • Qiagen large-construct DNA preparation kit (or equivalent)
  • Restriction enzymes: BglII, BamHI, SpeI, and NotI (NEB)
  • Qiaex resin or Qiaquick gel purification system spin columns (Qiagen)
  • 400 U/µl T4 DNA ligase and 10× T4 ligation buffer (NEB)
  • Electrocompetent DH10B E. coli cells (Epicentre)
  • SOC medium (see recipe)
  • LB agar plates (see appendix 2D) supplemented with 12.5 µg/ml chloramphenicol
  • Pulsed-field (CHEF-mapper, Bio-Rad; or comparable) or field-inversion (Bio-Rad) gel electrophoresis apparatus
  • Electroporator (Gene-Pulser II, Bio-Rad; or equivalent)
  • Additional equipment and reagents for restriction endocuclease digestion of DNA (cpmb unit 3.1), agarose gel electrophoresis (unit 2.7), transformation by electroporation (cpmb unit 1.8), and pulsed-field gel electrophoresis (unit 5.1)

Basic Protocol 2: Creation of BAC-Based Human Artificial Chromosome Vectors by Transposon-Mediated Modification of a Genomic BAC Target with a Higher-Order Alpha-Satellite Array

 Materials
  • Transposon donor vector (from Basic Protocol 1)
  • Restriction enzymes: PshAI and NotI (NEB)
  • 2% low-melting-point agarose (SeaKem) gel, preparative scale
  • GELase and GELase buffer (Epicentre)
  • EZ-Tn5 transposase enzyme and buffer (Epicentre)
  • Target genomic BAC (e.g., see Invitrogen; Basu et al., 2005b)
  • Mineral oil (Sigma; optional)
  • Electrocompetent DH10B E. coli (Epicentre)
  • SOC medium (see recipe)
  • LB agar plates (appendix 2D) supplemented with 12.5 µg/ml chloramphenicol
  • LB agar plates (appendix 2D) supplemented with 12.5 µg/ml chloramphenicol and 25 µg/ml kanamycin
  • LB medium (appendix 2D) supplemented with 12.5 µg/ml chloramphenicol
  • Qiagen large-construct DNA preparation kit (or equivalent)
  • Pulsed-field gel electrophoresis apparatus (CHEF-mapper, Bio-Rad, or equivalent)
  • 37° and 70°C water baths
  • Electroporator (Gene-pulser II, Bio-Rad, or equivalent)
  • Additional equipment and reagents for restriction endonuclease digestion of DNA (cpmb unit 3.1), pulsed-field electrophoresis (unit 5.1), spectrophotometric quantitation of DNA (appendix 3D), and transformation by electroporation (cpmb unit 1.8)
Looking for materials?  Suppliers Guide
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  • Figure 5.18.1
    Transposon vector with an 86-kb D17Z1-derived alpha-satellite array. Mammalian cell line selection is based on a puro cassette conferring resistance to puromycin and a neo/kan cassette conferring resistance to geneticin (neomycin). The transposon vector is flanked by 19-bp transposase recognition elements (ME). The line labeled 1 kb indicates the scale of the transposon vector. Reproduced from Basu et al., 2005a with permission from Oxford University Press.

    View Image
  • Figure 5.18.2
    Construction of a synthetic alpha-satellite array. (A) Outline of iterative scheme for synthesis of mutated HOR versions of D17Z1 alpha-satellite arrays. (B) Outline of scheme for directional multimerization of mutated HORs. (C) Pulsed-field gel electrophoresis (PFGE) analysis of intermediates in the construction of 1732 HOR/pBeloBAC constructs. Each intermediate was digested with NotI, which drops out the entire subcloned alpha-satellite array from the pBeloBAC vector backbone. Each lane shows the completed intermediate en route to 32 copies of the mutated HOR. The number beneath each lane corresponds to the number of copies of the HOR in each construct, and the arrows to the right indicate the size of the alpha-satellite insert and the vector backbone. The insert in lane 1 is 2.7 kb and therefore too small to be resolved by PFGE. Reproduced from Basu et al., 2005a with permission from Oxford University Press.

    View Image
  • Figure 5.18.3
    Seamless ligation with Type IIS restriction enzymes. An alpha-satellite monomer (monomer n) as well as its neighboring monomer (monomer n+1) are PCR amplified with primers incorporating a Type IIS endonuclease site (in this case SapI: 5¢-gct cttc n1-3¢/3¢-cga gaag n4-5¢), designed as illustrated. The overhangs are custom built such that, upon digestion with SapI, seamless ligation of neighboring monomers may be facilitated without the introduction of any extraneous sequences. Note that SapI will remove its own recognition sequence during this process.

    View Image

Literature Cited

Literature Cited
    Basu, J. and Willard, H.F. 2005. Artificial and engineered chromosomes: Non-integrating vectors for gene therapy. Trends Mol. Med. 11:251-258.
    Basu, J., Stromberg, G., Compitello, G., Willard, H.F., and Van Bokkelen, G. 2005a. Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays. Nucl. Acids Res. 33:587-596.
    Basu, J., Compitello, G., Stromberg, G., Willard, H.F., and Van Bokkelen, G. 2005b. Efficient assembly of de novo human artificial chromosomes from large genomic loci. BMC Biotech. 5:21.
    Chan, G.K., Liu, S.T., and Yen, T.J. 2005. Kinetochore structure and function. Trends Cell Biol. 15:589-598.
    Grimes, B.R., Schindelhauer, D., McGill, N.I., Ross, A., Ebersole, T. A., and Cooke, H.J. 2001. Stable gene expression from a mammalian artificial chromosome. EMBO Rep. 2:910-914.
    Goryshin, I.Y. and Reznikoff, W.S. 1998. Tn5 in vitro transposition. J. Biol. Chem. 273:7367-7374.
    Harrington, J.J., Van Bokkelen, G., Mays, R.W., Gustashaw, K., and Willard, H.F. 1997. Formation of de novo centromeres and construction of first generation human artificial microchromosomes. Nat. Genet. 15:345-355.
    Ikeno, M., Inagaki, H., Nagata, K., Morita, M., Ichinose, H., and Okazaki, T. 2002. Generation of human artificial chromosomes expressing naturally controlled guanosine triphosphate cyclohydrolase I gene. Genes Cells 7:1021-1032.
    Kotzamanis, G., Cheung, W., Abdulrazzak, H., Perez-Luz, S., Howe, S., Cooke, H., and Huxley, C. 2005. Construction of human artificial chromosome vectors by recombineering. Gene 351:29-38.
    Mejia, J.E. and Larin, Z. 2000. The assembly of large BACs by in vivo recombination. Genomics 70:165-170.
    Ohzeki, J., Nakano, M., Okada, T., and Masumoto, M. 2002. CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA. J. Cell Biol. 159:765-775.
    Waye, J.S. and Willard, H.F. 1986. Structure, organization, and sequence of alpha-satellite DNA from human chromosome 17: Evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome. Mol. Cell. Biol. 6:3156-3165
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.