Conversion Technology for the Separation of Maternal and Paternal Copies of Any Autosomal Chromosome in Somatic Cell Hybrids

W. Edward Highsmith1, Kevin J. Meyer1, Victoria M. Marley1, Robert B. Jenkins1

1 Division of Laboratory Genetics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 3.6
DOI:  10.1002/0471142905.hg0306s55
Online Posting Date:  October, 2007
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Abstract

Conversion Technology (CT) is a streamlined version of somatic cell hybrid preparation that was developed to be amenable for use as a mutation detection method and as a diagnostic tool for individual patients. It is also a powerful research tool. There are two broad categories of potential applications for CT: research applications in gene mapping and identification, and clinical applications in the detection of disease‐causing mutations in individual patients. CT may emerge as the gold standard for both mutation detection and haplotyping. It will likely prove valuable in mutation detection due to its ability to detect mutations that are not amenable to detection by sequencing (e.g., deep intron or promoter mutations). It will likely prove valuable in gene identification projects because genotyping of haploid chromosomes yields unequivocal haplotypes. Curr. Protoc. Hum. Genet. 55:3.6.1‐3.6.38. © 2007 by John Wiley & Sons, Inc.

Keywords: haplotype; mutation detection; somatic cell hybrids; Conversion Technology

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Fusion of Mouse E2 Cells and Human Transformed Lymphoblast Cells
  • Basic Protocol 2: Harvesting DNA for Genotyping
  • Basic Protocol 3: Identification of Haploid Clones by STR Genotyping Using the ABI 3730 DNA Analyzer
  • Basic Protocol 4: Expansion of Hybrid Colonies
  • Support Protocol 1: Analysis of Multiplex STR PCR Electropherograms Using GeneMarker Software
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fusion of Mouse E2 Cells and Human Transformed Lymphoblast Cells

  Materials
  • Mouse E2 cell line (contact ) grown in T150 cell culture flasks (for basic mammalian cell culture techniques see appendix 3G), passage number <10
  • Hanks' balanced salt solution (HBSS; appendix 2D)
  • TrypLE Express (Invitrogen)
  • Trypan blue stain
  • E2 cell medium (see recipe)
  • Patient EBV‐transformed cells to be fused (see appendix 3H)
  • Fusion medium (see recipe)
  • Methylcellulose medium (see recipe)
  • ClonaCell HY Medium E (Stem Cell Technologies)
  • Genotyping controls: parental cells (the original EBV cell line, four wells), a human control (two wells), and a water blank (two wells)
  • DMEM/HAT/G418 selection medium (see recipe)
  • T150 tissue culture flasks
  • CO 2 cell culture incubator (Forma Scientific), 37°C
  • 2‐ml vials
  • Hemacytometer, 0.1 mm deep (Reichert or similar) or Coulter Counter
  • BTX ECM 2001 Electro Cell Manipulator with BTX enhancer 400 BTX
  • ECM 2001 Electro Cell cuvettes (blue‐capped; Fisher)
  • 15‐ and 50‐ml conical tubes
  • 10‐ml serological pipets
  • 100‐mm petri dishes
  • 96‐well cell culture plates (Fisher or equivalent)
  • Multichannel pipettor

Basic Protocol 2: Harvesting DNA for Genotyping

  Materials
  • TrypLE Express (Invitrogen)
  • 96‐well culture plates with colonies of selected cells ( protocol 1)
  • 0.2 M NaOH/1 mM EDTA (see recipe)
  • Earle's balanced salt solution (EBSS; Gibco)
  • DMEM/HAT/G418 medium (see recipe)
  • Multichannel micropipettor
  • Multichannel vacuum apparatus
  • V‐well PCR plate

Basic Protocol 3: Identification of Haploid Clones by STR Genotyping Using the ABI 3730 DNA Analyzer

  Materials
  • 1 M Tris, pH 7.4 (Sigma)
  • TE buffer, pH 7.4 ( appendix 2D)
  • Cell plate containing lysate ( protocol 2)
  • PCR master mix for each chromosome: prepared according to Table 3.6.1 and stored frozen up to 1 year at −20°C
  • Hi‐Di formamide (Applied Biosystems)
  • ABI 3100/3730 400HD (ROX) size standard (Applied Biosystems)
  • Platinum Taq polymerase (Invitrogen)
  • 96‐well plate centrifuge (Sorvall Legend‐T or equivalent)
  • Thermal cycler (e.g., Thermo Hybaid MultiBlock System, Thermo Electron)
  • Heating block (optional)
  • 5‐ to 50‐µl and 0.5‐ to 10‐µl multichannel pipettors and reservoirs
  • ABI plates (MicroAmp Optical 96‐well reaction plates with septa, Applied Biosystems)
  • 8‐tube PCR strips with adhesive plate sealing sheet (preferred; e.g., Eppendorf) or 8‐cap strips
  • ABI 3100 or 3730 capillary electrophoresis instrument (Applied Biosystems) or similar instrument with fluorescence detector

Basic Protocol 4: Expansion of Hybrid Colonies

  Materials
  • Cells in 96‐well plate
  • TrypLE Express (Invitrogen)
  • DMEM/HAT/G418 selection medium (see recipe)
  • Liquid nitrogen tank
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • 12‐well culture plates
  • Cryovials
  • T25 and T75 tissue culture flasks
  • Cryovials
  • Sarstedt vials
  • 15‐ml centrifuge tubes
  • Cryomed controlled‐rate freezer (Thermo Fisher Scientific)
  • Vacuum bottle apparatus
  • Liquid nitrogen freezer
  • Additional reagents and equipment for performing (FISH) analysis (unit 4.5) and extracting genomic DNA (e.g., see appendix 3B)

Support Protocol 1: Analysis of Multiplex STR PCR Electropherograms Using GeneMarker Software

  Materials
  • GeneMarker software package (Soft Genetics)
  • PC running Microsoft Windows 2000, NT, or XP with at least a 1‐GHz Pentium III processor or equivalent, 128 MB RAM, and a 10‐GB hard drive
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Figures

Videos

Literature Cited

Literature Cited
   Casey, G., Lindor, N.M., Papadopoulos, N., Thibodeau, S.N., Moskow, J., Steelman, S., Buzin, C.H., Sommer, S.S., Collins, C.E., Butz, M., Aronson, M., Gallinger, S., Barker, M.A., Young, J.P., Jass, J.R., Hopper, J.L., Diep, A., Bapat, B., Salem, M., Seminara, D., and Haile, R. 2005. Conversion analysis for mutation detection in MLH1 and MSH2 in patients with colorectal cancer. J. Am. Med. Assoc. 293:799‐809.
   Douglas, J., Boehnke, M., Gillanders, E., Trent, J., and Gruber, S. 2001. Experimentally‐derived haplotypes substantially increase the efficiency of linkage disequilibrium studies. Nat. Genet. 28:361‐364.
   Jenkins, R., Blair, H., Ballman, K., Giannini, C., Arusell, R., Law, M., Flynn, H., Passe, S., Felten, S., Brown, P., Shaw, E., and Buckner, J. 2006a. A t(1;19)(q10;p10) mediates the combined deletions of 1p and 19q and predicts a better prognosis of patients with oligodendroglioma. Cancer Res. 66:9852‐9861.
   Jenkins, R.B., Flynn, H., Blair, H., Meyer, K.J., Schneider, P.S., Marley, V., Schowalter, K., and Highsmith, W.E. 2006b. Isolation of the T(1;9)(Q10;P10) associated with human oligodendrogliomas using Conversion Technology. Annual Meeting of the American Society of Human Genetics, abstr. 287. New Orleans. http://www.ashg.org/genetics/ashg06s/index.shtml.
   Laken, S., Papadopoulos, N., Petersen, G., Gruber, S., Hamilton, S., Giardiello, F., Brensinger, J., Vogenstein, B., and Zinzler, K. 1999. Analysis of masked mutations in familial adenomatous polyposis. Proc. Natl. Acad. Sci. U.S.A. 96:2322‐2326.
   Marley, V., Schowalter, K., Miller, M., Meyer, K., Schneider, P., Jenkins, R., and Highsmith, W. 2006. Validation of 22 autosome and X whole chromosome ABI PCR multiplex haplotyping assays for Conversion Technology. J. Mol. Diagn. 628:abstract G32.
   Papadopoulos, N., Leach, F.S., Kinzler, K.W., and Vogelstein, B. 1995. Monoallelic mutation analysis (MAMA) for identifying germline mutations. Nat. Genet. 11:99‐102.
   Schaid, D. 2002. Relative efficiency of ambiguous vs. directly measured haplotype frequencies. Genet. Epidemiol. 23:426‐443.
   Schouten, J.P., McElgunn, C.J., Waaijer, R., Zwijnenburg, D., Diepvens, F., and Pals, G. 2002. Relative quantification of 40 nucleic acid sequences by multiplex ligation‐dependent probe amplification. Nucl. Acids Res. 30:57
   Strachan, T. and Read, A.P. 1999. Human Molecular Genetics 2, 2nd ed. Wiley‐Liss, New York.
   Thomas, S., Porteous, D., and Visscher, P. 2004. Power of direct vs. indirect haplotyping in association studies. Genet. Epidemiol. 26:116‐124.
   Weiss, M. and Green, H. 1967. Human‐mouse hybrid cell lines containing partial complements of human chromosomes and functioning human genes. Proc. Natl. Acad. Sci. U.S.A. 58:1104‐1111.
   Yan, H., Papadopoulos, N., Marra, G., Perrera, C., Jiricny, J., Boland, C.R., Lynch, H.T., Chadwick, R.B., de la Chapelle, A., Berg, K., Eshleman, J.R., Yuan, W., Markowitz, S., Laken, S.J., Lengauer, C., Kinzler, K.W., and Vogelstein, B. 2000. Conversion of diploidy to haploidy. Nature 403:723‐724.
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