Abstract
We report on the use of flow karyotyping and fluorescence in situ hybridization (FISH) to characterize the human chromosomes in somatic cell hybrids. The identity, DNA content, and relative frequency of human chromosomes are derived from flow karyotypes, i.e., measurements of Hoechst and chromomycin fluorescence intensities of chromosomes by dual beam flow cytometry. Chromosome integrity is assessed by comparing the peak position of a human chromosome in the flow karyotypes of a hybrid cell line and its human donor. When human donor cells are unavailable, the peak position of a human chromosome in a hybrid line is compared to the range of peak positions among normal individuals. The relative frequency of human chromosomes in subclones or hybrids grown in culture is monitored using the volumes of peaks in flow karyotypes. FISH with biotinylated human genomic DNA or chromosome-specific repeat sequences as probe is used in conjunction with flow karyotyping to confirm the number of human chromosomes in hybrids. Some small rearrangements are detected by flow karyotyping and not by FISH. On the other hand, translocations between human and rodent chromosomes are detected by FISH and not always by flow karyotyping. Flow karyotyping and FISH were used to characterize over 100 hybrid lines donated by other laboratories. A hybrid set useful for the construction of chromosome-enriched gene libraries is presented. In this set, each of the 24 human chromosome types is present and intact, as judged by these techniques, in a line containing little or no other human material.
Similar content being viewed by others
Literature cited
Ruddle, F.H., and Creagan, R.P. (1975).Annu. Rev. Genet. 9407–490.
Van Dilla, M.A., Deaven, L.L., et al. (1986).Bio/Technology 4537–552.
Siciliano, M.J., and White, B.F. (1987).Methods Enzymol. 151169–194.
Friend, K.K., Donohue, B.P., Kucherlapati, R.S., and Ruddle, F.H. (1976).Exp. Cell Res. 9931–36.
Pinkel, D., Straume, T., and Gray, J.W. (1986).Proc. Natl. Acad. Sci. U.S.A. 859138–9142.
Tucker, J.D., Christensen, M.L., and Carrano, A.V. (1988).Cytogenet. Cell Genet. 48103–106.
Boyle, A., Lichter, P., and Ward, D.C. (1990).Genomics 7127–130.
Kievits, T., Devilee, P., Wiegant, J., Wapenaar, M.C., Cornelisse, C.J., van Ommen, G.J.B., and Pearson, P.L. (1990).Cytometry 11105–109.
Ledbetter, S.A., Nelson, D.L., Warren, S., and Ledbetter, D.H. (1990).Genomics 6475–481.
Lichter, P., Ledbetter, S.A., Ledbetter, D.H., and Ward, D.C. (1990).Proc. Natl. Acad. Sci. U.S.A. 666634–6638.
Trask, B., van den Engh, G., Nussbaum, R., Schwartz, C., and Gray, J.W. (1990).Cytometry 11184–195.
Gray, J.W., Trask, B., van den Engh, G., Silva, A., Lozes, C., Grell, S., Schonberg, S., Yu, L.-C., and Golbus, M.S. (1988).Am. J. Hum. Genet. 4249–59.
Gray, J.W., Langlois, R.G., Carrano, A.V., Burkhart-Schultz, K., and Van Dilla, M.A. (1979).Chromosoma 739–27.
Harris, P., Boyd, E., Young, B.D., and Ferguson-Smith, M.A. (1986).Cytogenet. Cell. Genet. 4114–21.
Harris, P., Cooke, A., Boyd, E., Young, B.D., and Ferguson-Smith, M.A. (1987).Hum. Genet. 76129–133.
Trask, B., van den Engh, G., Mayall, B., and Gray, J.W. (1989).Am. J. Hum. Genet. 45739–752.
Trask, B., van den Engh, G., and Gray, J.W. (1989).Am. J. Hum. Genet. 45753–760.
Carter, N.P., Ferguson-Smith, M.E., Affara, N.A., Briggs, H., and Ferguson-Smith, M.A. (1990).Cytometry 11202–207.
Franco, B., Lai, L.-W., Patterson, D., Ledbetter, D.H., Trask, B., van den Engh, G., Iannacone, S., Frances, S., Patel, P.I., Lupski, J.R.Hum. Genet. (in press).
van den Engh, G.J., Trask, B.J., Gray, J.W., Langlois, R., and Yu, L.-C. (1985).Cytometry 692–100.
van den Engh, G.J., Trask, B., Lansdorp, P., and Gray, J.W. (1988).Cytometry 9266–270.
van den Engh, G. and Stokdijk, W. (1989).Cytometry 10282–293.
van den Engh, G., Hanson, D., and Trask, B. (1990).Cytometry 11173–183.
Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
Cooke, J.J., and Hindley, J. (1979).Nucleic Acids Res. 63177–3197.
Higgins, M.J., Want, H., Shtromas, I., Haliotis, T., Roder, J.C., Holden, J.J.A., and White, B.N. (1985).Chromosoma 9377–86.
Burk, R., Szabo, P., O'Brien, S., Nash, W., Yu, L., and Smith, K. (1985).Chromosoma 92225–233.
Waye, J., and Willard, H. (1986).Mol. Cell. Biol. 63156–3165.
Thompson, L.H., Mooney, C.L., and Brookman, K.W. (1985).Mutat. Res. 150423–429.
Warburton, D., Gersen, S., Yu, M.-T., Jackson, C., Handelin, B., and Housman, D. (1990).Genomics 6358–366.
Miller, Y.E., Daniels, G.L., Jones, C., and Palmer, D.K. (1987).Am. J. Hum. Genet. 411061–1070.
ISCN (1985).An International System for Human Cytogenetic Nomenclature (eds.) Harnden, D.G., and Klinger, H.P., published in collaboration withCytogenet. Cell. Genet. (Karger, Basel).
Janssen, J.W.G., Collard, J.G., Tulp, A., Cox, D., Millington-Ward, A., and Pearson, P. (1986).Cytometry 7411–417.
Bennett, J.H., Hayman, D.L., and Hope, R.M. (1986).Nature 32359–60.
Koi, M., Shimizu, M., Morita, H., Yamada, H., and Oshimura, M. (1989).Jpn. J. Cancer Res. 80413–418.
Athwal, R.S., March, M., Searle, B.M., and Deo, S.S. (1985).Somat. Cell. Mol. Genet. 11177–187.
Carlock, L.R., Smith, D., and Wasmuth, J.J. (1986).Somat. Cell. Mol. Genet. 12163–174.
Thompson, L.H., Mooney, C.L., Burkhart-Schultz, K., Carrano, A.V., and Siciliano, M.J. (1985).Somat. Cell. Mol. Genet. 1187–92.
Leonard, S., Arbogast, D., Geyer, D., Jones, C., and Sinensky, M. (1986).Proc. Natl. Acad. Sci. U.S.A. 832187–2189.
Dana, S., and Wasmuth, J.J. (1982).Somat. Cell. Genet. 8245–264.
Jones, N.J., Stewart, S.A., and Thompson, L.H. (1990).Mutagenesis 515–23.
Jones, C., Kao, F.T., and Taylor, R.T. (1980).Cytogenet. Cell. Genet. 28181–194.
Rashidbaigi, A., Langer, J.A., Jung, V., Jones, C., Morse, H.G., Tischfield, J.A., Trill, J.J., Kung, H.F., and Pestka, S. (1986).Proc. Natl. Acad. Sci. U.S.A. 83384–388.
Cirullo, R.E., and Wasmuth, J.J. (1984).Somat. Cell. Mol. Genet. 10225–234.
Thompson, L.H., Bachinski, L.L., Stallings, R.L., Dolf, G., Weber, C.A., Westerveld, A., and Siciliano, M.J. (1989).Genomics 5670–679.
Thompson, L.H., Carrano, A.V., Sato, K., Salazar, E.P., White, B.F., Stewart, S.A., Minkler, J.L., and Siciliano, M.J. (1987).Somat. Cell. Mol. Genet. 13539–551.
Lugo, T.G., Handelin, B., Killary, A.M., Housman, D.E., and Fournier, R.E.K. (1987).Mol. Cell. Biol. 72814–2820.
Van Keuren, M.L., Hart, I.M., Kao, F.-T., Neve, R.L., Bruns, G.A.P., Kurnit, D.M., and Patterson, D. (1987).Cytogenet. Cell Genet. 44142–147.
Tasset, D.M., Hartz, J.A., and Kao, F.-T. (1988).Am. J. Hum. Genet. 42854–866.
Callen, D.F. (1986).Ann. Genet. 29235–239.
Sundar Raj, C.V., Church, R.L., Klobutcher, L.A., and Ruddle, F.H. (1977).Proc. Natl. Acad. Sci. U.S.A. 744444–4448.
van Tuinen, P., Rich, D.C., Summers, K.M., and Ledbetter, D.H. (1987).Genomics 1374–381.
Raziuddin, A., Sarkar, F.H., Dutkowski, R., Shulman, L., Ruddle, F.H., and Gupta, S.L. (1984).Proc. Natl. Acad. Sci. U.S.A. 815504–5508.
Fisher, J.H., Kao, F.T., Jones, C., White, R.T., Benson, B.J., and Mason, R.J. (1987).Am. J. Hum. Genet. 40503–511.
Fisher, J.H., Emrie, P.A., Drabkin, H.A., Kushnik, T., Gerber, M., Hofmann, T., and Jones, C. (1988).Am. J. Hum. Genet. 43436–441.
Jones, C., Patterson, D., and Kao, F.-T. (1981).Somat. Cell. Genet. 7399–409.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Trask, B.J., van den Engh, G., Christensen, M. et al. Characterization of somatic cell hybrids by bivariate flow karyotyping and fluorescencein situ hybridization. Somat Cell Mol Genet 17, 117–136 (1991). https://doi.org/10.1007/BF01232970
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01232970