J

Final Report DOE Grant No. DE-FG06-93ER61662 "Flow Karyotyping and Flow instrumentation Development"

P.I.: Gerrit J. van den Engh Department of Molecular Biotechnology University of Washington, WA 98

The project had three major aims: 1. Improvement of technology for high-speed cell and chromosome sorting; 2. The use of such instrumentation in genome analysis; 3. Apply the principles developed and the lessons learned to automated processes for

the genome program.

The work was a continuation of studies that were started at the Lawrence Livermore National Laboratory before the PI moved to the University of Washington. At Livermore, a high-speed sorter for the selection of human chromosomes was built. The instrument incorporated Livermore's advanced sorter technology. The engineering focused on improving robustness and reliability so that the full potential of high-speed sorting would become available to the biological research laboratory. The new instrument, dubbed MoFlo for modular flow cytometer, proved to be a very practical and efficient tool during the chromosome isolation phase of the gene-library project. Its reliability and ease of operation exceeded that of the commercial instruments. The technology was licensed to two companies. Systemix, Palo Alto, CA, realized the instrument's advantages for the purification of stem cells for bone marrow transplantation and licensed the instrument for in-house use. Cytomation, Fort Collins, CO, decided to manufacture the instrument for the research market.

PI'S move to Washington, in 1992, was instigated by several reasons. The environment in the national laboratories is an excellent environment for development of technology. Exploration of new uses and optimization for bio/medical applications requires an environment driven by biological rather than technical problems. The founding of the department of Molecular Biotechnology at the University of Washington by Leroy Hood, provided a unique opportunity to apply the new sorting technology to a variety of biological studies thereby testing its usefulness beyond chromosome sorting. DOE acknowledged these possibilities by funding the establishment of a flow sorting development / application group at the new department.

The program at the University of Washington has been very successful. The use with different biological systems has led to a great number of technological improvements. All electronic functions are now controlled by computer. The alignment is greatly facilitated which diminishes the requirements for highly skilled personnel. The overall lay-out is simplified and can easily be cleaned and sterilized. The latest embodiment of the instrument is divided over two rooms. All equipment requiring regular adjustment and repairs is located in a "machine room" to which technicians in "street clothes" have ready access. The part of the instrument that is in contact with the cells is located in a GMP (good manufacturing practise) environment and is operated by collaborators

DISCLAIMER

This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liabiiity or responsibility for the accuracy, completeness, or use- fulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any spe- cific commercial product, process, or service by trade name, trademark, rnanufac- turer, or otherwise does not necessarily constitute or imply its endorsement, m m - mendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.

wearing sterile gowns. This new set-up makes possible, for the first time, the responsible use of cell sorting to clinical applications.Systemix has obtained FDA approval for use of this instrument in clinical studies. The University of Washington is building a gene therapy lab in which the clinical-grade sorter will play a dominant role. These developments show that one of the major aims of the program: the transfer of the technology from the research lab to the clinic, has been achieved. his part of the program is now adequately supported by industry and no longer requires DOE support.

An advance sorting facility that has been built in Seattle and is used in a variety of programs. Barbara Trask and Ger van den Engh have used the instrument in the study of gross polymorphisms in human chromosomes. The use of the instrument has spread to other studies. Groups sorting prostate cells, single sperm cells for PCR analysis, Drosophila embryos, bacteria labeled with green fluorescent protein, human chromosomes , and many other cell types regularly use the facility. Some of the studies may be found in the literature list. This "service" is now supported in part by an NSF center grant. The remaining costs are carried by the users. Like the technology development, this aspect of the program has become self-supporting and no longer requires requires DOE support.

The technical development program has attracted a number of talented molecular biology and engineering students. Cell sorters incorporate a wide spectrum of technologies: lasers and optics, high-speed analog and digital electronics, computer interfacing, delicate fluidics, etc.. The design of robust and user-friendly equipment poses a formidable engineering challenge. Students can select a favorite topic and can test and develop their skills in a "real-world" engineering project. This setting turns out to be highly motivating. Biology and engineering students work in teams and appreciate each other disciplines and approaches. This collaboration has resulted in remarkably elegant engineering solutions. The core technology is now covered by 10 patents. Six more are in progress. Licensing to industry is in progress.

The engineering skills that are now represented in the cell sorter group have extended to other automation projects. Presently under development are: a highly-automated phase-sensitive gel scanner (project in collaboration with Oxford GlycoSystems); development of a sorter nozzle for arraying beads with a combinatorial library for drug discovery (collaboration with Pharmacopeia); the fabrication of microstructures that trap DNA molecules by inducted dipole moments (supported by DOE).

The program has met its goals. The high-speed sorting technology has been made reliable and has been transferred to industry and the clinic. A prospering sorting facility has been established in Seattle. This facility is very productive in a variety of biological studies. The technical skills developed during this project are now used in the development of other instruments for automating genome research. Most of the activities are now self-supporting.

Relevant Publications (including some from the early stages of the program that were carried out in Livermore).

t

Gray, J.W., Pinkel, D., Trask, B., van den Engh, G., Segraves, R., Collins, C., Andreeff, M., Haimi, J., and Perle, M.A. (1989). Application of Flow Karyotyping and Fluorescence In Situ Hybridization to Detection of Chromosome Aberrations. In: Prognostic Cytometry and Cyfopathology of Prostate Cancer (Karr, J.D., Coffee, D.S., Gardner, W., Eds.) Elsevier Sci. Publ. pp. 304-313.

Nooter, H., Oostrum, R., Jonker, R.R., van Dekken, H., Stokdijk, W., and van den Engh, G.J. (1 989). Effect of Cyclosporin A on Daunorubicin Accumulation in Multidrug-Resistant P388 Leukemia Cells Measured by Real-Time Flow Cytometry. Cancer Chemother. Pharmacol. 23, 296-300.

van den Engh, G. and Stokdijk, W. (1989). Parallel Processing Data Acquisition System for Multi-Laser Flow Cytometry and Cell Sorting. Cytometry 10, 282-293.

Herweijer, H., van den Engh, G., and Nooter, K. (1989). A Rapid and Sensitive Flow Cytometric Method for the Detection of Multidrug Resistant Cells. Cyfometry 10, 463-468

Van Dilla, M.A., Deaven, L.L., Albright, K.L., Allen, N.A., Bartholdi, M.F., Brown, N.C., Campbell, E.W., Carrano, A.V., Christensen, M., Clark, L.M., Cram. L.S., Dean, P.N., de Jong, P., Fawcett, J.J., Fuscoe, J.C., Gray, J.W., Hildebrand, C.E., Jackson, P.J., Jett, J.H., Killa, S., Longmire, J.L., Lozes, C.R., Luedemann, M.L., McNinch, J.S., Mendelsohn, M.L., Meyne, J., Meincke, L.J., Moyzis, R.K., Mullikin, J., Munk, A.C., Perlman, J., Pederson, L., Peters, D.C., Silva, A.J., Trask, B.J., and van den Engh, G. (1990). The National Laboratory Gene Library Project. In: Flow Cyfogenefics (J.W. Gray, Ed.) Academic Press, London, pp 257-274.

Gray, J.W. and van den Engh, G.J. (1989). Instrumentation for Chromosome Analysis and Sorting. In: Flow Cytogenetics (Gray, J.W., Ed.) Academic Press, London, pp 17-33.

Gray, J.W., Trask, B., and van den Engh, G. (1989). Bivariate Flow Karyotyping. In: Flow Cytogenetics (Gray, J.W., Ed.) Academic Press, London, pp 137-1 50

Nooter, K., Herweijer, H., Jonker, R.R., and van den Engh, G.J. (1989). On-line Flow Cytometry : A Versatile Method for Kinetic measurements. In: Methods in Cell Biology, Volume 33: Flow Cytometry. (Darzynkiewicz, Z. and Crissman H.H., Eds.), Academic Press, San Diego, CA .pp.

Trask, B. and van den Engh, G. (1989). Chromosome and Nuclei Isolation with the MgSO4 Procedure. In: Methods in Cell Biology, Volume 33: Flow Cyfometry (Darzynkiewicz, Z. and Chrissman, H.H., Eds.) Academic Press, San Diego, CA, pp 363-367.

Merry, D.E., Lesko, J.G., Sosnoski, D.M., Lewis, R.A., Lubinsky, M., Trask, B., van den Engh, G., Collins, F., and Nussbaum, R.L. (1989). Choroideremia and Deafness with Stapes Fixation: A Contiguous Gene Deletion Syndrome in Xq21. Am. J. Human Genetics 45, 530-540.

Schnur, R.E., Trask, B.J., van den Engh, G., Punnett, H.H., Kistenmacher, M., Naids, R., Tomeo, M., and Nussbaum, R.L. (1989). An Xp22 Microdeletion Associated with Ocular Albinism and Ichthyosis: Approximation of Breakpoints and Estimation of Deletion Size Using Cloned DNA Probes and Flow Cytometry. Am. J. Human Genetics 45, 706-720.

Trask, B., van den Engh, G., Mayall, B., and Gray, J.W. (1989). Chromosome Heteromorphism Quantified by High Resolution Bivariate Flow Karyotyping. Am. J. Human Genetics 45, 739-752.

Trask, B., van den Engh, G., and Gray, J. (1989). Inheritance of Chromosome Heteromorphisms Analyzed by High Resolution Bivariate Flow Karyotyping. Am. J. Human Genetics 45, 753-760.

Dubelaar, G.B.J., Groenewegen, A.C., Stokdijk, W., van den Engh, G.J., and Visser, J.W.M. (1 989). The Optical Plankton Analyser (OPA): A Flow Cytometer for Plankton Analysis. I1 Specifications. Cytometry 10, 529-539.

Gray, J.W., Pinkel, D., Trask, B., van den Engh, G., Segraves, R., Collins, C., Andreeff, M., Haimi, J., and Perle, M.A. (1 989). Application of Flow Karyotyping and Fluorescence In Situ Hybridization to Detection of Chromosome Aberrations. In: Proceedings of the Prouts Neck Symposium on Cytopathology/Flow Cytometry and Prognostic lndicators of Prostate Cancer(J.P. Parker, D.S. Coffey, and W. Gardner, Jr., Eds.) pp. 304-313.

Trask, B., van den Engh, G., Nussbaum, R., Schwartz, C., and Gray, J.W. (1990). . Quantification of the DNA Content of Structurally Abnormal X Chromosomes and X Chromosome Aneuploidy Using High Resolution Bivariate Flow Karyotyping. Cyfometry 11, 184-185.

van den Engh, G., Hanson, D., and Trask, B. (1990). A Computer Program for Analyzing Bivariate Flow Karyotypes. Cytornetry 11 , 173-1 83.

Gray, J.W., Kuo, W.-L., Liang, J., Pinkel, D., van den Engh, G., Trask, B., Tkachuk, D., Waldman, F., and Westbrook, C. (1990). Analytical Approaches to Detection and Characterization of Disease-Linked Chromosome Aberrations. Bone Marrow Transplantation 6 (Sup 1) 14-1 9.

van den Engh,G., and Trask, B.J. (1 990). Chromosomal Analysis by Flow Cytometry. Proc. Royal Micros. SOC, New Series Vol. 1 (Microgo, eds, H.Y. Elder and A. Hilger) pp. 501- 506.

Trask, B.J., van den Engh, G.J., Christensen, M., Massa, H., Gray, J.W., Van Dilla, M.V. (1991). Characterization of Somatic Cell Hybrids by Bivariate Flow Karyotyping and Fluorescence In Situ Hybridization. Somat. Cell Molec. Genet. 17, 1 17-1 36.

Franco, B., Lai, L.W., Patterson, D., Ledbetter, D.H., Trask, B.J., van den Engh,G.J., lannaccone, S., Frances, S., Patel, P.I., Lupski, J.R. (1 991). Molecular Characterization of a Patient with del(l)(q23-q25). Hum Genet, 87, 269-277.

Schmitz, A., Guilly, M.N., Trask, B., and van den Engh, G.J. (1992). Immunofluorescent Labeling of Centromere for Flow Cytometric Analysis. Cytometry 13:478-484.

van den Engh, G.J., Farmer, C. (1992). Photo-bleaching and Photon Saturation in Flow Cytometry. Cytometry 13:669-677.

McCabe, E.R.B., Towbin, J.A., van den Engh, G.J., Trask, B.J. (1992) Xp21 contiguous gene syndromes: Deletion quantitation with bivariate flow karyotyping allows mapping of patient breakpoints. Am. J. Hum. Genet. 51 :I 277-1 285.

Trask, B.J., Christensen, M., Fertitta, A., Bergmann, A., Ashworth, L., Branscomb, E., Carrano, A.V., van den Engh, G.J. (1992) Fluorescence in situ hybridization mapping of chromosome 19: Mapping and verification of cosmid contigs formed by restriction enzyme fingerprinting. Genomics 14:162-167.

van den Engh, G.J., Sachs, R., Trask, B. (1992) Estimating genomic distance from DNA sequence location in cell nuclei using a random walk model. Science 257:1410-1412.

van den Engh, G.J. (1 993) New applications of flow cytometry. Current Opinion in Biotechnology 4: 63-68.

Gurrieri, F., Trask, B.J., van den Engh, G., Krauss, C.M., Schinzel, A., Pettenati, M.J., Schindler, D., Dietz-Band, J., Vergnaud, G., Scherer, S.W., Tsui, L.-C., Muenke, M. (1993) Physical mapping of the holoprosencephaly critical region in 7q36. Nature Genetics 3: 247-251.

Bayer, J.A., van den Engh, G.J. (1993) In situ hybridization. in: Flow Cytomefry, new developments (Jacquemin-Sablon, A., Ed.) Springer-Verlag, Berlin, pp 269-276

Trask, B.J., Allen, S., Massa, H., Fertitta, A., Sachs, R., van den Engh, G.J., Wu, M. (1994) Studies of metaphase and interphase chromosomes using fluorescence in situ hybridization. Cold Spring Harbor Symp. Quant. Biol. 58: 767-775.

Silverman, G.A., Schneider, S.S., Massa, H.F., Flint, A., Lalande, M., Leonard, J.C., Overhauser, J., van den Engh, G., Trask, B.J. (1995) The 18q- syndrome: analysis by bivariate flow karyotyping and the PCR reveals a successive set of deletion breakpoints within 18q21.2-q22.2. Am. J. Hum. Genet. 56:926-937.

Hui, S.M., Trask, B.J., van den Engh, G.J., Bartuski, A.J., Smith, A., Flint, A., Lalande, M., Silverman, G.A. (1 995) Analysis of randomly-amplified flow-sorted chromosomes using the polymerase chain reaction. Genomics 26: 364-371.

Sachs, R.K., van den Engh, G., Trask, B., Yokota, H., Hearst, J.E. (1995) A random-walk/Giant- loop model for interphase chromosomes. Proc. Natl. Acad, Sci. U S A . 92:2710-2714.

Yokota, H., van den Engh, G., Mostert, M., Trask, B.J. (1995) Treatment of cells with alkaline borate buffer extends the capability of interphase FISH mapping. Genornics 25: 485-491.

Juyal, R.C., Greenberg, F., Mengden, G.A., Lupski, J.R., Trask, B.J., van den Engh, G., Lindsay, E., Christy, H., Chen, K . 6 , Baldini, A., Shaffer, L.G., Patel, P.I. (1995) Smith- Magenis syndrome deletion: A case with equivocal cytogenetic findings resolved by fluorescence in situ hybridization. Am. J. Med. Genet. 58:286-291.

Rouquier, S., Trask, B.J., Taviaux, S., van den Engh, G., Diriong, S., Lennon, G.G., Giorgi, D. (1 995) Direct selection of cDNAs using whole chromosomes. Nucl. Acids Res. 23: 441 5- 4420.

Yokota, H., van den Engh, G., Hearst, J.E., Sachs, R.K., Trask, B.J. (1995) Evidence for the organization of chromatin in Megabase pair-sized loops arranged along a random-walk path in the human GO/Gl interphase nucleus. J. Cell Biology 130: 1239-1 249.

Asbury, C.L., Esposito, R., Farmer, C., van den Engh, G. (1996) Fluorescence Spectra of DNA Dyes Measured in a Flow Cytometer. Cytometry, 24:234-242.

Mariella, R., van den Engh, G., Masquelier, D., Eveleth, G. (1996) Flow-stream waveguide for the collection of perpendicular light scatter in flow cytometry. Cytometry, 24:27-31.

Trask, B.J., Mefford, H., van den Engh, G., Massa, H.F., Juyal, R.C., Finucane, B., Abuelo, D., Witt, D.R., Magenis, E., Baldini, A., Greenberg, F., Lupski, J.R., Patel, P.I. (1996) Quantification by flow cytometry of chromosome-17 deletions in Smith-Magenis syndrome

. patients. Human Genetics 98:710-718.

Chong, S., Almqvist, E., Telenius, H., LaTray, L., Nichols, K., Bourdelat-Parks, B., Goldberg, Y.P., Richards, F., Silence, D., Greenberg, C., Ives, E., Van den Engh, G., Hughes, M., Hayden, M.R. (1997) Contribution of DNA sequence and CAG size to mutational frequencies of intermediate alleles for HD: evidence from single sperm analyses. Human Molecular Genetics, 6:301-309.

Lanyi, A., Li, B., Li, S., Talmadge, C.B., Brichacek, B., Davis, J.R., Kozel, B.A., Trask, B., van den Engh, G., Stanbridge, E.J., Nelson, D.L., Chinault, C., Heslop, H., Gross, T.G., Seemayer, T.A., Klein, G., Purtilo, D.T., Sumegi, J. (1 997) A yeast artificial chromosome (YAC) contig encompassing the critical region of the X-linked lymphoproliferative disease (XLP) locus. Genomics 39: 55-65.

Mefford, H., van den Engh, G., Friedman, C., Trask B.J. (1997) Analysis of the variation in chromosome size among diverse human populations by bivariate flow karyotyping. Human Genetics (in press).

Siegel, A.F., Roach, J.C., van den Engh, G. (1997) Expectation and variance of true and false fragment matches in DNA restriction mapping.J. Comp. Bo!. (in press)

Siegel, A.F., Roach, J.C., Magness, C., Thayer, E., Hood, L., van den Engh, G. (1997) Optimization of restriction fragment DNA mapping. J. Comp. Bo/. (in press)

Yokota, H., Singer, M.J., van den Engh, G.J., Trask, B.J. (1997) Regional differences in the compaction of chromatin in human GO/GI interphase nuceli. Chromosome Research 51 57-1 66.

Liu et al. (1 997) Cell/cell interaction in prostate gene regulation and cytodifferentiation. Proc. Nail. Acad. Sci. U.S.A. (in press)

Rouquier, S., Taviaux, S., Trask, B.J., Brand-Arpon, V., van den Engh, G., Demaille, J., Giorgi, D. (1 997) Distribution of Olfactory Receptor Genes in the Human Genome. (submitted to Nature Genetics).

Trask, B.J., Friedman, C., Martin-Gallardo, A., Rowen, L., Akinbami, C., Blankenship, J., Collins, C., Giorgi, D., ladonato, S., Johnson, F., Massa, H., Morrish, T., Naylor, S., Rouquier, S., Smith T., Wong, D., Youngblom, J., van den Engh, G.J. (1997) Members of the olfactory receptor gene family are contained in large blocks of DNA duplicated polymorphically near the ends of human chromosomes. (submitted to Nature Genetics April 28, 1997).

Basiji, D.A., Esposito, R., van den Engh,G. (1 997) Measurement of fluorescence-spectra and excited-state lifetimes by phase-sensitive detection in six DNA-binding probes. (submitted).

Asbury C.L., van den Engh, G.J., Manipulation of DNA using non-uniform oscillating electric fields (submitted).

Patents

van den Engh, G.J., and Stokdijk, W., Parallel Pulse Processing and Data Acquisition for High Speed, Low Error Flow Cytometry. U.S. Patent 5,150,313, September 22, 1992.

van den Engh, G. Flow Cytometer. U.S. Patent No. 5,464,581. November 7,1995. Sasaki, D.T., van den Engh, G.J., Buckle A.M., High speed flow cytometric separation of

Mariella, R.P., van den Engh, G.J., Northrup, M.A., Aqueous carrier waveguide in a flow

van den Engh, G.J., and Esposito, R.J., Multiple Sort Flow Cytometer. U.S. Patent No.

van den Engh, G.J., Sample introduction system for a flow cytometer. U.S. Patent

van den Engh, G.J., Flow cytometer jet monitor system. U.S. Patent 5,602,039, February 11,

Flow cytometer jet monitor system. U.S. Patent 5,602,039, February 1 1, 1997. van den Engh, G.J., Esposito, RJ., System for sensing droplet formation time delay in a flow

van den Engh, G.J., High speed flow cytometer droplet formation system. U.S. Patent

van den Engh, G.J. and Weigl, B., Fluorescent reporter beads for fluid analysis. U.S. patent

van den Engh, GJ., Particle separating apparatus and method, US Patent pending

viable cells. U.S. Patent No. 5,466,572, November 14, 1995.

cytometer. U.S. Patent No. 5,475,487, December 12, 1995.

5,483,469, January 9, 1996.

5,602,349, February 1 1, 1997.

1997.

cytometer. U.S. Patent No. 5,643,796, July 1, 1997.

pending.

pending .