Menashe Marcus (February 20, 1938-January 2, 1987) This volume is dedicated to the memory of Menashe Marcus, a major contributor to the concept and substance of this book, who died on Jan- uary 2, 1987 at the age of 48. Menashe was a scientific colleague, collabora- tor, and friend to many of the coauthors of this work. All who knew him were enriched by his kindness, generosity, wonderful sense of humor, and intellectual honesty. His professional life was spent at the Hebrew Univer- sity in Jerusalem. He was dedicated to the advancement of biological research in Israel through his own work, his efforts to introduce precise scientific terminology into modem Hebrew, and through his many success- ful and devoted students. He maintained strong professional and personal ties with the scientific community in the United States, and did his post- doctoral work at the Massachusetts Institute of Technology, with sabbati- cal appointments at Columbia University College of Physicians and Sur- geons, New York University School of Medicine, and the National Institutes of Health. His enthusiasm and vigorous support for the idea that xiii xiv the seeds sown in phage and bacterial genetics would bear fruit in the study of mammalian cells in culture has been borne out by the exciting develop- ments of recent years. Guided by this precept, he pioneered techniques for the isolation and analysis of cell cycle mutants of mammalian cells. His scientific colleagues and friends join his wife Nima and his daughter Nufar in mourning his premature death. He leaves a legacy of scientific achieve- ment which will be long remembered. MICHAEL M. GOTTESMAN Preface The use of the tools of molecular biology to isolate, identify, and map a mutant gene, thereby defining an important process in cellular metabo- lism, is no longer the sole province of the microbiologist. The recent amalgamation of classical somatic cell genetics with recombinant DNA and gene transfer technology has resulted in new approaches especially useful for the study of mutant cells. This volume illustrates how special techniques in molecular biology can be applied to the study of mutant somatic cells in culture. Basic protocols for the manipulation of recombi- nant DNA can be found in other Methods in Enzymology volumes: Re- combinant DNA, Parts A-F, Volumes 68, 100, 101,153, 154, and 155. The book is divided into five sections representing the chronological and conceptual development of molecular cell genetics. The first section describes the origins and use of several important tissue culture systems developed for the genetic analysis of both undifferentiated and differen- tiated cells. For additional discussion of cultured cell systems, the reader is referred to Cell Culture, Volume 58 of this series. The second section presents methodology useful for the isolation of mutant mammalian cells. The third section details new procedures for the mapping of mammalian genes defined either by somatic cell mutations or cloned DNA fragments. The fourth section describes novel techniques for the isolation of mutant genes, and the final section presents new approaches to the study of gene expression in cultured mammalian cells. I would like to thank William Jakoby for suggesting this project to me, Nathan Kaplan for his enthusiastic endorsement, Ira Pastan for continued support and encouragement, and my wife, Susan, and children, Daniel and Rebecca, for their forbearance. Special thanks are due to Robert Fleisch- mann for critical comments on some of the manuscripts, to Joyce Sharrar for excellent secretarial help, to my other colleagues in the Laboratory of Molecular Biology in the National Cancer Institute who provided a sound- ing board for ideas, and to the many contributors to this volume for their timely and clearly presented contributions. MICHAEL M. GOTTESMAN XV Contributors to Volume 151 Article numbers are in parentheses following the names of contributors. Affaliafions listed ate current. SHIN-1CHI AKIYAMA (4), Department of Cancer Chemotherapy, Institute of Cancer Research, Faculty of Medicine, Kago- shima University, 1208-1 Usuki-cho, Ka- goshima 890, Japan KEVIN ALBRIGHT (19), Experimental Pa- thology Group, Los Alamos National Lab- oratory, Los Alamos, New Mexico 87545 MARTY BARTHOLDI (19), Experimental Pa- thology Group, Los Alamos National Lab- oratory, Los Alamos, New Mexico 87545 DAVID B. BROWN (26), Department of Biol- ogy, Yale University, New Haven, Con- necticut 06511 PETER C. BROWN (7), Department of Biolog- ical Sciences, Stanford University, Stan- ford, California 94305 BARRY D. BRUCE (22), Howard Hughes Medical Institute, University of California, San Francisco, California 94143 SUSAN BUHL (5), Department of Cell Biol- ogy, Albert Einstein College of Medicine, Bronx, New York 10461 EVELYN CAMPBELL (19), Experimental Pa- thology Group, Los Alamos National Lab- oratory, Los Alamos, New Mexico 87545 CHARLES R. CANTOR (35), Department of Human Genetics and Development, Co- lumbia University, New York, New York 10032 ADELAIDE M. CAROTHERS (34), Institute of Cancer Research, Columbia University, New York, New York 10032 C. THOMAS CASKEY (38), Institute for Mo- lecular Genetics, Department of Medicine, Biochemistry and Cell Biology, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030 LAWRENCE A. CHASIN (34), Department of Biological Sciences, Columbia University, New York, New York 10027 ix DOUGLAS CHRITTON (19), Department of Surgery, Immunology Center, Loma Linda Medical Center, Loma Linda, Cali- fornia 92354 PHILIP COFHNO (2), Departments of Medi- cine and Microbiology and Immunology, University of California, San Francisco, San Francisco, California 94143 FRANCIS S. COLLINS (35), Departments of Internal Medicine and Human Genetics and the Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109 L. SCOTT CRAM (19), Experimental Pathol- ogy Group, Los Alamos National Labora- tory, Los Alamos, New Mexico 87545 G. J. DARLINGTON (3), Department of Pa- thology, Baylor College of Medicine, Houston, Texas 77030 LARRY L. DEAVEN (19), Experimental Pa- thology Group, Los Alamos National Lab- oratory, Los Alamos, New Mexico 87545 JAN-ERIK EDSTROM (37), Department of Ge- netics. University of Lund, S-22362 Lund, Sweden DAVID J. P. FITZGERALD (12), Laboratory of Molecular Biology, National Cancer In- stitute, National Institutes of Health, Be- thesda, Maryland 20892 ROBERT FLEISCHMANN (29), Laboratory of Molecular Biology, National Cancer Insti- tute, National Institutes of Health, Be- thesda, Maryland 20892 C. MICHAEL FORDIS (27), Laboratory of Mo- lecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 ARLETTE FRANCHI (11), Centre de Biochi- mie du CNRS, FacultO des Sciences, Uni- versitd de Nice, Parc Valrose, 06034 Nice, France x CONTRIBUTORS TO VOLUME 151 DEBORAH FRENCH (5), Department of Cell Biology, Albert Einstein College of Medi- cine, Bronx, New York 10461 GEORGE A. GAITANARIS (28), Institute of Cancer Research, College of Physicians and Surgeons, Columbia University, New York, New York 10032 SUSANNAH GAL (8), Laboratory of Molecu- lar Biology, National Cancer Institute, Na- tional Institutes of Health, Bethesda, Maryland 20892 GERALD A. GILLESPIE (35), Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510 STEPHEN P. GOFF (36), Department of Bio- chemistry and Molecular Biophysics, Co- lumbia University College of Physicians and Surgeons, New York, New York 10032 MAX E. GOTTESMAN (28), Institute of Cancer Research, Columbia University College of Physicians and Surgeons, New York, New York 10032 MICHAEL M. GOTTESMAN (1, 9, 24), Labo- ratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 MARY E. HARPER (40), Gen-Probe, San Diego, California 92121 JOSEPH HIRSCHBERG (13), Department of Genetics, Hebrew University of Jerusalem, Jerusalem 91904, Israel BRUCE H. HOWARD (27, 28, 29), Laboratory of Molecular Biology, National Cancer In- stitute, National Institutes of Health, Be- thesda, Maryland 20892 HEDWIG JAKOB (6), Unit~ de G~n~tique Cel- lulaire du Coll~ge de France et de I'Institut Pasteur, 75724 Paris, Cedex 15, France ROLF KAISER (37), Department of Radiation Biology, University of Bonn, 1)-5300 Bonn, Federal Republic of Germany MICHAEL E. KAMARCK (14), Department of Exploratory Research, Molecular Thera- peutics Inc., West Haven, Connecticut 06516 THERESA KELLY (5), Department of Cell Bi- ology, Albert Einstein College of Medicine, Bronx, New York 10461 YuN-FAI LAU (31), Howard Hughes Medi- cal Institute, and Departments of Physiol- ogy and Medicine, University of Califor- nia, San Francisco, California 94143 SIMON K. LAWRANCE (35), Scripps Clinic and Research Foundation, La Jolla, Cali- fornia 92037 ROGER V. LEBO (22), Department of Ob- stetrics, Gynecology, and Reproductive Sciences, and Howard Hughes Medical Institute, University of California, San Francisco, California 94143 PIN-FANG LIN (26), Pharmaceutical Re- search and Development Division, Bristol- Myers Company, Wallingford, Connecti- cut 06492 MARY LUEDEMANN (19), Experimental Pa- thology Group, Los Alamos National Lab- oratory, Los Alamos, New Mexico 87545 MENASHE MARCUS l (13), Department of Ge- netics, Hebrew University of Jerusalem, Jerusalem 91904, Israel LISA M. MARSELLE (40), Department of Anatomy, University of Massachusetts Medical School, Worcester, Massachusetts 01605 MARY McCoRMICK (28, 29, 33), Laboratory of Molecular Virology, National Cancer Institute, National Institutes of Health, Be- thesda, Maryland 20892 JOHN R. McGILL (21), Department of Ob- stetrics and Gynecology, The University of Texas Health Science Center, San An- tonio, Texas 78284 JULIE MEYNE (19), Experimental Pathology Group, Los Alamos National Laboratory, Los Alamo& New Mexico 87545 PAT MURPHY (26), Department of Human Genetics, Yale University, New Haven, Connecticut 06510 SUSAN L. NAYLOR (21), Department of Cel- lular and Structural Biology, The Univer. i Deceased. CONTRIBUTORS TO VOLUME 15 1 xi sity of Texas Health Science Center, San Antonio, Texas 78284 JEAN-FRANtTOIS NICOLAS (6), Unit~ de Gbn- btique Cellulaire du Colldge de France et de l'Institut Pasteur, 75724 Paris, Cedex 15, France HIROTO OKAYAMA (32), Laboratory of Cell Biology, National Institute of Mental Health, National Institutes of Health, Be- thesda, Maryland 20892 DAVID PATTERSON (10), Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado 80262 JACQUES POUYSSf~GUR (11), Centre de Bio- chimie du CNRS, Facult~ des Sciences, Universit~ de Nice, Parc Valrose, 06034 Nice, France DAN ROHME (37), Department of Genetics, University of Lurid, S-22362 Lund, Sweden IGOR B. RONINSON (25), Center for Genetics, University of Illinois College of Medicine, Chicago, Illinois 60612 DAVID S. RODS (7), Department of Biologi- cal Sciences, Stanford University, Stan- ford, California 94305 FRANK H. RUDDLE (26), Department of Bi- ology, Yale University, New Haven, Con- necticut 06511 PAUL J. SAXON (23), Department of Microbi- ology and Molecular Genetics, University of California, Irvine, California 92717 MATTHEW D. SCHARFF (5), Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 ROBERT T. SCHIMKE (7), Department of Bio- logical Sciences, Stanford University, Stanford, California 94305 JERRY W. SHAY (17), Department of Cell Biology, University of Texas Health Sciences Center at Dallas, Dallas, Texas 75235 MICHAEL J. SICILIANO (15), Department of Genetics, The University of Texas M. D. Anderson Hospital and Tumor Institute, Texas Medical Center, Houston, Texas 77030 CASSANDRA L. SMITH (35), Departments of Microbiology and Psychiatry, Columbia University, New York, New York 10032 GILBERT H. SMITH (39), Laboratory of Tumor Immunology and Biology, Na- tional Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 ERIC J. STANBRIDGE (23), Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92717 J. TIMOTHY STOUT (38), Institute for Molec- ular Genetics, Department of Medicine, Biochemistry and Cell Biology, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030 FLOYD H. THOMPSON (20), Arizona Cancer Center, University of Arizona, Tucson, Ar- izona 85724 JEFFREY M. TRENT (20), Arizona Cancer Center, University of Arizona, Tucson, Ar- izona 85 724 BRUCE R. TROE~ (30), Laboratory of Molec- ular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 GAIL URLAUB (34), Department of Biologi- cal Sciences, Columbia University, New York, New York 10027 HOWARD B. URNOVlTZ (16), Medical Re- search Institute, San Francisco, California 94115 ALEXANDER VARSHAVSKY (41), Depart- ment of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 CHARLES A. WALDREN (10), Department of Radiology, University of Colorado Health Sciences Center, Denver, Colorado 80262 SHERMAN M. WEISSMAN (35), Departments of Human Genetics, Medicine, and Molec- ular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510 THEODOOR VAN DAALEN WETTERS (2), De- partment of Microbiology and Immunol- xii CONTRIBUTORS TO VOLUME 151 ogy, University of California, San Fran- cisco, California 94143 BILLIE F. WHITE (15), Department of Ge- netics, The University of Texas M. D. An- derson Hospital and Tumor Institute, Texas Medical Center, Houston, Texas 77030 WOODRING E. WRIGHT (18), Department of Cell Biology, The University of Texas Southwestern Medical School, Dallas, Texas 75235 MASARU YAMAIZUMI (26), Research Insti- tute for Microbial Diseases, Osaka Univer- sity, Osaka, Japan BERNHARD U. ZAaEL (21), Department of Pediatrics, University of Mainz, Mainz D-6500, Federal Republic of Germany ULRICH ZXMMERMAIVN (16), Institute for Biotechnology, University of Wflrzburg, ROntgenring 11, 8700 W~rzburg, Federal Republic of Germany [ 1] CHINESE HAMSTER OVARY CELLS 3 [1 ] Chinese Hamster Ovary Cells By MICHAEL M. GOTTESMAN Chinese hamster ovary (CHO) cells have been extensively used for genetic analysis in tissue culture since the pioneering work of Puck, who first isolated this cell line.~ These cells have been used for the isolation of mutants affecting intermediary metabolism; DNA, RNA, and protein syn- thesis; membrane functions; and several more complex forms of cell be- havior such as cell growth and endocytosis. A recent compilation of CHO mutants lists more than 80 classes of mutants isolated using this cell line. 2 There are many reasons for the successful use of CHO cells in somatic cell genetics among which are (1) ease of growth with a doubling time of 12 hr and cloning efficiency in excess of 80%3; (2) simple karyotype with 21 large, easily recognized chromosomes4; (3) apparently high frequency of mutant phenotypes based on the "functional hemizygosity" of some of the CHO genome 5 as well as a high frequency of "segregation-like" events which unmask otherwise recessive mutations6; and (4) the ease with which CHO cells can be transfected with DNA. 7 Although these characteristics make CHO cells useful for the isolation of mutants affecting general cell functions, this line is not suitable for an analysis of most differentiated functions. There are two other disadvantages of these cells which should be borne in mind; namely, they are not derived from a fully inbred Chinese hamster line and hence mutant cell lines cannot be reintroduced back into the animal of origin, and they are not susceptible to infection by standard retroviruses which might be used as DNA vectors (see chapter by Goff[36]; this volume). T. T. Puck, S. J. Ciecuira, andA. Robinson, J. Exp. Med. 108, 945 (1985) 2 M. M. Gottesman, in "Molecular Cell Genetics" (M. M. Gottcsman, ed.), p. 887. Wiley, New York, 1985. 3 M. M. Gottesman, in "Molecular Cell Genetics" (M. M. Gottesrnan, ed.), p. 139. Wiley, New York, 1985. 4 M. J. Siciliano, R. L. Stallings, and G. M. Adair, in "Molecular Cell Genetics" (M. M. Gottesman, ed.), p. 95. Wiley, New York, 1985. 5 L. Siminovitch, Cell7, 1 (1976). 6 R. G. Worton and S. G. Grant, in "Molecular Cell Genetics" (M. M. Gottesman, ed.), p. 831. Wiley, New York, 1985. 7 I. Abraham, J. S. Tyagi, and M. M. Gottesman, Somatic Cell Genet. 8, 23 (1982). Copyright © 1987 by A~demic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 151 All rights ofreDroduction in any form r'-~erved. 4 CELL LINES FOR GENETIC ANALYSIS [ 1 ] History of CHO Lines Hu and Watson introduced the Chinese hamster into the United States as a laboratory animal in 1948. 8 They were first bred seriously by Yergan- ian starting in 1951. In 1957, one of his partially inbred female hamsters was given to Puck, who established a fibroblastic cell line from the ovary of this animal.~ The cell line was originally slightly aneuploid, 9 having either 23 or 21 chromosomes instead of the 11 pairs found in the Chinese hamster, and grew vigorously. One subline of the original isolate, called CHO-K1 (ATCC CCL 61) was maintained in Denver by Puck and Kao, whereas another subline was sent to Tobey at Los Alamos. This latter line was adapted to suspension growth by Thompson at the University of Toronto (CHO-S) in 1971 and has given rise to a number of Toronto subclones with similar properties including the line CHO Pro -5 used exten- sively by Siminovitch and numerous colleagues in Toronto, CHO GAT- of McBurney and Whitmore, subline 10001 of Gottesman at the NIH, and subline AA8 of Thompson. There are some differences in the karyotypes of the CHO-K1 and CHO-S cell lines, and CHO-S grows well in spinner and suspension culture, whereas CHO-K 1 does not. Both sublines seem to give rise readily to mutant phenotypes. The methodologies described in this chapter were developed for work with the CHO-S sublines, but most of the methods, with the exception of suspension culture, can be used for CHO-K1 cell lines. Growth of CHO Cells CHO cells are proline auxotrophs, unlike most other cultured cell lines, and require medium containing this nutrient, such as Ham's F l2 (for formulation, see Puck 9) or a-modified Eagle's medium (a-MEM) without ribonucleoside or deoxyribonucleosides (for formulation, see Gottesman3), both of which are commercially available. These rich media must in addition contain other limiting nutrients for CHO cells, since they support more rapid growth of CHO lines than is possible in MEM alone supple- mented with proline. We routinely use a-MEM supplemented with I0% fetal bovine serum (calf serum will work but will not support suspension growth) with penicillin (50 units/ml) and streptomycin (50 gg/ml). Fetal bovine sera must be prescreened and should support clonal growth of CHO S G. Yerganian, in"MolecularCellGenefics" (M.M. GoResman, ed.), p. 3. Wiley, New York, 1985. 9 T. T. Puck, in"MolecularCeUGenetics"(M.M. GoResrnan, ed.),p. 37. Wfley, NewYork, 1985. [1] CHINESE HAMSTER OVARY CELLS 5 cells at a concentration of 0.5%. Our usual protocol for screening sera involves the following tests: 1. Determine the cloning efficiency of CHO cells in different serum concentrations. Hate 200 CHO cells in medium containing 10, 5, 2, 1, 0.5, and 0.2% serum. After 7-10 days clones should be visible at all serum concentrations with the possible exception of 0.2%. The clones can be more easily visualized by staining with 0.5% methylene blue in 50% eth- anol. 2. Determine the doubling time of CHO cells in 10% serum. Hate 2 × l04 cells in eight 35-mm dishes or in eight individual wells of a 24-well multiwell dish (CoStar). After 16 hr, remove medium and add l ml of 0.25% trypsin, 0.2 M EDTA in PBS or Tris-dextrose buffer (TD buffer is NaC1, 8 g/liter; KC1, 0.38 g/liter; Na2HPO4, 0. l g/liter; Tris-HC1, 3 g/liter; and dextrose, 1.0 g/liter adjusted to pH 7.4 with HC1). Incubate at 37 ° for 30 min and add the suspended cells to 9 ml isotonic cell counting medium for counting. Repeat the trypsinization and cell counts every 24 hr for 3 more days at which time the cell monolayers should be confluent. CHO cells should double every 12 hr. Failure to double at this rate suggests a problem with medium, serum, growth conditions (see below), or infection with a microorganism such as mycoplasma. 3. Test fetal bovine serum for ability to support growth in suspension (see below). Only one of three random fetal bovine serum samples will support optimal cell growth in suspension. Poor sera will result in clump- ing of cells. 4. Confirm that the appearance of the cells growing in the lot of serum being tested is the same as their appearance in other serum lots. The cells should be fibroblastic and nonvacuolated. Membrane ruffling and bleb- bing is quite common, especially after the cells are initially plated. 5. Confirm that cells growing in the lot of serum being tested have the same biochemical and genetic phenotypes as in previous lots of serum used in the laboratory. If extensive gene transfer studies are anticipated, serum lots should be tested for ability to support DNA mediated gene transfer at good frequency (see chapter by Fordis and Howard [27], this volume). CHO cells grow optimally at 37 °3 and prefer a slightly alkaline pH (optimum pH is 7.4-7.8). 3 In bicarbonate-buffered medium such as ~-MEM, CO2 concentration should be approximately 5%. If higher CO2 concentrations are used, as would be the case when CHO cells are culti- vated in the same incubator with cells growing in MEM, the medium will be too acid and cells will not grow optimally. CHO cells are transformed and will overgrow at high cell density and die. For this reason, it is essential to split cells every few days. For main- [...]... derivatives of H4II EC3 Additional properties of the cells have been described by Weiss and coworkers and a number of these are also included in the table A large number of studies have been carried out on cell hybrids which employed derivative sublines of the H4II EC3 cells Some combinations include the fusion of rat hepatoma cells with mouse L cells 27 and mouse hepatoma cells? ° The analysis of differentiated... addition of DNA, wash cells with serum-free medium, drain, and immediately add 2.4 ml of the DNA-DEAE-dextran mixture/60 mm petri dish Incubate DNA and cells for 4 - 6 hr After incubation, aspirate DNA solution and shock cells with addition of 2 ml of 10% DMSO solution/petri dish Higher concentrations of DMSO may be tolerated by some cells Remove DMSO after 2 min; timing is critical Immediately wash cells. .. against losing cells that have been defrosted is to freeze them in 10% glycerol Although survival of cells frozen in glycerol is not as good as that of cells frozen in DMSO, cells frozen and defrosted in glycerol will survive at room temperature for several hours 3 Defrost cells by rapid immersion in a 37 ° water bath and, as soon as the last trace of ice is gone, dilution into a 20-fold excess of complete... [2] $49 MOUSET LYMPHOMACELLS 15 of the response and appropriate modification of the timing of these manipulations Mutagenesis Mutagenesis of $49 cells has generally been used by us to increase the frequency and predetermine the nature of mutations in selectable genes In addition, we have described a mutagen screening system utilizing $49 cells that distinguishes general classes of mutagenic mechanisms,... we split cells 1/50 to 1/100 every 3 - 4 days If dense monolayer cultures of CHO cells are needed for biochemical analysis (i.e., DNA or RNA extraction, or preparation of extracts for enzymatic analysis), 5 × 105 cells should be plated 72 hr prior to harvesting in a 100-mm tissue culture dish containing 15 ml medium or 1 × 106 cells 48 hr prior to harvesting For large quantities of cells CHO cells can... ethanol The frequency of ouabain-resistant mutants should increase by a factor of 10- to 100-fold after EMS treatment Storage of CHO Cells CHO cells are quite hardy and will survive most standard storage procedures We use the following protocol for routine freezing: 1 Prepare a dense monolayer culture of cells (5 X 106/100 m m dish) Trypsinize and suspend at a density of 1 × 106 cells/ ml in ice-cold... digestion of the liver with subsequent plating of the cells in the presence of hydrocortisone hemisuccinate in Hams F12 medium supplemented with 10% fetal bovine serum After cloning, the RL-PR-C line was established At the time of its description in 1980, the cells had undergone 326 population doublings and the karyotype of this strain showed a modal number of 42 which coincides with that of the normal... cells/ ml in 72 hr Mutagenesis of CHO Cells For most selections, it is necessary to mutagenize cells to get a reasonable frequency of mutants Because of its relative stability and ease of handling, we generally use cthylmcthane sulfonate (EMS) as a mutagen The following protocol should yield 10- to 100-fold increases in mutation rate: 1 Plate 5 × 105 CHO cells in each of three T-75 tissue culture flasks... frequencies of 10-30% Such survival frequencies can be obtained by exposure of the cells to 0.75/zg/ml ICR-191 or 500/zg/ml EMS for 24 hr each or to 2 gg/ml MNNG for 4.5 hr We measure survivorship by plating 100-200 cells per dish in nonselective medium immediately after mutagenesis Cultured mammalian cells require a period of time after mutagen treatment to express stable phenotypic alterations For $49 cells. .. maintenance of Hepa cells in vitro Passage of the cells at a 1 : 8 dilution weekly is a convenient protocol Hepa cells are removed from the growth surface by draining the supernatant medium, rinsing twice with 0.05% trypsin, and resuspending the cells in a serum-containing solution to stop the action of the enzyme Trypsinization longer than 3 - 5 min results in membrane damage and low yield upon replating Cells . properties of growing $49 cells. (1) The ability of cAMP to arrest wild-type but not cAMP-resistant mutant cells in the Gt phase of the cell cycle. (2) The extreme sensitivity of cells to white. P. Coffino, Cell 35, 311 (1983). [2] $49 MOUSE T LYMPHOMA CELLS 15 of the response and appropriate modification of the timing of these manip- ulations. Mutagenesis Mutagenesis of $49 cells. reasons for the successful use of CHO cells in somatic cell genetics among which are (1) ease of growth with a doubling time of 12 hr and cloning efficiency in excess of 80%3; (2) simple karyotype