Molecular Genetics of Mammalian Cells

586 Pages·1987·21.405 MB·English

by  Abelson J.N. (ed.), Simon M.E. (ed.), Gottesman M.M. (ed.)

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 colleague, scientific 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
iiix
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 daughterN ufar
in mourning his premature death. He leaves a legacy of scientific achieve-
ment which will be long remembered.
LEAHCIM M. NAMSETTOG
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 ni 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.
LEAHCIM M. NAMSETTOG
XV
Contributors to Volume 151
Article numbers are in parentheses following the names of contributors.
snoifailaffA detsil ate .tnerruc
SHIN-1CHI AKIYAMA (4), Department of SALGUOD (19), CHRITTON Department of
Cancer Chemotherapy, Institute of Cancer Surgery, Immunology Center, Loma
Research, Faculty of Medicine, Kago- Linda Medical Center, Loma Linda, Cali-
shima University, 1208-1 Usuki-cho, Ka- fornia 92354
goshima 890, Japan
COFHNO (2), PHILIP Departments of Medi-
KEVIN (19), ALBRIGHT Experimental Pa- cine and Microbiology and Immunology,
thology Group, Los Alamos National Lab- University of California, San Francisco,
,yrotaro Los Alamos, New Mexico 87545 San Francisco, California 94143
YTRAM IDLOHTRAB (19), Experimental Pa- SICNARF S. SNILLOC (35), Departments of
thology Group, Los Alamos National Lab- Internal Medicine and Human Genetics
,yrotaro Los Alamos, New Mexico 87545 and the Howard Hughes Medical Institute,
DIVAD B. NWORB (26), Department of Biol- University of Michigan Medical School,
ogy, Yale University, New Haven, Con- Ann Michigan Arbor, 48109
necticut 06511 L. TTOCS (19), CRAM Experimental Pathol-
RETEP C. NWORB (7), Department of -goloiB ogy Group, Los Alamos National Labora-
ical Sciences, Stanford University, Stan- tory, Los Alamos, New Mexico 87545
ford, California 94305 G. J. DARLINGTON (3), Department of Pa-
YRRAB D. BRUCE (22), Howard Hughes thology, Baylor College of Medicine,
Medical Institute, University of ,ainrofilaC Houston, Texas 77030
San Francisco, California 94143 YRRAL L. NEVAED (19), Experimental Pa-
SUSAN BUHL (5), Department of Cell Biol- thology Group, Los Alamos National Lab-
ogy, Albert Einstein College of Medicine, ,yrotaro Los Alamos, New Mexico 87545
Bronx, New York 10461 JAN-ERIK MORTSDE (37), Department of -eG
NYLEVE LLEBPMAC (19), Experimental Pa- netics. University of Lund, S-22362 Lund,
thology Group, Los Alamos National Lab- Sweden
,yrotaro Los Alamos, New Mexico 87545 DIVAD J. P. (12), FITZGERALD Laboratory
SELRAHC R. (35), CANTOR Department of of Molecular Biology, National In- Cancer
Human Genetics and Development, -oC stitute, National Institutes of Health, Be-
lumbia University, New York, New York thesda, Maryland 20892
10032 TREBOR NNAMHCSIELF (29), Laboratory of
EDIALEDA M. SREHTORAC (34), Institute of Molecular Biology, National Insti- Cancer
Cancer Research, Columbia University, tute, National Institutes of Health, Be-
New York, New York 10032 thesda, Maryland 20892
C. SAMOHT YEKSAC (38), Institute for Mo- C. MICHAEL FORDIS (27), Laboratory of Mo-
lecular Genetics, Department of Medicine, Biology, National lecular Institute, Cancer
Biochemistry and Cell Biology, Howard National Institutes of Health, Bethesda,
Hughes Medical Institute, Baylor College Maryland 20892
of Medicine, Houston, Texas 77030 ETTELRA (11), FRANCHI Centre de Biochi-
ECNERWAL A. (34), CHASIN Department of mie du CNRS, OtlucaF des Sciences, Uni-
Biological Sciences, Columbia University, versitd de Nice, Parc 06034 Valrose, Nice,
New York, New York 10027 France
ix
x CONTRIBUTORS TO VOLUME 151
HAROBED (5), FRENCH Department of Cell ASEREHT YLLEK (5), Department of Bi- Cell
Biology, Albert Einstein College of Medi- ,ygolo Albert Einstein College of Medicine,
cine, Bronx, New York 10461 Bronx, New York 10461
EGROEG A. SIRANATIAG (28), Institute of YuN-FAI UAL (31), Howard Hughes Medi-
Cancer Research, College of Physicians cal Institute, and Departments of Physiol-
and Surgeons, Columbia University, New ogy and Medicine, University of -rofilaC
York, New York 10032 nia, San Francisco, California 94143
HANNASUS GAL (8), Laboratory of Molecu- NOMIS K. ECNARWAL (35), Scripps Clinic
Biology, lar National Institute, Cancer Na- and Research Foundation, La Jolla, Cali-
tional Institutes of Health, Bethesda, fornia 92037
Maryland 20892 ROGER V. LEBO (22), Department of Ob-
DLAREG A. EIPSELLIG (35), Department of stetrics, Gynecology, and Reproductive
Human Genetics, Yale University School Sciences, and Howard Hughes Medical
of Medicine, New Haven, Connecticut Institute, University of California, San
06510 Francisco, California 94143
NEHPETS P. GOFF (36), Department of Bio- PIN-FANG LIN (26), Pharmaceutical Re-
chemistry and Molecular Biophysics, -oC search and Development Division, Bristol-
lumbia University College of Physicians Myers Company, Wallingford, Connecti-
and Surgeons, New York, New York 10032 cut 06492
XAM E. GOTTESMAN (28), Institute of MARY NNAMEDEUL (19), Experimental Pa-
Cancer Research, Columbia University thology Group, Los Alamos National Lab-
College of Physicians and Surgeons, New ,yrotaro Los Alamos, New Mexico 87545
York, New York 10032 EHSANEM SUCRAM (13), l Department of -eG
LEAHCIM M. NAMSETTOG (1, 9, 24), Labo- netics, Hebrew University of Jerusalem,
ratory of Molecular Biology, National Jerusalem 91904, Israel
Cancer Institute, National Institutes of LISA M. ELLESRAM (40), Department of
Health, Bethesda, Maryland 20892
Anatomy, University of Massachusetts
MARY E. HARPER (40), Gen-Probe, San Medical School, Massachusetts Worcester,
Diego, California 92121 01605
HPESOJ GREBHCSRIH (13), Department of YRAM McCoRMICK (28, 29, 33), Laboratory
Genetics, Hebrew University of Jerusalem, of Molecular Virology, National Cancer
Jerusalem 91904, Israel Institute, National Institutes of Health, Be-
thesda, Maryland 20892
ECURB H. DRAWOH (27, 28, 29), Laboratory
of Molecular Biology, National Cancer In- JOHN R. McGILL (21), Department of Ob-
stetrics and Gynecology, The University of
stitute, National Institutes of Health, Be-
thesda, Maryland 20892 Texas Health Science Center, San An-
tonio, Texas 78284
GIWDEH BOKAJ (6), Unit~ de Cel- G~n~tique
MEYNE JULIE (19), Experimental Pathology
lulaire du Coll~ge de France et de I'Institut
Group, Los Alamos National Laboratory,
Pasteur, Paris, 75724 Cedex ,51 France
Los Alamo& New Mexico 87545
ROLF RESIAK (37), Department of Radiation
PAT MURPHY (26), Department of Human
Biology, University of Bonn, Bonn, 1)-5300
Genetics, Yale University, New Haven,
Republic Federal of Germany
Connecticut 06510
LEAHCIM E. KCRAMAK (14), Department of NASUS L. ROLYAN (21), Department of Cel-
Exploratory Research, Molecular Thera- lular and Structural Biology, The Univer.
peutics Inc., West Haven, Connecticut
06516 i Deceased.
CONTRIBUTORS TO VOLUME 15 1 xi
sity of Texas Health Science Center, San ARDNASSAC L. (35), SMITH stnemtrapeD of
Antonio, Texas 78284 Microbiology and Psychiatry, Columbia
SIOTtNARF-NAEJ SALOCIN (6), Unit~ de -nbG ,ytisrevinU New York, New York 10032
btique Cellulaire du Colldge de France et GILBERT H. SMITH (39), Laboratory of
de l'Institut Pasteur, 75724 Paris, Cedex Tumor Immunology and Biology, Na-
,51 France tional Institute, Cancer National Institutes
HIROTO AMAYAKO (32), Laboratory of Cell of Health, Bethesda, Maryland 20892
Biology, National Institute of Mental ERIC J. EGDIRBNATS (23), Department of
Health, National Institutes of Health, Be- Microbiology and Molecular Genetics,
thesda, Maryland 20892 University of California, Irvine, California
DAVID NOSRETTAP (10), Eleanor Roosevelt 92717
Institute for Cancer Research, Denver,
J. YHTOMIT TUOTS (38), Institute for Molec-
80262 Colorado
ular Genetics, Department of Medicine,
SEUQCAJ RUG~fSSYUOP (11), Centre de Bio- Biochemistry and Cell Biology, Howard
chimie du CNRS, Facult~ des Sciences, Hughes Medical Institute, Baylor College
Universit~ de Nice, Parc Valrose, 06034 of Medicine, Houston, Texas 77030
Nice, France
DYOLF H. NOSPMOHT (20), Arizona Cancer
DAN (37), ROHME Department of ,sciteneG Center, University of Arizona, Ar- Tucson,
University of Lurid, S-22362 Lund, izona 85724
Sweden
YERFFEJ M. TRENT (20), Arizona Cancer
B. IGOR NOSNINOR (25), Center for Genetics, Center, University of Arizona, Ar- Tucson,
University of Illinois College of Medicine, izona 85 427
Illinois Chicago, 60612
ECURB R. (30), TROE~ Laboratory of Molec-
DIVAD S. (7), RODS Department of Biologi-
ular Biology, National Cancer Institute,
cal Sciences, Stanford University, Stan-
National Institutes of Health, Bethesda,
ford, California 94305
Maryland 20892
FRANK H. (26), RUDDLE Department of Bi-
GAIL BUALRU (34), Department of Biologi-
ology, Yale University, New Haven, Con-
cal Sciences, Columbia University, New
necticut 06511
York, New York 10027
PAUL J. NOXAS (23), Department of Microbi-
DRAWOH B. URNOVlTZ (16), Medical Re-
ology and Molecular Genetics, University
Institute, search San Francisco, California
of California, Irvine, California 92717
94115
WEHTTAM D. FFRAHCS (5), Department of
Cell Biology, Albert Einstein College of REDNAXELA YKSVAHSRAV (41), Depart-
Medicine, Bronx, New York 10461 ment of Biology, Massachusetts Institute
of Technology, Cambridge, Massachusetts
TREBOR T. EKMIHCS (7), Department of Bio-
02139
logical Sciences, Stanford University,
Stanford, California 94305 SELRAHC A. NERDLAW (10), Department of
YRREJ W. SHAY (17), Department of Cell Radiology, University of Health Colorado
Sciences Denver, 80262 Colorado Center,
Biology, University of Texas Health
Sciences Center at Dallas, Dallas, Texas NAMREHS M. NAMSSIEW (35), Departments
53257 of Human Medicine, Genetics, and Molec-
LEAHCIM J. ONAILICIS (15), Department of ular Biophysics and Biochemistry, Yale
Genetics, The University of Texas M. D. University School of Medicine, New
Anderson Hospital and Tumor Institute, Haven, Connecticut 06510
Texas Medical Center, Houston, Texas ROODOEHT NAV NELAAD SRETTEW (2), De-
77030 partment of Microbiology and Immunol-
xii CONTRIBUTORS TO VOLUME 151
ogy, University of California, San Fran- URASAM IMUZIAMAY (26), Research Insti-
cisco, California 94143 tute for Microbial Diseases, Osaka Univer-
EILLIB F. WHITE (15), Department of Ge- sity, Osaka, Japan
netics, The University of Texas M. D. An- DRAHNREB U. ZAaEL (21), Department of
derson Hospital and Tumor Institute, Pediatrics, University of Mainz, Mainz
Texas Medical Center, Houston, Texas D-6500, Federal Republic of Germany
77030 ULRICH NVIAMREMMXZ (16), Institute for
GNIRDOOW E. (18), WRIGHT Department of Biotechnology, University of Wflrzburg,
Cell Biology, The University of Texas ROntgenring ,11 8700 W~rzburg, Federal
Southwestern Medical School, Dallas, Republic of Germany
Texas 75235
1] [ CHINESE HAMSTER OVARY CELLS 3
[1 ] Chinese Hamster Ovary Cells
By LEAHCIM M. NAMSETTOG
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
21 hr and cloning efficiency in excess of (2) 80%3; simple karyotype with
12 large, easily recognizcehdr omosomes4; (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 infectiobny 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).
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History of CHO Lines
Hu and Watson introduced the Chinese hamster into the United States
as a laboratory animal in 8 1948. 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 12 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
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[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 ,01 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 %01 serum. Hate
2 × l04 cells in eight 35-mm dishes or in eight individual wells of a 24-well
multiwell dish (CoStar). After 61 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 21 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
sameb iochemical 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-