Expression of Recombinant Genes in Eukaryotic Systems

405 Pages·1999·10.925 MB·English

by  Glorioso J.C., Schmidt M.C., Abelson J.N. (eds.)

Preface
The ability to express recombinant proteins in eukaryotic cells has
greatly increased our ability to study protein function in a variety of settings.
Initial work in this field focused on gaining an understanding of the cis-acting
sequences necessary for mRNA expression, processing, and translation. As
the field has matured, the more sophisticated task of finely regulating gene
expression has taken center stage. Researchers want to be able to limit
expression to particular tissues and to determine the timing and level of
expression. In addition, exciting new technologies are being developed that
use small RNA molecules to regulate expression of endogenous genes. In
this volume of Methods ni Enzymology, we have brought together a number
of exciting new techniques for regulated gene expression in eukaryotic cells.
We hope that this volume will prove to be a valuable resource for
those who are looking for new methods to express recombinant proteins
in eukaryotic systems. We realize that no single volume can be complete,
and certainly there are methods for recombinant protein expression not
represented here. However, we have tried to bring together many novel
and creative methods currently being used in many top laboratories.
We thank the authors for the time and effort they devoted to describing
the current methods for recombinant protein expression in use in their labo-
ratories.
HPESOJ C. OSOIROLG
NITRAM C. TDIMHCS
iiix
l MICROARRAY-BASED EXPRESSION MONITORING 3
11 Fluorescence-Based Expression Monitoring
Using Microarrays
By ELIZABETH A. WINZELER, MARK SCHENA, and RONALD W. DAVIS
Introduction
The amount of DNA sequence available to researchers, along with the
need to access this great wealth of information experimentally, has been
increasing exponentially. Miniature nucleic acid arrays, often called "DNA
chips" or "microarrays," offer opportunities to collect much of the same
data that can be obtained with standard molecular biology hybridization
methods but in a highly parallel fashion. These microarrays contain dense
collections of nucleic acids either polymerase chain reaction (PCR) prod-
ucts or oligonucleotides that are either synthesized or deposited at fixed
spatial locations on specially prepared glass slides. When labeled DNA or
RNA samples are hybridized to the microarrays, the abundance of specific
target sequences in the sample can be estimated based on the observed
signal intensity at the physical position of the complementary probe or
probes. Pico- to femtomoles of thousands of different nucleic acid probes
can be arranged at densities of 400 to 250,000 elements/cm .2 With this
miniaturization and through the use of fluorescence the simultaneous analy-
sis of entire genomes becomes possible.
Although microarrays show great promise as tools for genotyping, map-
ping, and resequencing, 2,1 an equally important application for the microar-
ray is measuring transcript abundance. 4,3 Microarrays have been used to
simultaneously measure the mRNA expression levels for every gene in
Saccharomyces cerevisiae under several different growth conditions, v-5 to
characterize the differences between normal and metastatic tissues, 8 and
.M Chee, .R Yang, E. Hubbell, A. Berno, .X .C Huang, D. Stern, J. Winkler, D. J. Lockhart,
.M .S Morris, and .S .P Fodor, Science 274, 016 .)6991(
2 j. G. Hacia, .L .C Brody, .M .S Chee, .S .P Fodor, and .F .S Collins, Nature Gener ,41
144 .)6991(
3 .M Schena, D. Shalon, R. .W Davis, and .P O. Brown, Science 270, 764 .)5991(
4 D. J. Lockhart, H. Dong, .M .C Byrne, K. T. Follettie, .M .V Gallo, .M .S Chee, .M Mittmann,
.C Wang, .M Kobayashi, H. Horton, and E. .L Brown, Nature Biotechnol. ,41 5761 .)6991(
5 .L Wodicka, H. Dong, .M Mittmann, M.-H. Ho, and D. .J Lockhart, Nature Biotechnol. ,51
9531 .)7991(
6 j. .L DeRisi, .V R. Iyer, and .P O. Brown, Science 278, 086 .)7991(
7 .R J. Cho, .M J. Campbell, E. A. Winzeler, .L Steinmetz, A. Conway, .L Wodicka, .T G.
,grebsfloW A. E. Gabrielian, D. Landsman, D. J. Lockhart, and R. .W Davis, Mol. Cell .)8991(
8 j. DeRisi, .L Penland, .P O. Brown, .M .L Bittner, .P .S Meltzer, .M Ray, .Y Chen, .Y .A
,uS dna J. .M Trent, Nature Genet. ,41 754 .)6991(
Copyright © 1999 by Academic Press
All rights of reproduction in any form reserved.
METHODS IN ENZYMOLOGY, VOL. 306 0076-6879/99 $30.00
4 ANALYSIS OF GENE EXPRESSION 1
as a vehicle for gene discovery. °1,9 They can also be used to probe genome
content when DNA instead of RNA or cDNA is hybridized. 11,5 Global
gene expression profiles will be used in diagnostics and to look for drug
targets. 21 Data generated by array-based expression experiments will be
essential for understanding genetic regulatory networks and integrated
cellular responses.
As the availability of fluorescence-based microarray technology grows,
more applications for these tools will be discovered and new methods for
generating microarrays will be developed. The different types, their differ-
ent manufacturing processes, and different applications have been reviewed
extensively elsewhere. 51-31 This article describes the two basic types of
microarrays, protocols for fluorescently labeling messenger RNA from eu-
karyotic cells for hybridization to microarrays, and considerations involved
in experimental design and data analysis.
Nomenclature
The term DNA microarray (sometimes called cDNA microarray) will
be used to describe ordered collections of plasmid clones or DNA fragments
that have been attached to glass at densities of greater than 100 probe
elements/cm .2 Oligonucleotide microarrays will be distinguished from DNA
microarrays, not because their manufacture is necessarily different, but
because hybridization conditions are generally different. Oligonucleotide
or DNA arrays may also be fabricated on porous materials, such as nitrocel-
lulose or nylon membranes, but the spot density is generally lower. Al-
though these filter arrays can also be used to monitor expression using a
large number of probes, the target is usually radiolabeled and discussion
of such is outside the scope of this article. In addition, the word "target"
will describe the molecule whose characteristics are unknown, in this case
the messenger RNA or cDNA in solution, and the word "probe" will
describe the molecules that are affixed to the microarray. Unlike conven-
tional Southern or Northern methods, the target, not the probe, bears
the label.
9 M. Schena, D. Shalon, R. Heller, A. Chai, P. O. Brown, and R. W. Davis, Proc. Natl. Acad,
Sci. U.S.A. 93, 10614 (1996).
01 R. A. Heller, M. Schena, A. Chai, D. Shalon, T. Bedilion, J. Gilmore, D. E. Woolley, and
R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 94, 2150 (1997).
11 D. Lashkari, J. DeRisi, J. McCusker, A. Namath, C. Gentile, .S Hwang, P. Brown, and R.
Davis, Proc. Natl. Acad. Sci. U.S.A. 94, 13057 (1997).
21 L. H. Hartwell, P. Szankasi, C. J. Roberts, A. W. Murray, and .S H. Friend, Science 278,
1064 (1997).
31 A. Marshall and J. Hodgson, Nature Biotechnol. 16, 27 (1998).
41 M. Johnston, Curt. Biol. 8, R171 (1998).
51 G. Ramsay, Nature Biotechnol. 16, 40 (1998).
1 MICROARRAY-BASED EXPRESSION MONITORING 5
To construct DNA microarrays, nucleic acid probes (usually 500 to 2000
bases in size) are generated by PCR-amplifying plasmid library inserts
(using primers complementary to the vector portion of the library) or
portions of genomic DNA (using primers designed specifically for the open
reading frames or genes of interest). The PCR products are then deposited,
usually using a robotic microspotting device, at defined locations on a glass
slide. 61 The robotic spotting device is equipped with a set of tips that pick
up small amounts of PCR products (~0.2 tzl) from a microtiter plate by
capillary action and then sequentially dispense the product at specific loca-
tions on multiple slides by touching the tips to the slide surface in a way
that is equivalent to the mechanism by which ink is released from a quill
pen when the tip touches paper. When fine spotting pins are used, spots
containing as little as 1 ng of nucleic acid can be arrayed at densities of up
to 5000 elements/cm .2 Because each PCR reaction may produce a few
micrograms of probe, thousands of microarrays may be produced from a
single set of PCR reactions. Other robotic microfabrication technologies.
such as ink jetting, in which the probe solution is placed in a piezoelectric
controlled print head that can release microscopic droplets, are under devel-
opment. 7,
After the PCR products are spotted on the microarray, they are fixed
covalently to the surface and denatured. Fluorescently labeled mRNA,
cDNA, or cRNA is hybridized to the microarray. The fluorescence intensity
at each location on the slide is determined with a confocal microscope that
has been modified to scan the slide surface or with a charge-coupled device
(CCD) camera. 81 Detection by mass spectrometry or radioisotope is also
possible, but will not be considered here. The scanned image is then ana-
lyzed. An example of a scanned image of a DNA microarray is shown in
Fig. 1 (see color insert). Messenger RNA was prepared from human Jurkat
cells and hybridized to an array containing 248 human and sispodibarA
cDNAs using the procedures described here. As the amount of probe on
the array exceeds the amount of target, the observed signal at any given
position is a good estimate of the abundance of cognate target in the sample.
editoelcunogilO syarraorciM
Oligonucleotide microarrays consist of shorter (less than 50 bases) nu-
cleic acid fragments of known sequence, covalently attached to a derivatized
glass slide at defined locations. The oligonucleotides can be either prefabri-
cated and then attached using microdeposition technology (robotic printing
61 D. Shalon, .S J. Smith, and P. O. Brown, Genome Res. 6, 639 (1996).
71 A. M. Castellino, Genome Res. 7, 943 (1997).
81 M. Schena and R. Davis, in "PCR Methods Manual" (M. Innis, D. Gelfand, and J. Sninsky,
eds.), p. 445. Academic Press, San Diego, 1999.
6 ANALYSIS OF GENE EXPRESSION 1
or ink jetting) or synthesized in situ using techniques such as photolithogra-
phy or ink jetting of the individual dA, dC, dG, or dT phosphoramidite
monomersJ 7 Affymetrix (Santa Clara, CA) manufactures high-density oli-
gonucleotide microarrays using a light-directed combinatorial method. °2,91
With this method, photoreactive (versus chemically reactive) phosphor-
amidite monomers are used in oligonucleotide synthesis. A series of physical
photomasks that protect portions of the glass surface is designed based on
the sequences of the oligonucleotides that will be synthesized. By shining
a mercury light through the different photomasks no more than 001 (4 x
25) masks are need to make 25-mers, the synthesis can be controlled in a
spatially addressable fashion, allowing the production of microarrays whose
sequence diversity is limited only by the resolution of the mask (up to
400,000 different 25-mer probes in a 1.6-cm 2 area). Each element on the
array contains ~107 molecules of a particular probe sequence in a 50-/xm 2
area. One of the chief advantages of in situ methods over amplification/
microdeposition methods is that the sequences are selected from databases
using software, and no physical handling of the probes is required, reducing
the probability of probe cross-contamination or mix-up significantly.
As with DNA microarrays, the mRNA (or cDNA) is labeled and hybrid-
ized to the microarray. Hybridization occurs at lower temperatures, and
protocols usually call for the fragmentation of the labeled material before
hybridization to reduce the amount of secondary structure that forms at
the lower temperatures. The microarrays are washed and scanned, and
hybridization is detected by fluorescence.
Analysis of Gene Expression Using Microarrays
Preparation of Microarrays
Microarrays designed to use fluorescence detection and containing
probe sequences from different organisms can be purchased from suppliers
such as Affymetrix (Santa Clara, CA) or Synteni (Fremont, CA) and many
new producers are entering the market. The manufacture of certain types
of microarrays may be technically inaccessible to most laboratories. How-
ever, if a robotic spotting device is available, DNA microarrays can be
fabricated in the laboratory. These instruments can be built using commer-
cially available parts (see http://cmgm.stanford.edu/pbrown/) or can be pur-
chased from companies such as Molecular Dynamics (Sunnyvale, CA) or
91 S. P. A. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, and D. Solas, Science 251,
767 (1991).
02 A. C. Pease, D. Solas, E. J. Sullivan, M. T. Cronin, C. P. Holmes, and S. P. Fodor, Proc.
Natl. Acad. Sci. U.S.A. 91, 5022 (1994).
FIo. .1 Scanned image of a gene expression microarray. A fluorescent scan of a microarray
prepared by mechanical microspotting is shown. A total of 240 human blood cDNAs and 8
sispodibarA controls were amplified by PCR, purified, and arrayed on silylated microscope
slides in duplicate at 200-t~m spacing. The 496 element microarray was then hybridized with
a fluorescent probe prepared by enzymatic incorporation of Cy3-dCTP into cDNA by single-
round reverse transcription of poly(A) + mRNA from cultured Jurkat cells, essentially as
described in Table III. The microarray was scanned at 10-~m resolution with a confocal laser
detection system. Fluorescent data are depicted in a rainbow pseudocolor palette for ease of
visualization. Elements complementary to abundant messages are red, whereas black/dark
blue elements correspond to genes having low or undetectable transcript levels.
1 MICROARRAY-BASED EXPRESSION MONITORING 7
Telechem International (Sunnyvale, CA). Generating arrays with a spotting
device, although repetitive, is not prohibitively difficult (Tables I and II).
An example of a scanned image of an array generated with a robotic
spotting device is shown in Fig. .1 Hundreds or thousands of PCR reactions
need to be performed using either a large number of different templates
or a large number of different primers. Clones from any plasmid library
(ordered or unordered cDNA, genomic DNA, etc.) can be used as tem-
plates. In principle unamplified plasmid DNA could also be spotted, but
the presence of vector sequence present in all elements could increase back-
ground.
If the genomic DNA is to be used as template, different pairs of oligonu-
cleotide primers need to be designed and synthesized for each element
TABLE I
PCR NOITACIFILPMA FO SENOLC MORF C1MONEG DNA
RO cDNA SEIRARBIL
.1 Assemble PCR reactions in 96-well plate
Reagent Volume (/xl)
10x PCR buffer (15 mM Mg )+2 0.01
dNTP cocktail (2 mM each) 0.01
Primer 1 (100 pmol/tzl)" 0.1
Primer 2 (100 pmol//xl) 0.1
Genomic or plasmid DNA (10 ng//xl) b 0.1
H20 76.0
qaT DNA polymerase (5 U/tzl) 0.1
1~/001
.2 Amplify targets in 96- or 384-well format using 30 rounds of PCR (94 ,°
30 sec; 55 °, 30 sec; 72 °, 60 sec)
.3 Purify using PCR product purification kit and elute products with 001
1~ of 0.1× TE (pH 8.0)
.4 Dry products to completion in Speed-Vac
5. Resuspend each PCR product in 7.5 lxt 5× SSC (0.3-1.0 mg/ml DNA)
.6 Transfer to flat bottom 384-well plate (Nunc) for arraying
Suggested materials
PCR primers modified with a 5'-amino modifier C6 (Glen Research)
96-well thermal cycler (PCR system 9600-Perkin Elmer, Norwalk, CT)
96-well PCR plates (MicroAmp 96-well Perkin Elmer)
qaT DNA polymerase (Stratagene, La Jolla, CA)
PCR product purification kit (Telechem International)
Flat-bottom 384-well plates (Nunc, Naperville, IL)
" Use of generic primer pairs (-21-mers) to vector sequences allows high-
throughput processing.
h Plasmid DNA can be prepared by alkaline lysis and purified. The 96-
well REAL prep (Qiagen) facilitates rapid preparation.
8 ANALYSIS OF GENE EXPRESSION 1
TABLE II
GNIYARRAORCIM DNA SLIDE GNISSECORP
.1 Obtain silylated (free aldehyde) microscope slides (CEL Associates, Houston, TX)
2. Print cDNAs using microspotting device according to manufacturer's instructions
3. Allow printed arrays to dry overnight in slide box a
4. Soak slides twice in 0.2% (w/v) sodium dodecyl sulfate (SDS) for 2 min at room tem-
perature with vigorous agitation b
5. Soak slides twice in doubly distilled H20 for 2 min at room temperature with vigorous
agitation
6. Transfer slides into doubly distilled H20 at 95-100 ° for 2 min to allow DNA denatur-
ation
7. Allow slides to dry thoroughly at room temperature (~5 min)
8. Transfer slides into a sodium borohydride solution c for 5 min at room temperature to
reduce free aldehydes
9. Rinse slides three times in 0.2% SDS for 1 min each at room temperature
10. Rinse slides once in doubly distilled H20 for l min at room temperature
11. Submerge slides in doubly distilled H20 at 95-100 ° for 2 sec d
12. Allow the slides to air dry and store in the dark at 25 ° (stable for >1 year)
Suggested materials
Microspotting device (Telechem International, Molecular Dynamics, Cartesian)
Microscope slides (CEL Associates)
a Drying increases cross-linking efficiency. Several days or more is acceptable.
b This step removes salt and unbound DNA.
c Dissolve 1.0 g NaBH4 in 300 ml phosphate-buffered saline. Add 100 ml 100% ethanol
to reduce bubbling. Prepare just prior to use!
d Heating the slides aids greatly in the drying process.
on the microarray. The Whitehead Institute Primer program (http://www.
genome.wi.mit.edu/genome software/), which uses a nearest neighbor algo-
rithm to calculate melting temperatures, has been used successfully to
pick primers for the amplification of open reading frames from the yeast
genome. 12L1 Using this program, greater than %49 of PCR reactions gener-
ated useable products, 11 as detected by analyzing a small amount of product
on a gel. Ideally, primer pairs should be chosen so that they have similar
melting temperatures and so that the resulting PCR products are of similar
sizes. When possible, primers should be synthesized in the same format as
used for the PCR reactions (microtiter plates, either 96 or 384 well). Com-
puter scripts can be written that automate the process of primer selection
for groups of sequences or for an entire genome.
The success of PCR reactions can be checked by analyzing the products
on an agarose gel or by examining the microarray elements once they
have been spotted: fluorescein-labeled dNTPs can be included in the PCR
12 W. Rychlik, W. J. Spencer, and R. E. Rhoads, Nucleic Acids Res. 18, 6409 (1990).
1 MICROARRAY-BASED NOISSERPXE GNIROTINOM 9
reactions and the array can be scanned for fluorescein to determine the
reaction success. PCR reactions that fail are noted and repeated, although
failures are generally arrayed along with successes in order to ease handling
and documentation (if space is not an issue). It is generally not necessary
to quantitate the amount of product if two-color hybridization (described
later) will be performed. In addition, it is assumed that even with inefficient
reactions, the amount of product will exceed the amount of target. PCR
reactions are spotted onto glass that is derivatized chemically by treatment
with reactive aldehydes (Table II) or polycations, such as polylysine. 6
Preparation of Polyadenylated mRNA
Small differences in the environmental conditions to which a cell is
exposed can have a profound impact on the global pattern of transcription. 5
If two growth conditions are to be compared, extreme caution should be
taken to ensure that the cells from which the mRNA is isolated are treated
equivalently. For example, cells should ideally be grown in the same batch
of media, in tissue culture-treated plasticware, and should be harvested at
similar densities if possible. Various methods can be used to rapidly isolate
total RNA from cells, depending on the organism. Following isolation, the
polyadenylated RNA is usually purified from total RNA on oligo(dT) resin.
The amount of polyadenylated RNA needed will depend on the particular
microarray, the labeling method, and the size of the hybridization chamber,
but is generally between 0.5 and 10/zg. With 10/zg of labeled material,
transcripts that are present at one copy per mammalian cell or one copy
per every 20 yeast cells can be detected. If mRNA quantities are limited
(0.5 tzg or less), the mRNA sample can be amplified by an in vitro transcrip-
tion step as described later.
Two-Color Analysis
Because microarrays have typically been manufactured using relatively
simple instruments, ensuring that identical amounts of DNA are deposited
at different locations has been difficult. This theoretical limitation can be
overcome by using a two-color labeling strategy. 61 Here target derived from
mRNA from one condition is labeled with one fluor, whereas target from
a different condition is labeled with a second fluor. Similar amounts of
labeled material (usually eDNA) from the two samples are cohybridized
to the microarray and the fluorescence intensity at the two appropriate
emission wavelengths is determined. A good estimate of the relative differ-
ences in abundance of a target in the two samples can be obtained by
comparing the ratio of the fluorescence intensities at the two wavelengths.
By always using the same reference sample, microarrays produced using
10 ANALYSIS OF GENE EXPRESSION 1
different sets of PCR products and by different individuals can be compared.
Two-color strategies have been employed for mutation screening with oligo-
nucleotide microarrays 2 and could theoretically be used in expression analy-
sis, reducing the number of hybridizations that would need to be performed.
Array ngiseD
The design of arrays for gene expression experiments should include
appropriate controls for signal linearity and specificity. Probes to targets
whose abundance is well characterized and probes to genes from different
organisms should be included on the microarray, for example, human
probes on an Arabidopsis microarray or bacterial genes on a yeast microar-
ray. Target for these control probes can be generated by cloning the probe
into a vector containing a phage promoter, and a poly(A) tract, and then
generating polyadenylated mRNA by runoff ni vitro transcription using
the phage polymerase. This target can be added at various concentrations
or at various times during the mRNA isolation and hybridization. Probes
to ribosomal genes and to nontranscribed regions of the genome may also
be included. In addition, controls that allow different scans to be normalized
(as described later) should be arrayed. These may include multiple probes
to genes whose expression is not expected to vary in the experiment (in
yeast, probes complementary to the genes encoding actin and the TATA-
binding protein have been used) or spots of total genomic DNA (for mi-
croarray experiments)) 1
Direct Labeling of regnesseM RNA for noitazidirbyH
ot editoelcunogilO syarraorciM
One of the simplest methods for generating target is to label mRNA
directly. At least two different methods have been reported: the mRNA
is first fragmented to 30-50 base sizes by precipitating the RNA, resus-
pending the sample in a buffer containing magnesium ions 40 mM Tris-
acetate (pH 8.1), 001 mM potassium acetate, 30 mM magnesium acetate
and then heating the sample to 94 ° for 35 min. The RNA fragments are
then kinased by diluting the fragmentation reaction 2-fold and adding ATP
to 8/xM, dithiothreitol (DTr, to 3 mM), bovine serum albumin (BSA, 01
tzg/ml), and polynucleotide kinase. The reaction is incubated for 2 hr at
37 .° Then a biotinylated oligoribonucleotide (5'-biotin-AAAAAA 3') is
ligated directly to fragmented, kinased mRNA using T4 RNA ligase in a
buffer 50 mM Tris-HCl (pH 7.6), 01 mM DTT, and 1 mM ATP containing
a 10-fold molar excess of 5'-biotin-AAAAAA 3'. 4
Heat-denatured mRNA (or total RNA) can also be incubated with a
biotinylated psoralen derivative (35 tzM, from Schleicher and Schuell,