U.S. patent application number 09/817003 was filed with the patent office on 2002-01-17 for arrayed transfection method and uses related thereto.
Invention is credited to Sabatini, David M..
Application Number | 20020006664 09/817003 |
Document ID | / |
Family ID | 25222147 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020006664 |
Kind Code |
A1 |
Sabatini, David M. |
January 17, 2002 |
Arrayed transfection method and uses related thereto
Abstract
An arrayed transfection method of introducing nucleic acid of
interest into cells.
Inventors: |
Sabatini, David M.;
(Cambridge, MA) |
Correspondence
Address: |
ROPES & GRAY
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
25222147 |
Appl. No.: |
09/817003 |
Filed: |
March 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60193580 |
Mar 30, 2000 |
|
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60154737 |
Sep 17, 1999 |
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Current U.S.
Class: |
435/456 ;
435/455; 435/7.21 |
Current CPC
Class: |
C12N 15/87 20130101;
C12N 15/88 20130101; C12Q 1/6806 20130101; C12Q 1/6806 20130101;
C12Q 2565/518 20130101 |
Class at
Publication: |
435/456 ;
435/455; 435/7.21 |
International
Class: |
G01N 033/567; C12N
015/86; C12N 015/85 |
Claims
What is claimed is:
1. A reverse-transfection method of introducing DNA into eukaryotic
cells comprising: (a) depositing a DNA-containing mixture onto a
surface in discrete, defined locations, wherein the DNA-containing
mixture comprises DNA to be introduced into the eukaryotic cells
and a carrier protein and allowing the DNA-containing mixture to
dry on the surface, thereby producing a surface having the
DNA-containing mixture affixed thereon in discrete, defined
locations; and (b) plating the eukaryotic cells onto the surface in
sufficient density and under appropriate conditions for entry of
DNA in the DNA-containing mixture into eukaryotic cells, whereby
DNA in the DNA-containing mixture is introduced into the eukaryotic
cells.
2. The method of claim 1, wherein the DNA to be introduced is
contained in a vector; the carrier protein is gelatin; the slide is
a glass slide or a .SIGMA. poly-L-lysine slide and the eukaryotic
cells are mammalian cells.
3. The method of claim 2, wherein the vector is a plasmid or a
viral-based vector.
4. The method of claim 2, wherein the gelatin concentration in the
DNA-containing mixture is from about 0.05% to about 0.5%
5. A method of introducing DNA of interest into eukaryotic cells,
comprising: (a) depositing a carrier-DNA mixture onto a surface in
discrete, defined locations, wherein the carrier-DNA mixture
comprises DNA of interest and a carrier protein, and allowing the
carrier-DNA mixture to dry on the surface, thereby producing a
surface bearing the carrier-DNA mixture in discrete defined
locations; (b) covering the surface bearing the carrier-DNA mixture
with an appropriate amount of a lipid-based transfection reagent
and maintaining the resulting product under conditions appropriate
for complex formation between DNA in the carrier-DNA mixture and
the transfection reagent; (c) removing transfection reagent,
thereby producing a surface bearing DNA; (d) plating the eukaryotic
cells onto the surface bearing DNA, in sufficient density and under
appropriate conditions for entry of the DNA into the eukaryotic
cells, whereby DNA of interest is introduced into the cells.
6. The method of claim 5, wherein the carrier protein is gelatin
and the surface is the surface of a slide.
7. The method of claim 6, wherein the slide is a glass slide or a
.SIGMA. poly-L-lysine slide.
8. The method of claim 7, wherein the concentration of gelatin in
the vector-DNA mixture is from about 0.05% to about 0.5%.
9. The method of claim 8, wherein the concentration of gelatin is
from about 0.1% to about 0.2%.
10. The method of claim 5, wherein the DNA of interest is in an
expression vector and eukaryotic cells that contain DNA of interest
are maintained under conditions appropriate for expression of the
DNA, whereby DNA of interest is expressed.
11. The method of claim 10, further comprising identifying
eukaryotic cells in which a protein of interest is expressed,
comprising contacting eukaryotic cells on the surface with an
antibody which binds the protein of interest and detecting binding
of the antibody, wherein binding identifies eukaryotic cells in
which the protein of interest is expressed.
12. A method of introducing DNA of interest into eukaryotic cells,
comprising: (a) depositing a gelatin-DNA mixture onto a surface in
discrete, defined locations, wherein the gelatin-DNA mixture
comprises DNA of interest and a gelatin, and allowing the
gelatin-DNA mixture to dry on the surface, thereby producing a
surface bearing the gelatin-DNA mixture in discrete defined
locations; (b) covering the surface bearing the gelatin-DNA mixture
with an appropriate amount of a lipid-based transfection reagent
and maintaining the resulting product under conditions appropriate
for complex formation between DNA in the gelatin-DNA mixture and
the transfection reagent; (c) removing transfection reagent,
thereby producing a surface bearing DNA; (d) plating the eukaryotic
cells onto the surface bearing DNA, in sufficient density and under
appropriate conditions for entry of the DNA into the eukaryotic
cells, whereby DNA of interest is introduced into the cells.
13. The method of claim 12, wherein the surface is the surface of a
slide.
14. The method of claim 13, wherein the slide is a glass slide or a
.SIGMA. poly-L-lysine slide.
15. The method of claim 14, wherein the concentration of gelatin in
the vector-DNA mixture is from about 0.05% to about 0.5%.
16. The method of claim 15, wherein the concentration of gelatin is
from about 0.1% to about 0.2%.
17. The method of claim 12, wherein the DNA of interest is in an
expression vector and eukaryotic cells that contain DNA of interest
are maintained under conditions appropriate for expression of the
DNA, whereby DNA of interest is expressed.
18. The method of claim 17, further comprising identifying
eukaryotic cells in which a protein of interest is expressed,
comprising contacting eukaryotic cells on the surface with an
antibody which binds the protein of interest and detecting binding
of the antibody, wherein binding identifies eukaryotic cells in
which the protein of interest is expressed.
19. The method of claim 4, wherein the eukaryotic cells are
mammalian cells and are plated in (b) at high density onto the
surface bearing the vector-DNA mixture.
20. A method of introducing DNA of interest into eukaryotic cells,
comprising: (a) depositing a lipid-DNA mixture onto a surface in
discrete, defined locations, wherein the lipid-DNA mixture
comprises DNA of interest; a carrier protein; a sugar; a buffer
that facilitates DNA condensation and an appropriate lipid-based
transfection reagent and allowing the lipid-DNA mixture to dry on
the surface, thereby producing a surface bearing the lipid-DNA
mixture in defined locations; (b) plating the eukaryotic cells onto
the surface bearing the lipid-DNA mixture in sufficient density and
under appropriate conditions for entry of DNA of interest into the
eukaryotic cells, whereby DNA of interest is introduced into the
cells.
21. The method of claim 20, wherein the carrier protein is gelatin
and the surface is the surface of a slide.
22. The method of claim 21, wherein the slide is a glass slide or a
.SIGMA. poly-L-lysine slide.
23. The method of claim 22, wherein the concentration of gelatin in
the lipid-DNA mixture is from about 0.01% to about 0.05% and the
concentration of sucrose is from about 0.1M to about 0.4M.
24. The method of claim 20, wherein the DNA of interest is in an
expression vector and eukaryotic cells that contain DNA of interest
are maintained under conditions appropriate for expression of the
DNA, whereby DNA of interest is expressed.
25. A method of affixing DNA to a surface, to produce an array of
DNA in discrete, defined locations of known sequence or source,
comprising spotting of carrier-DNA mixture onto the surface in
discrete, defined locations and allowing the resulting surface
bearing the carrier-DNA mixture to dry sufficiently that the spots,
referred to as DNA-containing spots, remain affixed to the surface
under conditions in which the arrays are used.
26. A method of affixing DNA to a surface, to produce an array of
DNA in discrete, defined locations of known sequence or source,
comprising spotting of gelatin-DNA mixture onto the surface in
discrete, defined locations and allowing the resulting surface
bearing the gelatin-DNA mixture to dry sufficiently that the spots,
referred to as DNA-containing spots, remain affixed to the surface
under conditions in which the arrays are used.
27. A method of affixing DNA to a surface, to produce an array of
DNA in discrete, defined locations of known sequence or source,
comprising spotting a lipid-DNA mixture onto the surface in
discrete, defined locations to produce spots and allowing the
resulting surface bearing the lipid-DNA mixture to dry sufficiently
that the spots remain affixed to the surface under conditions in
which the arrays are used.
28. A method of producing an array on a surface of reverse
transfected cells that contain defined DNA, comprising: a) spotting
a carrier-DNA mixture spotting of gelatin-DNA mixture onto the
surface in discrete, defined locations and allowing the resulting
surface bearing the carrier-DNA mixture to dry sufficiently that
the spots, referred to as DNA-containing spots, remain affixed to
the surface under conditions in which the arrays are used; b)
covering the surface bearing the DNA-containing spots with an
appropriate amount of a lipid-based transfection reagent and
maintaining the resulting product under conditions appropriate for
complex formation between DNA in the spots and the transfection
reagent; c) removing transfection reagent, producing a surface
bearing DNA; d) adding cells in an appropriate medium to the
surface bearing DNA, to produce a surface bearing DNA and plated
cells; and e) maintaining the surface bearing DNA and plated cells
under conditions that result in entry of DNA into plated cells,
thus producing an array of reverse transfected cells that contain
defined DNA.
29. A method of producing an array on a surface of reverse
transfected cells that contain defined DNA, comprising: a) spotting
a gelatin-DNA mixture spotting of gelatin-DNA mixture onto the
surface in discrete, defined locations and allowing the resulting
surface bearing the gelatin-DNA mixture to dry sufficiently that
the spots, referred to as DNA-containing spots, remain affixed to
the surface under conditions in which the arrays are used; b)
covering the surface bearing the DNA-containing spots with an
appropriate amount of a lipid-based transfection reagent and
maintaining the resulting product under conditions appropriate for
complex formation between DNA in the spots and the transfection
reagent; c) removing transfection reagent, producing a surface
bearing DNA; d) adding cells in an appropriate medium to the
surface bearing DNA, to produce a surface bearing DNA and plated
cells; and e) maintaining the surface bearing DNA and plated cells
under conditions that result in entry of DNA into plated cells,
thus producing an array of reverse transfected cells that contain
defined DNA.
30. A method of producing on a surface an array of reverse
transfected cells that contain defined DNA, comprising: a) spotting
a lipid-DNA mixture onto the surface in discrete, defined
locations, to produce spots and allowing the resulting surface
bearing the lipid-DNA mixture to dry sufficiently that the spots
remain affixed to the surface under conditions in which the arrays
are used; b) plating cells on top of the surface produced in (a)
and maintaining the resulting surface, which contains dried
lipid-DNA mixture and cells to be reverse transfected, under
conditions appropriate for growth of cells and entry of DNA into
cells, thus producing an array of reverse transfected cells.
31. An array produced by the method of claim 25.
32. An array produced by the method of claim 26.
33. An array produced by the method of claim 27.
34. An array produced by the method of claim 28.
35. An array produced by the method of claim 29.
36. An array produced by the method of claim 30.
37. A method of forming a plurality of diverse transfection vectors
on a solid support, said support comprising a surface with a
plurality of preselected regions, said method comprising: a)
forming on each of said preselected regions a carrier-DNA mixture
having a different transfection vector; b) adding cells in an
appropriate medium to the surface bearing DNA, to produce a surface
bearing DNA and plated cells; and c) maintaining the surface
bearing DNA and plated cells under conditions that result in entry
of DNA into plated cells, thus producing an array of reverse
transfected cells that contain defined DNA.
Description
RELATED APPLICATIONS
[0001] This is a continuation-in-part of U.S. Ser. No. 09/______
which was filed Sep. 18, 2000, and also claims the benefit of U.S.
application Ser. No. 60/193,580, filed Mar. 30, 2000 and U.S.
application Ser. No. 60/154,737, filed Sep. 17, 1999. The entire
teachings of each of the above-referenced applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Genome and expressed sequence tag (EST) projects are rapidly
cataloging and cloning the genes of higher organisms, including
humans. The emerging challenge is to uncover the functional roles
of the genes and to quickly identify gene products with desired
properties. The growing collection of gene sequences and cloned
cDNAs demands the development of systematic and high-throughput
approaches to characterizing the gene products. The uses of DNA
microarrays for transcriptional profiling and of yeast two-hybrid
arrays for determining protein-protein interactions are recent
examples of genomic approaches to the characterization of gene
products (Schena, M., et al., Nature, 10:623 (2000)). Comparable
strategies do not exist to analyze the function, within mammalian
cells, of large sets of genes. Currently, in vivo gene analysis can
be done--on a gene-by-gene scale--by transfecting cells with a DNA
construct that directs the overexpression of the gene product or
inhibits its expression or function. The effects on cellular
physiology of altering the level of a gene product is then detected
using a variety of functional assays.
[0003] A variety of DNA transfection methods, such as calcium
phosphate coprecipitation, electroporation and cationic
liposome-mediated transfection (e.g., lipofection) can be used to
introduce DNA into cells and are useful in studying gene regulation
and function. Additional methods, particularly high throughput
assays that can be used to screen large sets of DNAs to identify
those encoding products with properties of interest, would be
useful to have available.
SUMMARY OF THE INVENTION
[0004] The present invention provides a strategy for high
throughput analysis of gene function in cells. One aspect of the
present invention provides methods and reagents for creating
transfected cell microarrays that are suitable for rapidly
screening large sets of nucleic acid constructs for those encoding
desired products or for causing cellular phenotypes of interest is
described. For instance, a spatially defined array of nucleic
acids, such as expression vectors, is used to generate an spatially
defined array of transfected cells. The cells can be screened for
the ability of a transfected nucleic acid to confer a particular
phenotype on the cell, and by reference to the position of the
cell(s) on the array, the identity of the nucleic acid can be
determined.
[0005] Accordingly, the present invention relates to a method,
referred to as a reverse transfection method, in which a defined
nucleic acid (a nucleic acid of known sequence or source), also
referred to as a nucleic acid of interest or a nucleic acid to be
introduced into cells, is introduced into cells in defined areas of
a lawn of eukaryotic cells, in which it will be expressed or will
itself have an effect on or interact with a cellular component or
function. Any suitable nucleic acid such as an oligonucleotide, DNA
and RNA can be used in the methods of the present invention. The
particular embodiments of the invention are described in terms of
DNA. However, it is to be understood that any suitable nucleic acid
is encompassed by the present invention.
[0006] In one embodiment, the present invention relates to a method
in which defined DNA (DNA of known sequence or source), also
referred to as DNA of interest or DNA to be introduced into cells,
is introduced into cells in defined areas of a lawn of eukaryotic
cells, in which it will be expressed or will itself have an effect
on or interact with a cellular component or function. In the
method, a mixture, defined below, comprising DNA of interest (such
as cDNA or genomic DNA incorporated in an expression vector) and a
carrier protein is deposited (e.g., spotted or placed in small
defined areas) onto a surface (e.g., a slide or other flat surface,
such as the bottoms of wells in a multi-welled plate) in defined,
discrete (distinct) locations and allowed to dry, with the result
that the DNA-containing mixture is affixed to the surface in
defined discrete locations.
[0007] Such locations are referred to herein, for convenience, as
defined locations. The DNA-containing mixture can be deposited in
as many discrete locations as desired. The resulting product is a
surface bearing the DNA-containing mixture in defined discrete
locations; the identity of the DNA present in each of the discrete
locations (spots) is known/defined. Eukaryotic cells, such as
mammalian cells (e.g., human, monkey, canine, feline, bovine, or
murine cells), bacterial, insect or plant cells, are plated
(placed) onto the surface bearing the DNA-containing mixture in
sufficient density and under appropriate conditions for
introduction/entry of the DNA into the eukaryotic cells and
expression of the DNA or its interaction with cellular components.
Preferably, the eukaryotic cells (in an appropriate medium) are
plated on top of the dried DNA-containing spots at high density
(e.g., 1.times.10.sup.5/cm.sup.2), in order to increase the
likelihood that reverse transfection will occur. The DNA present in
the DNA-containing mixture affixed to the surface enters eukaryotic
cells (reverse transfection occurs) and is expressed in the
resulting reverse transfected eukaryotic cells.
[0008] In one embodiment of the method, referred to as a
"gelatin-DNA" embodiment, the DNA-containing mixture, referred to
herein as a gelatin-DNA mixture, comprises DNA (e.g., DNA in an
expression vector) and gelatin, which is present in an appropriate
solvent, such as water or double deionized water. The mixture is
spotted onto a surface, such as a slide, thus producing a surface
bearing (having affixed thereto) the gelatin-DNA mixture in defined
locations. The resulting product is allowed to dry sufficiently
that the spotted gelatin-DNA mixture is affixed to the slide and
the spots remain in the locations to which they have become
affixed, under the conditions used for subsequent steps in the
method. For example, a mixture of DNA in an expression vector and
gelatin is spotted onto a slide, such as a glass slide coated with
.SIGMA. poly-L-lysine (e.g., Sigma, Inc.), for example, by hand or
using a microarrayer. The DNA spots can be affixed to the slide by,
for example, subjecting the resulting product to drying at room
temperature, at elevated temperatures or in a vacuum-dessicator.
The length of time necessary for sufficient drying to occur depends
on several factors, such as the quantity of mixture placed on the
surface and the temperature and humidity conditions used.
[0009] The concentration of DNA present in the mixture will be
determined empirically for each use, but will generally be in the
range of from about 0.01 .mu.g/.mu.l to about 0.2 .mu.g/.mu.l and,
in specific embodiments, is from about 0.02 .mu.g/.mu.l to about
0.10 .mu.g/.mu.l. Alternatively, the concentration of DNA present
in the mixture can be from about 0.01 .mu.g/.mu.l to about 0.5
.mu.g/.mu.l, from about 0.01 .mu.g/.mu.l to about 0.4 .mu.g/.mu.l
and from about 0.01 .mu.g/.mu.l to about 0.3 .mu.g/.mu.l.
Similarly, the concentration of gelatin, or another carrier
macromolecule, can be determined empirically for each use, but will
generally be in the range of 0.01% to 0.5% and, in specific
embodiments, is from about 0.05% to about 0.5%, from about 0.05% to
about 0.2% or from about 0.1% to about 0.2%. The final
concentration of DNA in the mixture (e.g., DNA in gelatin) will
generally be from about 0.02 .mu.g/.mu.l to about 0.1 .mu.g/.mu.l
and in a specific embodiment described herein, DNA is diluted in
0.2% gelatin (gelatin in water) to produce a final concentration of
DNA equal to approximately 0.05 .mu.g/.mu.l.
[0010] If the DNA used is present in a vector, the vector can be of
any type, such as a plasmid or viral-based vector, into which DNA
of interest (DNA to be expressed in reverse transfected cells) can
be introduced and expressed (after reverse transfection) in
recipient cells. For example, a CMV-driven expression vector can be
used. Commercially available plasmid-based vectors, such as pEGFP
(Clontech) or pcDNA3 (Invitrogen), or viral-based vectors can be
used. In this embodiment, after drying of the spots containing the
gelatin-DNA mixture, the surface bearing the spots is covered with
an appropriate amount of a lipid-based transfection reagent and the
resulting product is maintained (incubated) under conditions
appropriate for complex formation between the DNA in the spots (in
the gelatin-DNA mixture) and the lipid-based transfection reagent.
In one embodiment, the resulting product is incubated for
approximately 20 minutes at 25.degree. C. Subsequently,
transfection reagent is removed, producing a surface bearing DNA
(DNA in complex with transfection reagent), and cells in an
appropriate medium are plated onto the surface. The resulting
product (a surface bearing DNA and plated cells) is maintained
under conditions that result in entry of the DNA into plated
cells.
[0011] A second embodiment of the method is referred to as a
"lipid-DNA" embodiment. In this embodiment, a DNA-containing
mixture (referred to herein as a lipid-DNA mixture) which comprises
DNA (e.g., DNA in an expression vector); a carrier protein (e.g.,
gelatin); a sugar, such as sucrose; a buffer that facilitates DNA
condensation and an appropriate lipid-based transfection reagent is
spotted onto a surface, such as a slide, thus producing a surface
bearing the lipid-DNA mixture in defined locations. The resulting
product is allowed to dry sufficiently that the spotted lipid-DNA
mixture is affixed to the slide and the spots remain in the
locations to which they have become affixed, under the conditions
used for subsequent steps in the method. For example, a lipid-DNA
mixture is spotted onto a slide, such as a glass slide coated with
.SIGMA. poly-L-lysine (e.g., Sigma, Inc.), for example, by hand or
using a microarrayer. The DNA spots can be affixed to the slide as
described above for the gelatin-DNA method.
[0012] The concentration of DNA present in the mixture will be
determined empirically for each use, but will generally be in the
range of 0.5 .mu.g/.mu.l to 1.0 .mu.g/.mu.l. A range of sucrose
concentrations can be present in the mixture, such as from about
0.1M to about 0.4M. Similarly, a range of gelatin concentrations
can be present in the mixture, such as from about 0.01% to about
0.05%. In this embodiment, the final concentration of DNA in the
mixture will vary and can be determined empirically. In specific
embodiments, final DNA concentrations range from about 0.1
.mu.g/.mu.l to about 2.0 .mu.g/.mu.l. If a vector is used in this
embodiment, it can be any vector, such as a plasmid, or viral-based
vector, into which DNA of interest (DNA to be expressed in reverse
transfected cells) can be introduced and expressed (after reverse
transfection), such as those described for use in the gelatin-DNA
embodiment.
[0013] After drying is complete (has occurred to a sufficient
extent that the DNA remains affixed to the surface under the
conditions used in the subsequent steps of the method), eukaryotic
cells into which the DNA is to be reverse transfected are placed on
top of the surfaces onto which the DNA-containing mixture has been
affixed. Actively growing cells are generally used and are plated,
preferably at high density (such as 1.times.10.sup.5/cm.sup.2 ), on
top of the surface containing the affixed DNA-containing mixture in
an appropriate medium, such as Dulbecco's Modified Eagles Medium
(DMEM) containing 10% heat-inactivated fetal serum (IFS) with
L-glutamine and penicillin/streptomycin (pen/strep). Other media
can be used and their components can be determined based on the
type of cells to be transfected. The resulting slides, which
contain the dried lipid-DNA mixture and cells into which the DNA is
to be reverse transfected, are maintained under conditions
appropriate for growth of the cells and entry of DNA, such as an
entry of an expression vector containing the DNA, into cells. In
the present method, approximately one to two cell cycles are
sufficient for reverse transfection to occur, but this will vary
with the cell type and conditions used and the appropriate length
of time for a specific combination can be determined empirically.
After sufficient time has elapsed, slides are assessed for reverse
transfection (entry of DNA into cells) and expression of the
encoded product or effect of the introduced DNA on
reverse-transfected cells, using known methods. This can be done,
for example, by detecting immunofluorescence or enzyme
immunocytochemistry, autoradiography, in situ hybridization or
other means of detecting expression of the DNA or an effect of the
encoded product or of the DNA itself on the cells into which it is
introduced. If immunofluorescence is used to detect expression of
an encoded protein, an antibody that binds the protein and is
fluorescently labeled is used (e.g., added to the slide under
conditions suitable for binding of the antibody to the protein) and
the location (spot or area of the surface) containing the protein
is identified by detecting fluorescence. The presence of
fluorescence indicates that reverse transfection has occurred and
the encoded protein has been expressed in the defined location(s)
which show fluorescence. The presence of a signal, detected by the
method used, on the slides indicates that reverse transfection of
the DNA into cells and expression of the encoded product or an
effect of the DNA in recipient cells has occurred in the defined
location(s) at which the signal is detected. As described above,
the identity of the DNA present at each of the defined locations is
known; thus, when expression occurs, the identity of the expressed
protein is also known.
[0014] Thus, the present invention relates, in one embodiment, to a
method of expressing defined DNA, such as cDNA or genomic DNA, in
defined locations or areas of a surface onto which different DNAs,
such as DNA in a vector, such as an expression vector, has been
affixed, as described herein. Because each area of the surface has
been covered/spotted with DNA of known composition, it is a simple
matter to identify the expressed protein. In addition, the present
method is useful to identify DNAs whose expression alters (enhances
or inhibits) a pathway, such as a signaling pathway in a cell or
another property of a cell, such as its morphology or pattern of
gene expression. The method is particularly useful, for example, as
a high-throughput screening method, such as in a microarray format.
It can be used in this format for identifying DNAs whose expression
changes the phosphorylation state or subcellular location of a
protein of interest or the capacity of the cell to bind a reagent,
such as a drug or hormone ligand. In a second embodiment, which is
also useful as a high-throughput screening method, DNA reverse
transfected into cells has an effect on cells or interacts with a
cellular component(s) without being expressed, such as through
hybridization to cellular nucleic acids or through antisense
activity.
[0015] Also the subject of this invention are arrays, including
microarrays, of defined DNAs spotted onto (affixed to) a surface
and array: including microarrays of reverse transfected cells
spotted to (affixed to) a surface by the method described herein.
Such arrays can be produced by the gelatin-DNA embodiment or the
lipid-DNA embodiment of the present method. Arrays of this
invention are surfaces, such as slides (e.g., glass or .SIGMA.
poly-L-lysine coated slides) or wells, having affixed thereto
(bearing) in discrete, defined locations DNAs, such as cDNAs or
genomic DNA, or cells containing DNA of interest introduced into
the cells by the reverse transfection method described herein.
[0016] A method of making arrays of the present invention is also
the subject of this invention. The method comprises affixing DNAs
or reverse transfected cells onto a surface by the steps described
herein for the gelatin-DNA embodiment or the lipid-DNA
embodiment.
[0017] A DNA array of the present invention comprises a surface
having affixed thereto, in discrete, defined locations, DNA of
known sequence or source by a method described herein. In one
embodiment, DNA is affixed to a surface, such as a slide, to
produce an array (e.g., a macro-array or a micro-array) by spotting
a gelatin-DNA mixture, as described herein, onto the surface in
distinct, defined locations (e.g., by hand or by using an arrayer,
such as a micro-arrayer) and allowing the resulting surface bearing
the gelatin-DNA mixture to dry sufficiently that the spots remain
affixed to the surface under conditions in which the arrays are
used. In an alternative embodiment, DNA is affixed to a surface,
such as a slide, to produce an array by spotting a lipid-DNA
mixture, as described herein, onto the surface in distinct defined
locations (e.g., by hand or by using an arrayer, such as a
micro-arrayer) and allowing the resulting surface bearing the
lipid-DNA mixture to dry sufficiently that the spots remain affixed
to the surface under the conditions in which the arrays are used.
This result in production of a surface bearing (having affixed
thereto) DNA-containing spots.
[0018] An array of reverse transfected cells can also be produced
by either embodiment described herein. In the gelatin-DNA
embodiment, the steps described above for producing DNA arrays are
carried out and subsequently, the surface bearing the
DNA-containing spots is covered with an appropriate amount of a
lipid-based transfection reagent and the resulting product is
maintained (incubated) under conditions appropriate for complex
formation between DNA in the spots and the reagent. After
sufficient time (e.g., about 20 minutes at 25.degree. C.) for
complex formation to occur, transfection reagent is removed,
producing a surface bearing DNA and cells in an appropriate medium
are added. The resulting product (a surface bearing DNA and plated
cells) is maintained under conditions that result in entry of DNA
into plated cells, thus producing an array (a surface bearing an
array) of reverse transfected cells that contain defined DNA and
are in discrete, defined locations on the array. Such cell arrays
are the subject of this invention.
[0019] In the lipid-DNA embodiment, the steps described above for
producing DNA arrays are carried out and subsequently (after drying
is sufficient to affix the DNA-containing spots to the surface,
such as a slide or well bottom), cells are plated on top of the
surface bearing the DNA-containing spots and the resulting slides,
which contain the dried lipid-DNA mixture and cells to be reverse
transfected, are maintained under conditions appropriate for growth
of the cells and entry of DNA into the cells, thus producing an
array (a surface bearing an array) of reverse transfected cells
that contain defined DNA and are in discrete, defined locations on
the array. Such arrays are the subject of this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a schematic representation of one embodiment of
the present method of reverse transfection, in which cDNA (HA-GST,
HA-FKBP12 or myc-FRB) in an expression vector (prk5) was introduced
into cells by the following procedures: combining cDNA in an
expression vector, a lipid-based transfection reagent and a carrier
protein, to produce a mixture; spotting the mixture onto a glass
slide; allowing the spotted mixture to dry on the slide surface;
plating human embryonic kidney (HEK 293T) cells into which cDNA is
to be introduced onto the slide; maintaining the resulting slide
under conditions appropriate for reverse transfection to occur; and
detecting immunofluorescence using a fluorescently labeled antibody
that binds HA but not myc, demonstrating the presence and location
of expressed cDNA.
[0021] FIG. 2 shows the results of reverse transfection of HEK293T
cells with HA-GST, as demonstrated using anti-HA
immunofluorescence.
[0022] FIG. 3 shows the results of reverse transfection of HEK293T
cells with pBABE EGFP, as demonstrated by detecting endogenous
fluorescence of EGFP.
[0023] FIG. 4A is a schematic for making transfected cell
microarrays using a well-less transfection of plasmid DNAs in
defined areas of a lawn of mammalian cells. Plasmid DNA dissolved
in an aqueous gelatin solution is printed on a glass slide using a
robotic arrayer. The slide is dried and the printed array covered
with a lipid transfection reagent. After removal of the lipid, the
slide is placed in a culture dish and covered with cells in media.
The transfected cell microarray forms in 1-2 days and is then ready
for downstream assays. An alternative method in which the lipid is
added to the DNA/gelatin solution prior to printing is also
described.
[0024] FIG. 4B is a GFP-expressing microarray made from a slide
printed in a 12>8 pattern with a GFP expression construct.
[0025] FIG. 4C is a higher magnification image obtained with
fluorescence microscopy of the cell cluster boxed in FIG. 4B. Scale
bar equals 100 .mu.m.
[0026] FIG. 4D is a graph of GFP cDNA (picograms) versus mean
signal intensity +/-S.D. showing expression levels of clusters in a
transfected cell microarray are proportional, over a four-fold
range, to the amount of plasmid DNA printed on the slide. Arrays
were printed with elements containing the indicated amounts of the
GFP construct. Amount of DNA assumes a one nanoliter printing
volume. After transfection, the mean +/-S.D. of the fluorescence
intensities of the cell clusters were determined. Arrays were
prepared as described in Example 3 except that the concentration of
the GFP expression plasmid was varied from 0.010-0.050 .mu.g/.mu.l
while the total DNA concentration was kept constant at 0.050
.mu.g/.mu.l with empty vector (prk5). Cell clusters were
photographed and the signal intensity quantitated with Image Quant
(Fuji). The fluorescent image is from a representative
experiment.
[0027] FIG. 4E is a scan image showing that by printing mixtures of
two plasmids, cotransfection is possible with transfected cell
microarrays. Arrays with elements containing expression constructs
for HA-GST, GFP or both were transfected and processed for anti-myc
immunofluorescence. For immunofluorescence staining the cells were
fixed as described in Example 3, permeabilized in 0.1% Triton X-100
in PBS for 15 minutes at room temperature and probed with primary
and secondary antibodies as described. Primary antibodies were used
for 1 hour at room temperature at the following concentrations:
1:500 anti-HA ascites (BaBCo), 2 .mu.g/ml anti-myc 9E-10
(Calbiochem), 2 .mu.g/ml anti-V5 (Invitrogen), or 10 .mu.g/ml 4G10
anti-phosphotyrosine (Upstate Biotechnologies). The secondary
antibody used was Cy3 .mu.g/ml labeled anti-mouse antibody (Jackson
Immunoresearch) at 3.1 .mu.g/ml for 40 minutes at room temperature.
Panels labeled Cy3 and GFP show location of clusters expressing
HA-GST and GFP, respectively. Merged panel shows superimposition of
Cy3 and GFP signals and yellow color indicates co-expression. Scale
bar equals 100 .mu.m.
[0028] FIG. 4F is an enlarged view of boxed area of scan image from
FIG. 4E.
[0029] FIG. 5A is a laser scan showing detection of the receptor
for FK506. Arrays with elements containing expression constructs
for GFP, myc-FKBP12 or both were printed and transfected with
HEK293 cells. 5 nM dihydro-FK506 [propyyl-.sup.3H] (NEN) was added
to the culture media 1 hour prior to fixation and processing for
immunofluorescence and autoradiography. Slides were process for
anti-myc immunofluorescence, scanned at 5 .mu.m resolution and
photographed using a fluorescent microscope, and then exposed to
tritium sensitive film (Hyperfilm, Amersham) for 4 days.
Autoradiographic emulsion was performed as described by the
manufacturer (Amersham). Laser scans show expression pattern of GFP
and FKBP12 and superimposition of both (merged). Film
autoradiography detects binding of tritiated FK506 to the same
array (autorad film).
[0030] FIG. 5B is a higher magnification image obtained by
fluorescent microscopy of an FKBP12-expressing cluster (FKBP12).
Emulsion autoradiography detects, with cellular resolution, binding
of tritiated FK506 to the same cluster (autorad emulsion).
[0031] FIG. 5C is a scan showing detected components of tyrosine
kinase signaling cascades. 192 V5-epitope-tagged cDNAs in
expression vectors were printed in two 8.times.12 subgrids named
array 1 and 2. For ease of determining the coordinates of cell
clusters within the arrays a border around each array was printed
with the GFP expression construct. After transfection, separate
slides were processed for anti-V5 or anti-phosphotyrosine
immunofluorescence and Cy3 and GFP fluorescence detected. Merged
images of array 1 show location of clusters expressing V5-tagged
proteins (left panel) and having increased levels of
phosphotyrosine (right panel). No DNA was printed in coordinates
F10-12.
[0032] FIG. 5D show two examples of the morphological phenotypes
detectable in the transfected cell microarrays described in FIG.
5C. Clusters shown are E8 and F7 from array 2.
[0033] FIG. 6 shows a transfection array that has been transferred
to a nitrocellulose filter.
DETAILED DESCRIPTION OF THE INVENTION
[0034] I. Overview
[0035] The growing collection of gene sequences and cloned cDNAs
demands the development of systematic and high-throughput
approaches to characterizing the gene products. The uses of DNA
microarrays for transcriptional profiling and of two-hybrid assays
for determining protein-protein interactions are recent examples of
genomic approaches to the characterization of gene products.
Comparable strategies have not previously existed to analyze the
function, particular within mammalian cells, of large sets of
genes. Currently, in vivo analysis can be done, on a gene-by-gene
scale, by expressing with cells a nucleic acid construct that
directs the overexpression of a gene product or inhibits its
synthesis or function.
[0036] The present invention relates to a microarray-driven gene
expression system for the functional analysis of many gene products
in parallel. Cells are cultures on a solid surface printed in
defined locations with different nucleic acid constructs which can
be taken up by the cells. The effects on cellular physiology by the
product of the transfection array can be detected. Rather than
having to recover the transfected construct to ascertain its
identity, the identity is determined by the position of the
transfectant of interest on the array. The subject assay can be
particular useful where cell is the read-out used to identify a
construct of interest.
[0037] A microarray-based system was developed to analyze the
function in cells of many genes in parallel. Cells are cultured on
a glass slide printed in defined locations with solutions
containing different DNAs. Cells growing on the printed areas take
up the DNA, creating spots of localized transfection within a lawn
of non-transfected cells. By printing sets of complementary DNAs
(cDNAs) cloned in expression vectors, micorarrays which comprise
groups of live cells that express a defined cDNA at each location
can be made. Transfected cell microarrays can be of broad utility
for the high-throughput expression cloning of genes, particularly
in areas such as signal transduction and drug discovery. For
example, as shown herein, transfected cell microarrays can be used
for the unambiguous identification of the receptor for the
immunosuppressant FK506 and components of tyrosine kinase
pathways.
[0038] The present invention relates to a method of introducing
defined DNAs into cells at specific discrete, defined locations on
a surface by means of a reverse transfection method. That is, the
present method makes use of DNAs, of known sequence and/or source,
affixed to a surface (DNA spots), such as a slide or well bottom,
and growing cells that are plated onto the DNA spots and maintained
under conditions appropriate for entry of the DNAs into the cells.
The size of the DNA spots and the quantity (density) of the DNA
spots affixed to the surface can be adjusted depending on the
conditions used in the methods. For example, the DNA spots can be
from about 100 .mu.m to about 200 .mu.m in diameter and can be
affixed from about 200 .mu.m to about 500 .mu.m apart on the
surface. The present method further includes identification or
detection of cells into which DNA has been reverse transfected. In
one embodiment, DNA introduced into cells is expressed in the
cells, either by an expression vector containing the DNA or as a
result of integration of reverse transfected DNA into host cell
DNA, from which it is expressed. In an alternative embodiment of
the present method, DNA introduced into cells is not expressed, but
affects cell components and/or function itself. For example,
antisense DNA can be introduced into cells by this method and
affect cell function. For example, a DNA fragment which is
anti-sense to an mRNA encoding a receptor for a drug can be
introduced into cells via reverse transfection. The anti-sense DNA
will decrease the expression of the drug receptor protein, causing
a decrease in drug binding to cells containing the anti-sense DNA.
In the method, a mixture comprising DNA of interest (such as cDNA
or genomic DNA incorporated in an expression vector) and a carrier
protein is deposited (e.g., spotted or placed in small defined
areas) onto a surface (e.g., a slide or other flat surface, such as
the bottoms of wells in a multi-welled plate) in defined, discrete
(distinct) locations and allowed to dry, with the result that the
DNA-containing mixture is affixed to the surface in defined
discrete locations.
[0039] Detection of effects on recipient cells (cells containing
DNA introduced by reverse transfection) can be carried out by a
variety of known techniques, such as immunofluorescence, in which a
fluorescently labeled antibody that binds a protein of interest
(e.g., a protein thought to be encoded by a reverse transfected DNA
or a protein whose expression or function is altered through the
action of the reverse transfected DNA) is used to determine if the
protein is present in cells grown on the DNA spots.
[0040] The methods of this invention are useful to identify DNAs of
interest (DNAs that are expressed in recipient cells or act upon or
interact with recipient cell constituents or function, such as DNAs
that encode a protein whose function is desired because of
characteristics its expression gives cells in which it is
expressed). They can be used in a variety of formats, including
macro-arrays and micro-arrays. They permit a DNA array to be
converted into a protein or cell array, such as a protein or cell
microarray.
[0041] II. Definitions Before further description of the invention,
certain terms employed in the specification, examples and appended
claims are, for convenience, collected here.
[0042] As used herein, the term "nucleic acid" refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA). "Complementary DNA" or a
"cDNA" as used herein includes recombinant genes synthesized by
reverse transcription of mRNA and from which intervening sequences
(introns) have been removed.
[0043] As used herein, the terms "heterologous nucleic acid" and
"foreign nucleic acid" refer to a nucleic acid, e.g., DNA or RNA,
that does not occur naturally as part of the genome in which it is
present or which is found in a location or locations in the genome
that differs from that in which it occurs in nature. Heterologous
DNA is not endogenous to the cell into which it is introduced, but
has been obtained from another cell. Examples of heterologous
nucleic acid include, but are not limited to, DNA that encodes test
polypeptides, receptors, reporter genes, transcriptional and
translational regulatory sequences, selectable or traceable marker
proteins, such as a protein that confers drug resistance. Examples
of heterologous RNA include, but are not limited to, anti-sense RNA
sequences, ribozymes, and double-stranded RNA (for inducing
sequence-specific RNA interference).
[0044] As used herein, the terms "target nucleic acid" and "target
sequence" refer to the component of a transfection array, e.g., the
portion or portions of a nucleic acid being transfected into the
host cells, which is of interest with respect to its ability to
confer a change in the phenotype of the host cells. In general,
though not always, the target nucleic acid will that portion(s) of
the nucleic acid of the transfection array that is varied from one
portion of the array to the next. The target nucleic acid can be a
coding sequence for a protein, a "coding" sequence for an RNA
molecule (e.g., which is transcribed into an anti-sense RNA
sequence, a ribozyme or double-stranded RNA), or a regulatory
sequence (e.g., as part of a reporter construct), to name but a few
examples.
[0045] The term "feature", as it is used in describing a
transfection array, refers to an area of a substrate having a
homogenous collection of a target sequence (or sequences in the
case of certain co-transfection embodiments). One feature is
different than another feature if the target sequences of the
different features have different nucleotide sequences.
[0046] The term "loss-of-function", as it refers to the effect of a
target sequence, refers to those target sequences which, when
expressed in a host cell, inhibit expression of a gene or otherwise
render the gene product thereof to have substantially reduced
activity, or preferably no activity relative to one or more
functions of the corresponding wild-type gene product.
[0047] As used herein, a "desired phenotype" refers to a particular
phenotype for that the user of the subject method seeks to have
selectively conferred on the host cell line upon expression of a
target sequence.
[0048] As used herein, the term "vector" refers to a nucleic acid
molecule capable of being transporting into and/or maintained
within a cell. Preferred vectors are those capable of autonomous
replication. In the present specification, "plasmid" and "vector"
are used interchangeably as the plasmid is the most commonly used
form of vector.
[0049] As used herein, the term "operatively linked" refers to the
functional relationship of a nucleic acid sequence with regulatory
and effector nucleotide sequences, such as promoters, enhancers,
transcriptional and translational start and stop sites, and other
signal sequences. For example, operative linkage of DNA to a
promoter refers to the physical and functional relationship between
the DNA and the promoter such that the transcription of such DNA is
initiated from the promoter by an RNA polymerase that specifically
recognizes, binds to, and transcribes the DNA.
[0050] As used herein, the term "expression" refers to any number
of steps comprising the process by which polynucleic acids are
transcribed into RNA, and (optionally) translated into peptides,
polypeptides, or proteins. If the polynucleic acid is derived from
genomic DNA, expression may, if an appropriate eukaryotic host cell
or organism is selected, include splicing of the RNA.
[0051] As used herein, "recombinant cells" include any cells that
have been modified by the introduction of heterologous nucleic
acid. Control cells include cells that are substantially identical
to the recombinant cells, but do not express one or more of the
proteins encoded by the heterologous nucleic acid.
[0052] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein.
[0053] The terms "recombinant protein", "heterologous protein" and
"exogenous protein" are used interchangeably throughout the
specification and refer to a polypeptide which is produced by
recombinant DNA techniques, wherein generally, DNA encoding the
polypeptide is inserted into a suitable expression vector which is
in turn used to transform a host cell to produce the heterologous
protein. That is, the polypeptide is expressed from a heterologous
nucleic acid.
[0054] As used herein, "cell surface receptor" refers to molecules
that occur on the surface of cells, interact with the extracellular
environment, and transmit or transduce the information regarding
the environment intracellularly in a manner that may modulate
intracellular second messanger activities or transcription of
specific promoters, resulting in transcription of specific
genes.
[0055] As used herein, "extracellular signals" include a molecule
or a change in the environment that is transduced intracellularly
via cell surface proteins that interact, directly or indirectly,
with the signal. An extracellular signal or effector molecule
includes any compound or substance that in some manner alters the
activity of a cell surface protein. Examples of such signals
include, but are not limited to, molecules such as acetylcholine,
growth factors and hormones, lipids, sugars and nucleotides that
bind to cell surface and/or intracellular receptors and ion
channels and modulate the activity of such receptors and channels.
The term also include as yet unidentified substances that modulate
the activity of a cellular receptor, and thereby influence
intracellular functions. Such extracellular signals are potential
pharmacological agents that may be used to treat specific diseases
by modulating the activity of specific cell surface receptors.
"Orphan receptors" is a designation given to a receptors for which
no specific natural ligand has been described and/or for which no
function has been determined.
[0056] As used herein, a "reporter gene construct" is a nucleic
acid that includes a "reporter gene" operatively linked to at least
one transcriptional regulatory sequence. Transcription of the
reporter gene is controlled by these sequences to which they are
linked. The activity of at least one or more of these control
sequences is directly or indirectly regulated by the target
receptor protein. Exemplary transcriptional control sequences are
promoter sequences. A reporter gene is meant to include a
promoter-reporter gene construct which is heterologously expressed
in a cell.
[0057] "Signal transduction" is the processing of physical or
chemical signals from the cellular environment through the cell
membrane, and may occur through one or more of several mechanisms,
such as acitvation/inactivation of enzymes (such as proteases, or
other enzymes which may alter phosphorylation patterns or other
post-translational modifications), activation of ion channels or
intracellular ion stores, effector enzyme activation via guanine
nucleotide binding protein intermediates, formation of inositol
phosphate, activation or inactivation of adenylyl cyclase, direct
activation (or inhibition) of a transcriptional factor and/or
activation.
[0058] The term "modulation of a signal transduction activity of a
receptor protein" in its various grammatical forms, as used herein,
designates induction and/or potentiation, as well as inhibition of
one or more signal transduction pathways downstream of a
receptor.
[0059] The term "autocrine cell", as used herein, refers to a cell
which produces a substance which can induce a phenotypic response
within the same cell as produces the substance.
[0060] III. Transfection Arrays
[0061] The target nucleic acid used in the transfection arrays of
the present invention can be, for example, DNA, RNA or modified or
hybrid forms thereof. The target nucleic acid may be from any of a
variety of sources, such as nucleic acid isolated from cells, or
that which is recombinantly produced or chemically synthesized.
[0062] For example, the transfection array can include coding
sequence from cDNAs or genomic DNA. In addition to native
sequences, the coding sequences can include those which have been
mutated relative to the native sequence, e.g., a coding sequence
that differs from a naturally occurring sequence by deletion,
substitution or addition of at least one residue. It can correspond
to full length or partial sequences, can be antisense in
orientation, or can represent a non-coding sequence.
[0063] In other embodiments, all or a portion of the target nucleic
acid sequence can be synthesized chemically. In such a manner,
random and semi-random sequence can be introduced into the target
sequences, as well as modified forms of nucleotides and nucleotide
linkages, such as the use of modified backbones, methylated
nucleotides and the like.
[0064] The target nucleic acid sequences can be present as part of
a larger vector, such as an expression vector (e.g., a plasmid or
viral-based vector), but it need not be. The nucleic acid of the
array can be introduced into cells in such a manner that at least
the target sequence becomes integrated into the genomic DNA and is
expressed, or the target sequence remains extrachromosomal (e.g.,
is maintained episomally).
[0065] The nucleic acid for use in the transfection arrays of the
present invention can be linear or circular, double stranded or
single stranded, and can be of any size. In certain preferred
embodiments, especially where traditional expression vectors are
used, the target sequence is from about 200 nt to about 10 kb in
size, more preferably from about 200 nt to about 5 kb, and even
more preferably 200 nt to 2 kb. In such embodiments, the arrayed
nucleic acid, e.g., which includes the target sequence, can be from
about 1 kb to about 15 kb, and more preferably from about 5 kb to
about 8 kb.
[0066] In certain preferred embodiments, the transfection array is
made up of a variegated library of expression vectors. Ligating a
polynucleotide coding sequence or other transcribable sequences an
expression vector can be carried out using standard procedures.
Similar procedures, or modifications thereof, can be readily
employed to prepare arrays of expression vectors in accord with the
subject invention.
[0067] In general, it will be desirable that the vector be capable
of replication in the host cell. It may be a DNA which is
integrated into the host genome, and thereafter is replicated as a
part of the chromosomal DNA, or it may be DNA which replicates
autonomously, as in the case of a episomal plasmid. In the latter
case, the vector will include an origin of replication which is
functional in the host. In the case of an integrating vector, the
vector may include sequences which facilitate integration, e.g.,
sequences homologous to host sequences, or encoding integrases. The
use of retroviral long terminal repeats (LTR) or adenoviral
inverted terminal repeats (ITR) in the construct of the
transfection array can, for example, facilitate the chromosomal
integration of the construct.
[0068] Appropriate cloning and expression vectors for use with
bacterial, fungal, yeast, and mammalian cellular hosts are known in
the art, and are described in, for example, Powels et al. (Cloning
Vectors: A Laboratory Manual, Elsevier, N.Y., 1985). Such vectors
may be readily adapted for use in the present invention. The
expression vectors may comprise non-transcribed elements such as an
origin of replication, a suitable promoter and enhancer linked to
the gene to be expressed, and other 5' or 3' flanking
nontranscribed sequences, and 5' or 3' nontranslated sequences,
such as necessary ribosome binding sites, a poly-adenylation site,
splice donor and acceptor sites, and transcriptional termination
sequences.
[0069] Certain preferred mammalian expression vectors contain both
prokaryotic sequences, to facilitate the propagation of the vector
in bacteria (such as in an amplification step after recovery from
the array), and one or more eukaryotic transcription units for
expressing the target sequence in eukaryotic host cells. The
pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,
pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples
of mammalian expression vectors which can be readily adapted for
use in the subject method. Some of these vectors are modified with
sequences from bacterial plasmids, such as pBR322, to facilitate
replication and drug resistance selection in both prokaryotic and
eukaryotic cells. Alternatively, derivatives of viruses, such as
the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,
pREP-derived and p205) and the like, can be used to derive the
subject arrays. The various methods employed in the preparation of
the plasmids are well known in the art. For other suitable
expression systems for both prokaryotic and eukaryotic cells, as
well as general recombinant procedures, see Molecular Cloning A
Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis
(Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.
[0070] Particularly preferred vectors contain regulatory elements
that can be linked to the target sequence for transfection of
mammalian cells, and include are cytomegalovirus (CMV)
promoter-based vectors such as pcDNA1 (Invitrogen, San Diego,
Calif.), MMTV promoter-based vectors such as pMAMNeo (Clontech,
Palo Alto, Calif.) and pMSG (Pharmacia, Piscataway, N.J.), and SV40
promoter-based vectors such as pSVO (Clontech, Palo Alto,
Calif.).
[0071] A number of vectors exist for the expression of recombinant
proteins in yeast, where that is the host cell used in connection
with the array. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and
YRP17 are cloning and expression vehicles useful in the
introduction of genetic constructs into S. cerevisiae (see, for
example, Broach et al. (1983) in Experimental Manipulation of Gene
Expression, ed. M. Inouye Academic Press, p. 83, incorporated by
reference herein). These vectors can replicate in E. coli due the
presence of the pBR322 ori, and in S. cerevisiae due to the
replication determinant of the yeast 2 micron plasmid. Moreover, if
yeast are used as a host cell, it will be understood that the
expression of a gene in a yeast cell requires a promoter which is
functional in yeast. Suitable promoters include the promoters for
metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.
Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et
al., J. Adv. Enzyme Req. 7, 149 (1968); and Holland et al.
Biochemistry 17, 4900 (1978)), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phospho-fructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phospho-glucose isomerase, and glucokinase.
[0072] In some instances, it may be desirable to derive the host
cell using insect cells. In such embodiments, the transfection
array can be derived from, for example, a baculovirus expression
system. Examples of such baculovirus expression systems include
pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),
pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived
vectors (such as the .beta.-gal containing pBlueBac III).
[0073] Where the source of target sequence for the array are
naturally occurring, those sequences can be isolated from any cell
or collection of cells. For instance, the target sequences can be
isolated from the cells of either adult tissue or organs or
embryonic tissue or organs at any given developmental stage
(including oocyte, blastocyte, etc.). The cells can be derived from
healthy tissue or diseased tissue. In the case of a solid organ,
the cell sample can be obtained by, e.g., biopsy. For blood, lymph
and other bodily fluids, the cells can be isolated from the fluid
component, e.g., by filtration, affinity purification,
centrifuigation or any other technique known in the art. The cells
can be isolated to include a specific subset of phenotypes of cells
from a given tissue, or can include be derived to include all or a
substantial portion of cells representative of the tissue. For
instance, the cells can be derived from an organ where the cells
are particularly of epithelial, mesenchymal or endothelial origin.
Subsets of cells can be isolated, for example, by use of cell
surface markers or careful sectioning of a tissue.
[0074] In certain preferred embodiments, the target sequence are
cDNA sequences derived from mRNA isolated from a cell or cells of
interest. There are a variety of methods known in the art for
isolating RNA from a cellular source, any of which may be used to
practice the present method. The Chomczynski method, e.g.,
isolation of total cellular RNA by the guanidine isothiocyanate
(described in U.S. Pat. No. 4,843,155) used in conjunction with,
for example, oligo-dT strepavidin beads, is an exemplary mRNA
isolation protocol. The RNA, as desirable, can be converted to cDNA
by reverse transcriptase, e.g., poly(dT)-primered first strand cDNA
synthesis by reverse transcriptase, followed by second strand
synthesis (DNA pol I).
[0075] Likewise, there are a wide range of techniques for isolating
genomic DNA which are amenable for use in a variety of embodiments
of the subject method. In preferred embodiments, it will be
desirable to isolate only a portion of the total genomic DNA on the
basis of the chemical and/or physical state in which it is present
in a collection of cells. For instance, transcriptionally active
and/or potentially active genes can be distinguished by several
criteria from inactive sequences. In higher eukaryoties, gene
activation is accompanied by an increased general sensitivity to
endonucleases like DNase I or micrococcal nuclease. This increased
sensitivity probably reflects both the partial decondensation of
chromatin. In addition, gene activation usually causes a
coreplication domain that extends much beyond the decondensation
domain. Chromatin digestion by DNase I, for example, will produce
smaller digestion fragments from those areas of the genome which
have undergone decondensation relative to areas of condensed
chromatin structure (Galas et al. (1987) Nucleic Acids Res.
5:3157), e.g., the smaller fragments will be enriched for genomic
sequences from genes in activated states.
[0076] Likewise, changes in methylation status of a gene provides
another mechanism by which potential for expression can be altered,
and may serve as a criteria for selecting certain genomic sequences
as target nucleic acids. Thus, genomic DNA can be treated with
methyl-sensitive restriction enzymes (such as DpnI) in order to
produce endonuclease fragments of genes dependent on the
methylation state of the surrounding genomic sequences.
[0077] In certain embodiments, the subject array can be made of a
library of related, mutated sequences, such as a library of mutants
of a particular protein, or libraries of potential promoter
sequences, etc. There are a variety of forms of mutagenesis that
can be utilized to generate a combinatorial library. For example,
homologs of protein of interest (both agonist and antagonist forms)
can be generated and isolated from a library by screening using,
for example, alanine scanning mutagenesis and the like (Ruf et al.
(1994) Biochemistry 33:15 65-1572; Wang et al. (1994) J. Biol.
Chem. 269:3095-3099; Balint et al. (1993) Gene 137:109-118;
Grodberg et al. (1993) Eur. J. Biochem. 218:597-601; Nagashima et
al. (1993) J. Biol. Chem. 268:2888-2892; Lowinan et al. (1991)
Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science
244:1081-1085), by linker scanning mutagenesis (Gustin et al.
(1993) Virology 193:653-660; Brown et al. (1992) Mol. Cell Biol.
12:2644-2652; McKnight et al. (1982) Science 232:316); by
saturation mutagenesis (Meyers et al. (1986) Science 232:613); by
PCR mutagenesis (Leung et al. (1989) Method Cell Mol Biol 1:11-19);
or by random mutagenesis (Miller et al. (1992) A Short Course in
Bacterial Genetics, CSHL Press, Cold Spring Harbor, N.Y.; and
Greener et al. (1994) Strategies in Mol Biol 7:32-34).
[0078] In another embodiment, the transfection array provides a
library of small gene fragments as the target sequences, e.g.,
sequences which may encode dominant-acting .sctn.synthetic genetic
elements (SGEs), e.g., molecules that interfere with the function
of genes from which they are derived (antagonists) or that are
dominant consitutively active fragments (agonists) of such genes.
SGEs that can be identified by the subject method include, but are
not limited to, polypeptides, inhibitory antisense RNA molecules,
ribozymes, nucleic acid decoys, and small peptides.
[0079] The SGEs identified by the present method may function to
inhibit the function of an endogenous gene at the level of nucleic
acids, e.g., by an antisense or decoy mechanism, or by encoding a
polypeptide that is inhibitory through a mechanism of interference
at the protein level, e.g., a dominant negative fragment of the
native protein. On the other hand, certain SGEs may function to
potentiate (including mimicing) the function of an endogenous gene
by encoding a polypeptide which retains at least a portion of the
bioactivity of the corresponding endogenous gene, and may in
particular instances be constitutively active.
[0080] In one embodiment, the initial SGE library is generated from
total cDNA, that may be further fragmented, and provided in the
form of an expression library. Preferably, the inserts in the
library will range from about 100 bp to about 700 bp and more
preferably, from about 200 bp to about 500 bp in size.
[0081] For cDNA-derived libraries, the nucleic acid library can be
a normalized library containing roughly equal numbers of clones
corresponding to each gene expressed in the cell type from which it
was made, without regard for the level of expression of any
gene.
[0082] The initial SGE libraries can be generated to include both
sense and antisense coding (and non-coding sequences) sequences.
Transcription of the SGE sequence in the subtractive and target
cells will create antisense RNA that may inhibit transcription of
the corresponding endogenous gene. Translation of appropriate
protein coding sequences in the transcribed RNA can produce
full-length and truncated forms of endogenous proteins, as well as
short peptides, the differential biological effects of that are
assessed in the subtractive and target cells.
[0083] U.S. Pat. No. 5,702,898 describes a method to normalize a
cDNA library constructed in a vector capable of being converted to
single-stranded circles and capable of producing complementary
nucleic acid molecules to the single-stranded circles comprising:
(a) converting the cDNA library in single-stranded circles; (b)
generating complementary nucleic acid molecules to the
single-stranded circles; (c) hybridizing the single-stranded
circles converted in step (a) with complementary nucleic acid
molecules of step (b) to produce partial duplexes to an appropriate
Cot; (e) separating the unhybridized single-stranded circles from
the hybridized single-stranded circles, thereby generating a
normalized cDNA library.
[0084] In certain embodiments, the SGE library can be a subtractive
cDNA library. Many strategies have been used to create subtractive
libraries, and can be readily adapted for use in the present
method. One approach is based on the use of directionally cloned
cDNA libraries as starting material (Palazzolo and Meyerowitz,
(1987) Gene 52:197; Palazzolo et al. (1989) Neuron 3:527; Palazzolo
et al. (1990) Gene 88:25). In this approach, cDNAs prepared from a
first source tissue or cell line are directionally inserted
immediately downstream of a bacteriophage T7 promoter in the
vector. Total library DNA is prepared and transcribed in vitro with
T7 RNA polymerase to produce large amounts of RNA that correspond
to the original mRNA from the first source tissue. Sequences
present in both the source tissue and another tissue or cells, such
as normal tissue, are subtracted as follows. The in vitro
transcribed RNA prepared from the first source is allowed to
hybridize with cDNA prepared from either native mRNA or library RNA
from the second source tissue. The complementarity of the cDNA to
the RNA makes it possible to remove common sequences as they anneal
to each other, allowing the subsequent isolation of unhybridized,
presumably tissue-specific, cDNA. This approach is only possible
using directional cDNA libraries, since any cDNA sequence in a
non-directional library is as likely to be in the "sense"
orientation as the "antisense" direction (sense and antisense are
complementary to each other). A cDNA sequence unique to a tissue
would be completely removed during the hybridization procedure if
both sense and antisense copies were present.
[0085] In one directional cloning strategy, which can be used to
generate an initial SGE library, a DNA sequence encoding a specific
restriction endonuclease recognition site (usually 6-10 bases) is
provided at the 5' end of an oligo(dT) primer. This relatively
short recognition sequence does not affect the annealing of the
12-20 base oligo(dT) primer to the mRNA, so the cDNA second strand
synthesized from the first strand template includes the new
recognition site added to the original 3' end of the coding
sequence. After second strand cDNA synthesis, a blunt ended linker
molecule containing a second restriction site (or a partially
double stranded linker adapter containing a protruding end
compatible with a second restriction site) is ligated to both ends
of the cDNA. The site encoded by the linker is now on both ends of
the cDNA molecule, but only the 3' end of the cDNA has the site
introduced by the modified primer. Following the linker ligation
step, the product is digested with both restriction enzymes (or, if
a partially double stranded linker adapter was ligated onto the
cDNA, with only the enzyme that recognizes the modified primer
sequence). A population of cDNA molecules results which all have
one defined sequence on their 5' end and a different defined
sequence on their 3' end.
[0086] A related directional cloning strategy developed by Meissner
et al. (1987) PNAS 84:4171), requires no sequence-specific modified
primer. Meissner et al. describe a double stranded palindromic
BamHI/HindIII directional linker having the sequence
d(GCTTGGATCCAAGC), that is ligated to a population of
oligo(dT)-primed cDNAs, followed by digestion of the ligation
products with BamHI and HindIII. This palindromic linker, when
annealed to double stranded form, includes an internal BamHI site
(GGATCC) flanked by 4 of the 6 bases that define a HindIII site
(AAGCTT). The missing bases needed to complete a HindIII site are
d(AA) on the 5' end or d(TT) on the 3' end. Regardless of the
sequence to which this directional linker ligates, the internal
BamHI site will be present. However, HindIII can only cut the
linker if it ligates next to an d(AA):d(TT) dinucleotide base pair.
In an oligo(dT)-primed strategy, a HindIII site is always generated
at the 3' end of the cDNA after ligation to this directional
linker. For cDNAs having the sequence d(TT) at their 5' ends
(statistically 1 in 16 molecules), linker addition will also yield
a HindIII site at the 5' end. However, because the 5' ends of cDNA
are heterogeneous due to the lack of processivity of reverse
transcriptases, cDNA products from every gene segment will be
represented in the library.
[0087] In other embodiments, the SGE library is generated from
genomic DNA fragments. Preferably, the inserts in the library will
range from about 100 bp to about 700 bp and more preferably, from
about 200 bp to about 500 bp in size. Such SGE libraries, in
addition to encoding polypeptide and antisense molecules that may
be functional SGEs in the test method, may also "encode" decoy
molecules, e.g., nucleic acid sequences which correspond to
regulatory elements of a gene and which can inhibit expression of
the gene by sequestering, e.g., transcriptional factors, and
thereby competing for the necessary components to express the
endogenous gene.
[0088] In yet another embodiment, the SGE library is generated by
randomly fragmenting a single gene to obtain a random fragment
expression library derived exclusively from the gene of interest.
As a practical matter, such a library will contain a much greater
variety of SGEs derived from the gene of interest than will a
random fragment library prepared from total cDNA. Consequently, the
likelihood of obtaining optimized SGEs, that have a differential
activity according to the present method, from the single gene
random fragment library is much higher.
[0089] In one embodiment, purified DNA corresponding to the gene or
genome to be suppressed is first randomly fragmented by enzymatic,
chemical, or physical procedures. In a preferred embodiment, random
fragments of DNA are produced by treating the DNA with a nuclease,
such as DNase I. The random DNA fragments are incorporated as
inserts in a SGE library. For general principles of DNase I partial
digestion and library construction see Molecular Cloning, A
Laboratory Manual, Sambrook et al., Eds., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1989). In certain embodiments
the inserted fragment may be expressed as part of a fusion protein.
In other embodiments the inserted fragment alone may be expressed.
In another embodiment, ribozyme-encoding sequences may be inserted
directly adjacent to the insert to allow for selection of most
efficient ribozyme-antisense clones. In still other embodiments the
gene suppression element library may be further modified by random
mutagenesis procedures known in the art. The inserted fragments may
be expressed from either a constitutive or an inducible
promoter.
[0090] In still another embodiment, the subject method is carried
out with a library encoding a variegated population of small
peptides, e.g., 4-25 amino acid residues in length. The library can
be generated from coding sequences of total cDNA, or single genes,
or can be random or semi-random in sequence. Small peptide
fragments, corresponding to only a minute portion of a protein, can
inhibit the function of that protein in vivo.
[0091] In still other embodiments, the subject method is carried
out with a transfection array which, when the target sequence is
transcribed in the host cell, gives rise to double stranded RNA,
e.g., for use in identifying dsRNA constructs which produce a
particular phenotype by RNA interference.
[0092] Libraries of coding sequences, whether encoding random
peptides or full length proteins, may be expressed in many ways,
including as portions of chimeric (fusion) proteins. In some
instances it may be necessary to introduce an unstructured
polypeptide linker region between portions of a chimeric protein
derived from different proteins. This linker can facilitate
enhanced flexibility of the chimeric protein allowing each portion
to fold correctly and retain appropriate biological activity in the
host cell. The linker can be of natural origin, such as a sequence
determined to exist in random coil between two domains of a
protein. Alternatively, the linker can be of synthetic origin. For
instance, the sequence (Gly.sub.4Ser).sub.3 can be used as a
synthetic unstructured linker. Linkers of this type are described
in Huston et al. (1988) PNAS 85:4879; and U.S. Pat. Nos. 5,091,513
and 5,258,498. Naturally occurring unstructured linkers of human
origin are preferred as they reduce the risk of immunogenicity.
[0093] Where secretion of, e.g., a peptide library is desired, the
peptide library can be engineered for secretion by including a
secretion signal sequence as part of a fusion protein with the
peptide.
[0094] In certain preferred embodiments, the transfection array
provides, in a single array, e.g., preferably at least 10 different
sequences, more preferably at least 100, 1000 or even 10,000
different, discrete sequences.
[0095] Preferably, target sequences are arrayed in an addressable
fashion, such as rows and columns where the substrate is a planar
surface.
[0096] If each feature size is about 100 microns on a side, each
chip can have about 10,000 target sequence addresses (features) in
a one centimeter square (cm.sup.2) area. In certain preferred
embodiments, the transfection array provides a density of at least
10.sup.3 different features per square centimeter (10.sup.3
sequences/cm.sup.2), and more preferably at least 10.sup.4
features/cm.sup.2, 10.sup.5 features/cm.sup.2, or even at least
10.sup.6 features/cm.sup.2. Of course, lower densities are
contemplated, such as at least 100 features/cm.sup.2.
[0097] In certain embodiments, the transfection array provides
multiple different target sequences in each feature, e.g., in order
to promote co-transfection of the host cells with at least two
different target sequences. Co-transfection refers to the
simultaneous introduction of two or more plasmids or other DNA or
nucleic acid constructs into the same cell. If the plasmids or
nucleic acid constructs direct the expression of a gene product,
such as a protein, RNA or other gene product, the cell will then
express both gene products at the same time.
[0098] Co-transfections can be performed with transfected cell
microarrays if the solution spotted on the surface where reverse
transfection occurs contains more than one plasmid or nucleic acid
construct. Of course, the collection of different target sequences
in one feature should be distinct from other features of the array.
The co-transfection features can include, for example, 2-10
different target sequences per feature, 10-100 different target
sequences per feature, or even more than 100 different target
sequences per feature.
[0099] The capacity to co-transfect cells in a transfected cell
microarray has many important uses. These include but are not
limited to the ability to: infer the expression of a gene product
by detecting the expression of a co-transfected plasmid encoding a
marker protein (e.g. GFP, luciferase, beta-galactosidase, or any
protein to which a specific antibody is available), express all the
components of a multi-subunit complex (e.g. the T-cell receptor) in
the same cells, express all the components of a signal transduction
pathway (e.g. MAP kinase pathway) in the same cells, and express
all the components of a pathway that synthesizes a small molecule
(e.g. polyketide synthetase). In addition, the capacity to
co-transfect allows the creation of microarrays with combinatorial
combinations of co-expressed plasmids. This capacity is
particularly useful for implementing mammalian two-hybrid assays in
which plasmids encoding bait and prey proteins are co-transfected
into the same cells by spotting them in one feature of the
microarray.
[0100] The capacity to co-transfect is also useful when the goal is
to promote differentiation of the transfected cells along a certain
tissue lineage. For example, combination of genes can be expressed
in a stem or early progenitor cells that will force the
differentiation of the cells into endothelial, liver, heart,
pancreatic, lymphoid, islet, brain, lung, kidney or other cell
types. In this fashion, arrays can be made with primary-like cells
that can be used to examine interactions of protein or small
molecules that are cell-type specific.
[0101] Furthermore, combinations of cDNAs can be printed in
different patterns on the surface on which reverse transfection
occurs. Patterns include, but are not limited to, bulls-eyes,
squares, rectangles of varying heights and widths, and lines of
single cell thickness. By printing, in particular patterns,
combinations of cDNAs that cause differentiation of cells into
different tissue types, this technology can be used to obtain
arrays with distinct cell types in distinct locations. This
capacity can be useful when trying to create tissue-like structures
on the array, such as blood capillaries and stromal structures, or
when studying the response of one cell type to the protein
secretions of another cell type. For example, a secreting cell type
can be created in the center of a bulls-eye pattern and responder
cell types of different tissues can be created on the edge of
bulls-eye. The response of the responder cells to the secretions of
the center cell can then be examined.
[0102] Arrays containing mixtures of plasmids at each feature could
be constructed, merely to illustrate, by mixing plasmids before
printing, printing in serial, printing with masks, or printing with
patterned printheads. For example, plasmids could be mixed in a
container before printing and printed as a homogenous mixture.
Alternatively, plasmids could be printed on top of one another or
close to one another. In this method, the exact composition of the
mixture containing each plasmid could be modified to control the
sequencing and timing of their entry into a cell, e.g. slower or
faster release mixtures. Masks with different patterns of holes or
print heads with different configurations could also be used to
print combinations of plasmids. For example, different enzymes
involved in polyketide synthesis could be combined to generate to
different polyketides.
[0103] The carrier for use in the methods of the present invention
can be, for example, gelatin or an equivalent thereof. In certain
embodiments, the carrier is a hydrogel, such a polycarboxylic acid,
cellulosic polymer, polyvinylpyrrolidone, maleic anhydride polymer,
polyamide, polyvinyl alcohol, or polyethylene oxide.
[0104] Any suitable surface which can be used to affix the nucleic
acid containing mixture to its surface can be used. For example,
the surface can be glass, plastics (such as
polytetrafluoroethylene, polyvinylidenedifluoride, polystyrene,
polycarbonate, polypropylene), silicon, metal, (such as gold),
membranes (such as nitrocellulose, methylcellulose, PTFE or
cellulose), paper, biomaterials (such as protein, gelatin, agar),
tissues (such as skin, endothelial tissue, bone, cartilage),
minerals (such as hydroxylapatite, graphite). -. Additional
compounds may be added to the base material of the surface to
provide functionality. For example, scintillants can be added to a
polystyrene substrate to allow Scintillation Proximity Assays to be
performed. The substrate may be a porous solid support or
non-porous solid support. The surface can have concave or convex
regions, patterns of hydrophobic or hydrophilic regions,
diffraction gratings, channels or other features. The scale of
these features can range from the meter to the nanometer scale. For
example, the scale can be on the micron scale for microfluidics
channels or other MEMS features or on the nanometer scale for
nanotubes or buckyballs. The surface can be planar, planar with
raised or sunken features, spherical (e.g. optically encoded
beads), fibers (e.g. fiber optic bundles), tubular (both interior
or exterior), a 3-dimensional network (such as interlinking rods,
tubes, spheres) or other shapes. The surface can be part of an
integrated system. For instance, the surface can be the bottom of a
microtitre dish, a culture dish, a culture chamber. Other
components such as lenses, gratings, electrodes can be integrated
with the surface. In general, the material of the substrate and
geometry of the array will be selected based on criteria that it be
useful for automation of array formation, culturing and/or
detection of cellular phenotype.
[0105] In still other embodiments, the solid support is a
microsphere (bead), especially a FACS sortable bead. Preferably,
each bead is an individual feature, e.g., having a homogenous
population of target sequences and distinct from most other beads
in the mixture, and one or more tags which can be used to the
identify any given bead and therefore the target sequence it
displays. The identity of any given target sequence that can induce
a FACS-detectable change in cells that adhere to the beads can be
readily determined from the tag(s) associate with the bead. For
example, the tag can be an electrophoric tagging molecules that are
used as a binary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926).
Exemplary tags are haloaromatic alkyl ethers that are detectable as
their trimethylsilyl ethers at less than femtomolar levels by
electron capture gas chromatography (ECGC). Variations in the
length of the alkyl chain, as well as the nature and position of
the aromatic halide substituents, permit the synthesis of at least
40 such tags, which in principle can encode 240 (e.g., upwards of
10.sup.12) different molecules. A more versatile system has,
however, been developed that permits encoding of essentially any
combinatorial library. Here, the compound would be attached to the
solid support via the photocleavable linker and the tag is attached
through a catechol ether linker via carbene insertion into the bead
matrix (Nestler et al. (1994) J Org Chem 59:4723-4724). This
orthogonal attachment strategy permits the FACS sorting of the
cell/bead entities and subsequent decoding by ECGC after oxidative
detachment of the tag sets from isolated beads. In other
embodiments, the beads can be tagged with two or more fluorescently
active molecules, and the identity of the bead is defined by the
ratio of the various fluorophores.
[0106] In still another embodiment, the transfection array can be
disposed on the end of a fiber optic system, such as a fiber optic
bundle. Each fiber optic bundle contains thousands to millions of
individual fibers depending on the diameter of the bundle. Changes
in the phenotype of cells applied to the transfection array can be
detected spectrometrically by conductance or transmittance of light
over the spatially defined optic bundle. An optical fiber is a clad
plastic or glass tube wherein the cladding is of a lower index of
refraction than the core of the tube. When a plurality of such
tubes are combined, a fiber optic bundle is produced. The choice of
materials for the fiber optic will depend at least in part on the
wavelengthes at which the spectrometric analysis of the transfected
cells is to be accomplished.
[0107] In addition, the surface can be coated with, for example, a
cationic moiety. The cationic moiety can be any positively charged
species capable of electrostatically binding to negatively charged
polynucleotides. Preferred cationic moieties for use in the carrier
are polycations, such as polylysine (e.g., poly-L-lysine),
polyarginine, polyornithine, spermine, basic proteins such as
histones (Chen et al. (1994) FEBS Letters 338:167-169), avidin,
protamines (see e.g., Wagner et al. (1990) PNAS 87: 3410-3414),
modified albumin (i.e., N-acylurea albumin) (see e.g., Huckett et
al. (1990) Chemical Pharmacology 40: 253-263) and polyamidoamine
cascade polymers (see e.g., Haensler et al. (1993) Bioconjugate
Chem. 4:372-379). A preferred polycation is polylysine (e.g.,
ranging from 3,800 to 60,000 daltons). Alternatively, the surface
itself can be positively charged (such as gamma amino propyl silane
or other alkyl silanes).
[0108] The surface can also be coated with molecules for additional
functions. For instance, these molecules can be capture reagents
such as antibodies, biotin, avidin, Ni-NTA to bind epitopes,
avidin, biotinylted molecules, or 6-His tagged molecules.
Alternatively, the molecules can be culture reagents such as
extracellular matrix, fetal calf serum, collagen.
[0109] The present invention also encompasses methods of making
arrays which comprise nucleic acid affixed to a surface such that
when cells are plated onto the surface bearing the arrayed nucleic
acid, the nucleic acid can be introduced (is introducible) into the
cells (i.e., the nucleic acid can move from the surface into the
cells). The present invention also encompasses a nucleic acid array
comprising a surface having affixed thereto, in discrete, defined
locations, nucleic acid of known sequence or source by a method
described herein.
[0110] In certain embodiments, once the microarrays of transfected
cells have formed (i.e. cDNAs in the spots have entered cells and
the cells have expressed the encoded gene products), the
microarrays can be transferred onto a variety of surfaces. Surfaces
can be flexible or non-flexible and porous or non-porous. The
surfaces can be flat or patterned with concave or convex regions,
patterns of hydrophobic or hydrophilic regions, diffraction
gratings, channels or other features. The scale of these features
can range from the meter to the nanometer scale. Examples of
surfaces include but are not limited to, glass, plastics (such as
polytetrafluoroethylene, polyvinylidenedifluoride, polystyrene,
polyearbonate, polypropylene), silicon, metal, (such as gold),
membranes (such as nitrocellulose, methylcellulose, PTFE or
cellulose, polyvinylidene fluoride (PVDF)), paper, biomaterials
(such as protein, gelatin, agar), tissues (such as skin,
endothelial tissue, bone, cartilage), minerals (such as
hydroxylapatite, graphite). Furthermore, many of these surfaces can
be derivatized to provide additional functionalities. For example,
scintillants can be added to a polystyrene substrate to allow
Scintillation Proximity Assays to be performed. In another example,
nitrocellulose membranes can be covalently modified with metal
chelators that immoblize metals, such as nickel or cobalt, and
allow the selective binding of proteins carrying a specific amino
acid sequence, such as a hexa-histidine tag (6.times.His).
[0111] Transfers can be performed so that 1) the entire cellular
material on the microarray is transferred (i.e. both the endogenous
and recombinant materials made by the cells (RNA or protein)), or
2) so that only the recombinant material is transferred. The
transfer of the microarray to another surface is accomplished by
directly contacting the microarray to the other surface and
allowing the material to move to the new surface under the
influence of a force, such as capillary forces (commonly referred
to as `blotting`), electric or magnetic fields, vacuum suction
forces, or other forces. The material binds to the new surface
through an interaction mediated by hydrophobic, hydrophillic, Van
der Waals, ionic or other forces, or through specific
receptor-ligand interactions (e.g. antibody-epitope interactions)
or by becoming entangled in the molecular structure of the other
surface.
[0112] The ability to transfer cellular material from the
microarrays to another surface has many important uses. These
include, but are not limited to, the capacity to detect cellular
phenotypes or protein properties using techniques normally
performed on specific surfaces and the capacity to in parallel
purify the recombinant gene products expressed in the microarray.
Examples of techniques normally performed on specific surfaces
include western blotting, far-western blotting, southwestern
blotting, surface plasmon resonance (SPR), mass spectroscopy, and
others. These techniques normally require the immobilization of
native or denatured proteins on nitrocellulose, nylon, paper,
polyvinylidene fluoride (PVDF), or gold or other metal surfaces or
membranes. Southwestern blotting is used to detect the interaction
of a nucleic acid (such as DNA or RNA) with a protein. After
transfer to an appropriate membrane, microarrays of cells
expressing a collection of DNA binding proteins, such as
transcription factors, could be used to identify binding proteins
for genomic DNA sequence elements.
[0113] The transfer of microarrays to other surfaces is also useful
for the in parallel purification of the recombinant proteins
expressed on the microarray. In one embodiment of this approach,
all the recombinant proteins expressed on the microarray contain an
amino acid sequence that is a ligand for a specific protein or
chemical reagent (e.g. an epitope recognized by a polyclonal or
monoclonal antibody or a hexa-histidine tag recognized by a nickel
affinity matrix). Micorarrays expressing these proteins are then
transferred by direct contact to a surface that has been
derivatized with the reagent that binds the ligand (e.g. a
nitrocellulose membrane to which an anti-epitope monoclonal
antibody is bound or a nitrocellulose membrane derivatized with a
metal chelator that allows the binding of nickel to its surface).
After the material has bound to the new surface, the surface is
washed with an appropriate buffer that does not disrupt the
specific interaction but eliminates non-specific interactions with
the surface. Non-specific interactions include but are not limited
to the interactions of any cellular components that do not contain
the specific ligand recognized by the surface to which the
microarray has been transferred. The microarray of recombinant
proteins can then used to detect the interaction of other proteins
or small molecules with the array. The binding of proteins or small
molecules with the microarray can be detected with autoradiography,
fluorescence, mass spectroscopy, immunofluorescence, or
calorimetry.
[0114] Below is a proof of concept example for the transfer to a
nitrocelluloes membrane of a microarray of cells expressing
epitope-tagged proteins and growing on a glass slide.
[0115] Microarrays are transferred onto nitrocellulosemembranes and
the proteins detected with standard western blotting protocols. The
figure is an example of an array of myc-tagged proteins detected
via enhanced chemiluminescence using a standard anti-myc western
blotting protocol. The middle two rows (horizontally) are printed
with half the amount of the expression construct as the top and
bottom rows. The signal was detected with Kodak X-OMAT AR film and
each spot is .about.150 um in diameter.
[0116] To illustrate, when the microarrays are ready to be
processed (usually 1-2 days after transfection), forceps are used
to lift the slide from the culture dish and quickly rinse it with
PBS (phosphate buffered saline) in a Coplin Jar. After the rinse,
excess PBS is removed from the slide by briefly blotting its edge
with an absorbent paper towel. The slide is then placed with the
cells facing up on a flat surface, immobilized with tape and
allowed to dry for 2-3 minutes (this time can vary depending on how
much PBS remains on the slide). A nitrocellulose membrane about two
to three times the area of the slide(0.45 .mu.m pure nitrocellulose
membrane; cat. 162-0116, BioRad) is then very carefully place on
the slide, in a similar manner as is done for traditional plaque
lifts (i.e. center first). At this time it is very important to not
permit any horizontal movement of the membrane or slide at this
step. The membrane is kept on the slide for 1-3 minutes or until
the PBS has wetted the entire area of the membrane that covers the
slide. It is important to not press down on or roll a pin over the
membrane as this will invariably cause the membrane to slip and
destroy the array. Also, it is important to not allow all the
moisture on the slide to be transferred to the membrane as this
will cause the membrane to stick to the slide and it will tear when
it is lifted off. After transfer, the nitrocellulose membrane is
carefully lifted off the slide surface with forceps and allowed it
to air dry for 2 hours. After drying the membrane is dipped into a
pH 11 CAPS-methanol transfer buffer ( 2.2 g/l CAPS, 10% methanol,
pH 11) for 1-2 minutes and placed in a standard western blot
blocking solution. The membrane is then processed with primary and
secondary antibodies as in any standard western blotting
protocol.
[0117] IV. Cells
[0118] Suitable host cells for generating the subject assay include
prokaryotes, yeast, or higher eukaryotic cells, including plant and
animal cells, especially mammalian cells. Prokaryotes include gram
negative or gram positive organisms.
[0119] In certain preferred embodiments, the subject method is
carried out using cells derived from higher eukaryotes, e.g.,
metazoans, and in especially preferred embodiments, are mammalian
cells, and even more preferably are primate cells such as human
cells. Other preferred species of mammalian cells include canine,
feline, bovine, porcine, mouse and rat. For instance, such cells
can be hematopoietic cells, neuronal cells, pancreatic cells,
hepatic cells, chondrocytes, osteocytes, or myocytes. The cells can
be fully differentiated cells or progenitor/stem cells.
[0120] Moreover, the cells can be derived from normal or diseased
tissue, from differentiated or undifferentiated cells, from
embryonic or adult tissue.
[0121] The cells may be dispersed in culture, or can be tissues
samples containing multiple cells which retain some of the
microarchitecture of the organ.
[0122] In certain embodiments, the transfection array of the
subject invention is used to transfect a cell that can be
co-cultured with a target cell. A biologically active protein
secreted by the cells expressing genes from the transfection array
will diffuse to neighboring target cells and induce a particular
biological response, such as to illustrate, proliferation or
differentiation, or activation of a signal transduction pathway
which is directly detected by other phenotypic criteria. Likewise,
antagonists of a given factor can be selected in similar fashion by
the ability of the cell producing a functional antagonist to
protect neighboring cells from the effect of exogenous factor added
to the culture media. The host and target cells can be in direct
contact, or separated by, e.g., a cell culture insert (e.g.
Collaborative Biomedical Products, Catalog #40446).
[0123] If yeast cells are used, the yeast may be of any species
which are cultivable and in the transfection array can be
maintained upon transfection. Suitable species include Kluyverei
lactis, Schizosaccharomyces pombe, and Ustilaqo maydis;
Saccharomyces cerevisiae is preferred. Other yeast which can be
used in practicing the present invention are Neurospora crassa,
Aspergillus niger, Aspergillus nidulans, Pichia pastoris, Candida
tropicalis, and Hansenula polymorpha. The term "yeast", as used
herein, includes not only yeast in a strictly taxonomic sense,
i.e., unicellular organisms, but also yeast-like multicellular
fungi or filamentous fungi.
[0124] The choice of appropriate host cell will also be influenced
by the choice of detection signal. For instance, reporter
constructs can provide a selectable or screenable trait upon
gain-of-function or loss-of-function induced by a target nucleic
acid. The reporter gene may be an unmodified gene already in the
host cell pathway, or it may be a heterologous gene (e.g., a
"reporter gene construct"). In other embodiments, second messenger
generation can be measured directly in a detection step, such as
mobilization of intracellular calcium or phospholipid metabolism,
in which case the host cell should have an appropriate starting
phenotype for activation of such pathways.
[0125] The host cells are plated (placed) onto the surface bearing
the transfection array in sufficient density and under appropriate
conditions for introduction/entry of the nucleic acid into the
cells. Preferably, the host cells (in an appropriate medium) are
plated on the array at high density (e.g., on the order of
0.5-1.times.10.sup.5/cm.sup.2), in order to increase the likelihood
that transfection will occur. For example, the density of cells can
be from about 0.3.times.10.sup.5/cm.sup.2 to about
3.times.10.sup.5/cm.sup.2, and in specific embodiments, is from
about 0.5.times.10.sup.5/cm to about 2.times.10.sup.5/cm.sup.2 and
about 0.5.times.10.sup.5/cm.sup.2 to about
1.times.10.sup.5/cm.sup.2. The appropriate conditions for
introduction/entry of DNA into cells will vary depending on the
quantity of cells used.
[0126] In certain embodiments, the host cells can engineered to
express other recombinant genes. For instance, the host cells can
be engineered with a reporter gene construct, and the ability of
members of the transfection array to alter the level of expression
of the reporter gene can be assessed. Merely to illustrate, the
transfection array can be assessed for members which encode
transcriptional activators or transcriptional repressors of the
reporter gene, and may include native and non-native sequences. For
instance, the host cell can be transfected with reporter gene
construct including a promoter sequence for which a protein which
binds that sequence is sought. The transfection array can encode a
library of potential DNA binding domains fused to a polymerase
activation domain. Members of the library are selected by their
ability to induce expression of the reporter gene. Conversely, the
DNA binding specificity of a DNA binding protein can be determined
by arraying a library of reporter gene constructs which are
variegated with respect to the sequence of a transcriptional
regulatory element. The cell also expresses the DNA binding
protein, e.g., which naturally or by engineering includes a
transcriptional activation domain. Those members of the reporter
gene construct library which include appropriate regulatory
sequences are expressed, and the position of those constructs in
the array used to determine the consensus sequence for the DNA
binding protein.
[0127] In other instances, the host cells can be engineered so as
to have a loss-of-function or gain-of-function phenotype, and the
ability of the ability of members of the transfection array to
counteract such a phenotype is assessed.
[0128] In still other instances, the host cells are engineered to
express a recombinant cell surface receptor, and the transfection
array encodes a variegated library of gene products or peptides,
and the ability of one or more members of that library to induce or
inhibit signal transduction by the receptor is assessed. For
instance, the transfection array can provide a library of secreted
peptides, and the ability of a given peptide to induce signal
transduction is detected by the conversion of the cell to an
autocrine phenotype.
[0129] V. Detection
[0130] A variety of methods can be used to detect the consequence
of uptake, and in many embodiments, expression (at least
transcription) of the target sequences. In a general sense, the
assay provides the means for determining if the target sequence is
able to confer a change in the phenotype of the cell relative to
the same cell but which lacks the target sequence. Such changes can
be detected on a gross cellular level, such as by changes in cell
morphology (membrane ruffling, rate of mitosis, rate of cell death,
mechanism of cell death, dye uptake, and the like). In other
embodiments, the changes to the cell's phenotype, if any, are
detected by more focused means, such as the detection of the level
of a particular protein (such as a selectable or detectable
marker), or level of mRNA or second messenger, to name but a few.
Changes in the cell's phenotype can be determined by assaying
reporter genes (beta-galactosidase, green fluorescent protein,
beta-lactamase, luciferase, chloramphenicol acetyl transferase),
assaying enzymes, using immunoassays, staining with dyes (e.g.
DAPI, calcofluor), assaying electrical changes, characterizing
changes in cell shape, examining changes in protein conformation,
and counting cell number. Other changes of interest could be
detected by methods such as chemical assays, light microscopy,
scanning electron microscopy, transmission electron microscopy,
atomic force microscopy, confocal microscopy, image reconstruction
microscopy, scanners, autoradiography, light scattering, light
absorbance, NMR, PET, patch clamping, calorimetry, mass
spectrometry, surface plasmon resonance, time resolved
fluorescence. Data could be collected at single or multiple time
points and analyzed by the appropriate software.
[0131] For example, immunofluorescence can be used to detect a
protein. Alternatively, expression of proteins that alter the
phosphorylation state or subcellular localization of another
protein, proteins that bind with other proteins or with nucleic
acids or proteins with enzymatic activity can be detected.
[0132] In one embodiment, the screen can be for the inability to
grow or survive when a parasite or infectious agent is added to the
cell of interest. In this case the selection would be for
knock-outs that are targeting genes that are specifically essential
for some aspect of viral or parasitic function within a cell that
are only essential when that cell is infected. Since some viral
infection result in the induction of survival factors (such as
CrmA, p35) it is likely that at least some cell functions are
different and potentially selectively needed during viral, parasite
growth.
[0133] Another type of screening method means is for the expression
of a specific factor that can be measured and this measurement can
be adapted for a screen. This factor can be anything that is
accessible to measurement, including but not limited to, secreted
molecules, cell surface molecules, soluble and insoluble molecules,
binding activities, activities that induce activities on other
cells or induce other organic or inorganic chemical reactions.
These interactions can be detected by Time Resolved Fluorescence,
Surface Plasmon Resonance, Scintillation Proximity Assays,
autoradiography, Fluorescence Activated Cell Sorting, or other
methods.
[0134] Still another screening method is for changes in cell
structure that are detected by any means that could be adapted for
a selection scheme. This includes, but is not limited to,
morphological changes that are measured by physical methods such as
differential sedimentation, differential light scattering,
differential buoyant density, differential cell volume selected by
sieving, atomic force microscopy, electron microscopy.
[0135] When screening for bioactivity of test compounds,
intracellular second messenger generation can be measured directly.
Such embodiments are useful where, for example, the arrayed library
is being screened for target sequences which activate or inactivate
a particular signaling pathway. A variety of intracellular
effectors have been identified as being receptor- or ion
channel-regulated, including adenylyl cyclase, cyclic GMP,
phosphodiesterases, phosphoinositidases, phosphoinositol kinases,
and phospholipases, as well as a variety of ions.
[0136] In one embodiment, the GTPase enzymatic activity by G
proteins can be measured in plasma membrane preparations by
determining the breakdown of .gamma..sup.32P GTP using techniques
that are known in the art (For example, see Signal Transduction: A
Practical Approach. G. Milligan, Ed. Oxford University Press,
Oxford England). When receptors that modulate cAMP are tested, it
will be possible to use standard techniques for cAMP detection,
such as competitive assays which quantitate [.sup.3H]cAMP in the
presence of unlabelled cAMP.
[0137] Certain receptors and ion channels stimulate the activity of
phospholipase C which stimulates the breakdown of
phosphatidylinositol 4,5, bisphosphate to 1,4,5-IP3 (which
mobilizes intracellular Ca++) and diacylglycerol (DAG) (which
activates protein kinase C). Inositol lipids can be extracted and
analyzed using standard lipid extraction techniques. DAG can also
be measured using thin-layer chromatography. Water soluble
derivatives of all three inositol lipids (IPI, IP2, IP3) can also
be quantitated using radiolabelling techniques or HPLC.
[0138] The other product of PIP2 breakdown, DAG can also be
produced from phosphatidyl choline. The breakdown of this
phospholipid in response to receptor-mediated signaling can also be
measured using a variety of radiolabelling techniques.
[0139] The activation of phospholipase A2 can easily be quantitated
using known techniques, including, for example, the generation of
arachadonate in the cell.
[0140] In various cells, e.g., mammalian cells, specific proteases
are induced or activated in each of several arms of divergent
signaling pathways. These may be independently monitored by
following their unique activities with substrates specific for each
protease.
[0141] In the case of screening for ligands to certain receptors
and ion channels, it may be desirable to screen for changes in
cellular phosphorylation. Such assay formats may be useful when the
host cell expresses a receptor of interest, such as a receptor
kinase or phosphatase, and the arrayed library is being screened
for peptide sequences which can act in an autocrine fashion. For
example, immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad.
Sci. USA 81:7426-7430) using anti-phosphotyrosine,
anti-phosphoserine or abti-phosphothreonine antibodies. In
addition, tests for phosphorylation could be also useful when the
receptor itself may not be a kinase, but activates protein kinases
or phosphatase that function downstream in the signal transduction
pathway.
[0142] In yet another embodiment, the signal transduction pathway
of the targeted receptor or ion channel upregulates expression or
otherwise activates an enzyme which is capable of modifies a
substrate which can be added to the cell. The signal can be
detected by using a detectable substrate, in which case lose of the
substrate signal is monitored, or altenatively, by using a
substrate which produces a detectable product. In preferred
embodiments, the conversion of the substrate to product by the
activated enzyme produces a detectable change in optical
characteristics of the test cell, e.g., the substrate and/or
product is chromogenically or fluorogenically active. In an
illustrative embodiment the signal transduction pathway causes a
change in the activity of a proteolytic enzyme, altering the rate
at which it cleaves a substrate peptide (or simply activates the
enzyme towards the substrate). The peptide includes a fluorogenic
donor radical, e.g., a fluorescence emitting radical, and an
acceptor radical, e.g., an aromatic radical which absorbs the
fluorescence energy of the fluorogenic donor radical when the
acceptor radical and the fluorogenic donor radical are covalently
held in close proximity. See, for example, U.S. Ser. Nos.
5,527,681, 5,506,115, 5,429,766, 5,424,186, and 5,316,691; and
Capobianco et al. (1992) Anal Biochem 204:96-102. For example, the
substrate peptide has a fluorescence donor group such as
1-aminobenzoic acid (anthranilic acid or ABZ) or
aminomethylcoumarin (AMC) located at one position on the peptide
and a fluorescence quencher group, such as lucifer yellow, methyl
red or nitrobenzo-2-oxo-1,3-diazole (NBD), at a different position
near the distal end of the peptide. A cleavage site for the
activated enzyme will be diposed between each of the sites for the
donor and acceptor groups. The intramolecular resonance energy
transfer from the fluorescence donor molecule to the quencher will
quench the fluorescence of the donor molecule when the two are
sufficiently proximate in space, e.g., when the peptide is intact.
Upon cleavage of the peptide, however, the quencher is separated
from the donor group, leaving behind a fluorescent fragment. Thus,
activation of the enzyme results in cleavage of the detection
peptide, and dequenching of the fluorescent group.
[0143] In a preferred embodiment, the enzyme which cleaves the
detection peptide is one which is endogenous to the host cell. For
example, the barl gene of yeast encodes a protease, the expression
of which is upregulated by stimulation of the yeast pheromone
pathway. Thus, host cells which have been generated to exploit the
pheromone signal pathway for detection can be contacted with a
sutable detection peptide which can be cleaved by barl to release a
fluorogenic fragment, and the level of barl activity thus
determined.
[0144] In still other embodiments, the detectable signal can be
produced by use of enzymes or chromogenic/fluorscent probes whose
activities are dependent on the concentration of a second
messanger, e.g., such as calcium, hydrolysis products of inositol
phosphate, cAMP, etc. For example , the mobilization of
intracellular calcium or the influx of calcium from outside the
cell can be measured using standard techniques. The choice of the
appropriate calcium indicator, fluorescent, bioluminescent,
metallochromic, or Ca++-sensitive microelectrodes depends on the
cell type and the magnitude and time constant of the event under
study (Borle (1990) Environ Health Perspect 84:45-56). As an
exemplary method of Ca++ detection, cells could be loaded with the
Ca++ sensitive fluorescent dye fura-2 or indo-l, using standard
methods, and any change in Ca++ measured using a fluorometer.
[0145] As certain embodiments described above suggest, the signal
transduction activity for which an agonist or antagonist is sought
in the arrayed library can be measured by detection of a
transcription product, e.g., by detecting transcriptional
activation (or repression) of an indicator gene(s). Detection of
the transcription product includes detecting the gene transcript,
detecting the product directly (e.g., by immunoassay) or detecting
an activity of the protein (e.g., such as an enzymatic activity or
chromogenic/fluorogenic activity); each of which is generally
referred to herein as a means for detecting expression of the
indicator gene. The indicator gene may be an unmodified endogenous
gene of the host cell, a modified endogenous gene, or a part of a
completely heterologous construct, e.g., as part of a reporter gene
construct.
[0146] In one embodiment, the indicator gene is an unmodified
endogenous gene. For example, the instant method can rely on
detecting the transcriptional level of such endogenous genes as the
c-fos gene (e.g., in mammalian cells) or the Bar1 or Fus1 genes
(e.g., in yeast cells) in response to such signal transduction
pathways as originating from G protein coupled receptors.
[0147] In certain instances, it may be desirable to increase the
level of transcriptional activation of the endogenous indicator
gene by the signal pathway in order to, for example, improve the
signal-to-noise of the test system, or to adjust the level of
response to a level suitable for a particular detection technique.
In one embodiment, the transcriptional activation ability of the
signal pathway can be amplified by the overexpression of one or
more of the proteins involved in the intracellular signal cascade,
particularly enzymes involved in the pathway. For example,
increased expression of Jun kinases (JNKs) can potentiate the level
of transcriptional activation by a signal in an MEKK/JNKK pathway.
Likewise, overexpression of one or more signal transduction
proteins in the yeast pheromone pathway can increase the level of
Fus1 and/or Bar1 expression. This approach can, of course, also be
used to potentiate the level of transcription of a heterologous
reporter gene as well.
[0148] In other embodiments, the sensitivity of an endogenous
indicator gene can be enhanced by manipulating the promoter
sequence at the natural locus for the indicator gene. Such
manipulation may range from point mutations to the endogenous
regulatory elements to gross replacement of all or substantial
portions of the regulatory elements. In general, manipulation of
the genomic sequence for the indicator gene can be carried out
using techniques known in the art, including homologous
recombination.
[0149] In still another embodiment, a heterologous reporter gene
construct can be used to provide the function of an indicator gene.
Reporter gene constructs are prepared by operatively linking a
reporter gene with at least one transcriptional regulatory element.
If only one transcriptional regulatory element is included it must
be a regulatable promoter. At least one the selected
transcriptional regulatory elements must be indirectly or directly
regulated by the activity of the selected cell-surface receptor
whereby activity of the receptor can be monitored via transcription
of the reporter genes.
[0150] Many reporter genes and transcriptional regulatory elements
are known to those of skill in the art and others may be identified
or synthesized by methods known to those of skill in the art.
[0151] Examples of reporter genes include, but are not limited to
CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979),
Nature 282: 864-869) luciferase, and other enzyme detection
systems, such as beta-galactosidase; firefly luciferase (deWet et
al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase
(Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al.
(1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et
al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J.
Mol. Appl. Gen. 2: 101), human placental secreted alkaline
phosphatase (Cullen and Malim (1992) Methods in Enzymol.
216:362-368); .beta.-lactamase or GST.
[0152] Transcriptional control elements for use in the reporter
gene constructs, or for modifying the genomic locus of an indicator
gene include, but are not limited to, promoters, enhancers, and
repressor and activator binding sites. Suitable transcriptional
regulatory elements may be derived from the transcriptional
regulatory regions of genes whose expression is linked to the
desired phenotype sought from the arrayed library.
[0153] In the case of receptors which modulate cyclic AMP, a
transcriptional based readout can be constructed using the cyclic
AMP response element binding protein, CREB, which is a
transcription factor whose activity is regulated by phosphorylation
at a particular serine (S133). When this serine residue is
phosphorylated, CREB binds to a recognition sequence known as a CRE
(cAMP Responsive Element) found to the 5' of promotors known to be
responsive to elevated cAMP levels. Upon binding of phosphorylated
CREB to a CRE, transcription from this promoter is increased.
[0154] Phosphorylation of CREB is seen in response to both
increased cAMP levels and increased intracellular Ca levels.
Increased cAMP levels result in activation of PKA, which in turn
phosphorylates CREB and leads to binding to CRE and transcriptional
activation. Increased intracellular calcium levels results in
activation of calcium/calmodulin responsive kinase II (CaM kinase
II). Phosphorylation of CREB by CaM kinase II is effectively the
same as phosphorylation of CREB by PKA, and results in
transcriptional activation of CRE containing promotors.
[0155] Therefore, a transcriptionally-based readout can be
constructed in cells containing a reporter gene whose expression is
driven by a basal promoter containing one or more CRE. Changes in
the intracellular concentration of Ca++ (a result of alterations in
the activity of the receptor upon engagement with a ligand) will
result in changes in the level of expression of the reporter gene
if: a) CREB is also co-expressed in the cell, and b) either an
endogenous or heterologous CaM kinase phosphorylates CREB in
response to increases in calcium or if an exogenously expressed CaM
kinase II is present in the same cell. In other words, stimulation
of PLC activity may result in phosphorylation of CREB and increased
transcription from the CRE-construct, while inhibition of PLC
activity may result in decreased transcription from the
CRE-responsive construct.
[0156] In preferred embodiments, the reporter gene is a gene whose
expression causes a phenotypic change which is screenable or
selectable. If the change is selectable, the phenotypic change
creates a difference in the growth or survival rate between cells
which express the reporter gene and those which do not. If the
change is screenable, the phenotype change creates a difference in
some detectable characteristic of the cells, by which the cells
which express the marker may be distinguished from those which do
not. Selection is preferable to screening in that it can provide a
means for amplifying from the cell culture those cells which
express a test polypeptide which is a receptor effector.
[0157] The marker gene is coupled to the receptor signaling pathway
so that expression of the marker gene is dependent on activation of
the receptor. This coupling may be achieved by operably linking the
marker gene to a receptor-responsive promoter. The term
"receptor-responsive promoter" indicates a promoter which is
regulated by some product of the target receptor's signal
transduction pathway.
[0158] Alternatively, the promoter may be one which is repressed by
the receptor pathway, thereby preventing expression of a product
which is deleterious to the cell. With a receptor repressed
promoter, one screens for agonists by linking the promoter to a
deleterious gene, and for antagonists, by linking it to a
beneficial gene. Repression may be achieved by operably linking a
receptor- induced promoter to a gene encoding mRNA which is
antisense to at least a portion of the mRNA encoded by the marker
gene (whether in the coding or flanking regions), so as to inhibit
translation of that mRNA. Repression may also be obtained by
linking a receptor-induced promoter to a gene encoding a DNA
binding repressor protein, and incorporating a suitable operator
site into the promoter or other suitable region of the marker
gene.
[0159] In the case of yeast, suitable positively selectable
(beneficial) genes include the following: URA3, LYS2, HIS3, LEU2,
TRP1; ADE,1, 2, 3, 4, 5, 7, 8; ARG1, 3, 4, 5, 6, 8; HIS1,4, 5;
ILV1, 2, 5; THR1, 4; TRP2, 3, 4, 5; LEU1, 4,MET2, 3, 4, 8, 9, 14,
16, 19; URA1, 2, 4, 5, 10; HOM3, 6, ASP3; CHO1; ARO 2, 7; CYS3;
OLE1; IN01, 2, 4; PRO1, 3 Countless other genes are potential
selective markers. The above are involved in well-characterized
biosynthetic pathways. The imidazoleglycerol phosphate dehydratase
(IGP dehydratase) gene (HIS3) is preferred because it is both quite
sensitive and can be selected over a broad range of expression
levels. In the simplest case, the cell is auxotrophic for histidine
(requires histidine for growth) in the absence of activation.
Activation leads to synthesis of the enzyme and the cell becomes
prototrophic for histidine (does not require histidine). Thus the
selection is for growth in the absence of histidine. Since only a
few molecules per cell of IGP dehydratase are required for
histidine prototrophy, the assay is very sensitive.
[0160] The marker gene may also be a screenable gene. The screened
characteristic may be a change in cell morphology, metabolism or
other screenable features. Suitable markers include
beta-galactosidase (Xgal, C.sub.12FDG, Salmon-gal, Magenta-Gal
(latter two from Biosynth Ag)), alkaline phosphatase, horseradish
peroxidase, exo-glucanase (product of yeast exbl gene;
nonessential, secreted); luciferase; bacterial green fluorescent
protein; (human placental) secreted alkaline phosphatase (SEAP);
and chloramphenicol transferase (CAT). Some of the above can be
engineered so that they are secreted (although not
.beta.-galactosidase). A preferred screenable marker gene is
beta-galactosidase; yeast cells expressing the enzyme convert the
colorless substrate Xgal into a blue pigment. Again, the promoter
may be receptor-induced or receptor-inhibited.
[0161] VI. Exemplary Uses
[0162] A. Target identification.
[0163] The binding partners for molecules such as drugs, hormones,
interleukins, or secreted proteins can be identified by incubating
the compounds of interest with an array that overexpresses
potential targets within each array feature or combinations of
potential targets within each cell of an array feature. Binding
could be detected by methods such as SPR, SPA, TRF, or
autoradiography. In addition, the binding partners for cells could
be identified by incubating the cell of interest with arrays or
color-encoded beads. For instance, migratory or free-floating test
cells could be incubated with an array, allowed to migrate or bind,
and then the binding or migration detected by standard methods,
e.g. expressing GFP or other markers in the test cells.
Alternatively, the test cells could be mixed with a collection of
color-encoded beads, each expressing a distinct DNA construct with
a unique color code, e.g. a unique ratio of red to green dyes.
Binding could then be detected by fluorescence activated cell
sorting or other methods.
[0164] The array could also be used to identify the targets of an
organism's immune response to cancer, an infectious or autoimmune
disease, exposure to chemicals, or environmental changes. An array
expressing target proteins could be incubated with sera from the
organism. Binding of antibodies could be detected by labeling the
sera or using the appropriate secondary antibody. The identified
targets of the immune response could be used to design vaccines
against tumors or infectious diseases, immunosuppressive drugs,
anti-infective drugs or others.
[0165] In other embodiments, the present invention faciliates drug
target discovery by permitting the identification of an endogenous
gene whose inhibit or activation may be of therapeutic value. The
strategy relies, in part, on the ability of small gene fragments to
encode dominant-acting snthetic genetic elements (SGEs), e.g.,
molecules that interfere with the function of genes from which they
are derived (antagonists) or that are dominant consitutively active
fragments (agonists) of such genes. SGEs that can be identified by
the subject method include, but are not limited to, polypeptides,
inhibitory antisense RNA molecules, ribozymes, nucleic acid decoys,
and small peptides. For instance, a gene whose activity is
inactivated by an identified SGE can itself be used as a target for
drug development, e.g., to identify other agents, such as small
molecules and natural extracts, which can also inhibit the function
of the endogenous gene. Thus, another aspect of the present
invention provides drug screening assays for detecting agonists or
antagonists, as appropriate, of a gene (or gene product thereof)
that corresponds to a selected SGE. Likewise, the identification of
an SGE that can inhibit a particular pathological phenotype will
indicate diagnostic assays that can assess loss-of-function or
gain-of-function mutations, as appropriate, to the corresponding
endogenous gene.
[0166] In other embodiments, the use of transcription arrays which
give rise to dsRNA in the host cell can be used to assess the
loss-of-function of a particular gene. "RNA interference",
"post-transcriptional gene silencing", "quelling"--these different
names describe similar effects that result from the overexpression
or misexpression of transgenes, or from the deliberate introduction
of double-stranded RNA into cells (reviewed in Fire A (1999) Trends
Genet 15:358-363; Sharp P A (1999) Genes Dev 13:139-141; Hunter C
(1999) Curr Biol 9:R440-R442; Baulcombe D C (1999) Curr Biol
9:R599-R601; Vaucheret et al. (1998) Plant J 16:651-659). The
injection of double-stranded RNA into a cell can act systemically
to cause the post-transcriptional depletion of the homologous
endogenous RNA (Fire et al. (1998) Nature 391: 80681 1; and
Montgomery et al. (1998) PNAS 95:15502-15507). RNA interference,
commonly referred to as RNAi, offers a way of specifically and
potently inactivating a cloned gene, and is proving a powerful tool
for investigating gene function.
[0167] To illustrate, the subject method contemplates (a)
constructing a cDNA or genomic transfection array including cDNA or
genomic DNA in an orientation relative to a promoter(s) capable of
initiating transcription of the cDNA or genomic DNA to double
stranded RNA; (b) introducing the transfection array into cells by
the subject method, (c) identifying and isolating cells in which a
member of the transfection array confers a particular phenotype,
and (D) identifying the gene sequence from the library which gave
rise to the dsRNA construct responsible for conferring the
phenotype.
[0168] B. Target validation
[0169] The expression pattern of potential genes of interest could
be tested by constructing an array where each spot contains a
construct fusing regulatory sequences from the genes of interest
with a reporter gene. The regulatory sequences could be involved in
transcription, RNA processing or translation. The reporter gene
could be GFP, beta galactosidase, luciferase, beta lactamase or
other genes. The expression of the genes of interest could be
tested by incubating the array with different combinations of
conditions and cell lines and then assaying for the activity of the
reporter gene. Genes with the appropriate expression patterns could
then be studied further as potential drug targets.
[0170] The function of the gene of interest could be tested by
making arrays where DNA constructs modify the function of the gene
of interest and assaying the phenotype. These modifications could
be derived from methods such as overexpression, knockout
constructs, dominant negative mutants, anti-sense RNA, ribozyme RNA
or others. The resulting phenotypic change could be assayed under
different environmental conditions, genetic backgrounds and cell
types. For instance, genes which activate or inhibit a pathway
could be identified by examining the phenotype of cells on an array
where each feature overexpresses or underexpresses a gene of
interest. Genes with the appropriate phenotypes could then be
studied further as potential drug targets.
[0171] The function of a gene of interest could also be inferred by
identifying the binding partners for a protein of interest. For
instance, an array expressing proteins of interest could be tested
for DNA-binding, RNA-binding, protein-binding, nucleotide binding
or other functions by incubating the array with the appropriately
labeled molecule and/or detection system. Different classes of
proteins, e.g. DNA-binding proteins, could be identified and the
sequences examined for the discovery of novel binding motifs.
Alternatively, a two hybrid or three hybrid system could be used to
identify potential protein, RNA or other classes of binding
partners in vivo. For instance, the gene of interest could be
cloned into the appropriate "bait" vector and stably transfected in
a cell line with the appropriate reporter construct. The
interaction of the gene of interest with other potential partners
could be tested by using this cell line in an array of constructs
where test proteins are cloned into the appropriate "test" vector.
Alternatively, an array of affinity tagged constructs (e.g.
6.times.His, epitopes, avidin) could be transferred to an affinity
membrane, e.g. (Ni-NTA, anti-epitope antibody, biotinylated).
Associated proteins could be detected and identified by mass spec
or other methods. Proteins with the appropriate binding partners
could then be further investigated as potential targets.
[0172] The function of a gene of interest could also be inferred by
identifying its post-translational modifications. An array
expressing proteins of interest could be tested for
phosphorylation, sulfation, ubiquitination, glycosylation or other
post-translational modifications by incubation with the appropriate
labeling or detection reagent such as radiolabeled precursors,
anti-phosphoamino acid antibodies, anti-ubiquitin, lectins or other
specific detection reagents. Alternatively, post-translational
modifications could be detected by transferring the array to an
affinity membrane and then using mass spectrometry.
[0173] Subcellular localization of a protein could be investigated
by making an array where each feature contains a DNA construct with
the protein of interest fused to an epitope tag, GFP or other
marker. After transfection and cell growth, immunofluorescence
could be performed with a microscope, high resolution scanner or
other detection method to determine whether the proteins of
interest localized to the nucleus, cytoplasm, membrane,
extracellular or other compartments. Proteins with the appropriate
subcellular localization could then be further investigated as
potential targets.
[0174] C. Screening
[0175] Large molecule therapeutics (such as proteins, nucleic
acids, sugars) could be identified by making an array of the
appropriate constructs and screening for the desired phenotype. For
instance, a screen for secreted proteins could involve an array
where cells expressing secreted proteins are mixed with tester
cells with the potential for an assayable response to the secreted
proteins. After transfection and growth, the response of the tester
cells could be measured to identify features producing secreted
proteins with the desired effect.
[0176] Multiplexed screening could be performed by making arrays on
the bottom of each well of a microtiter dish. The binding of
molecules to an array of 100 or more potential targets in the
bottom of each well. These targets could be pharmacogenomic
variants, families of proteins, or other collections of proteins.
The binding could then be assayed by a scanner, plate reader or
other instrument, (e.g. Cellomics ArrayScan II (registered
trademark)).
[0177] Arrays could also be used to characterize compound
libraries. Binding of compound mixtures to targets in the array
could be characterized to provide an overall assessment the
diversity of the mixture. High diversity mixtures would bind to
more targets than low diversity mixtures. The mixture could be, for
example, a combinatorial library or natural product extract.
[0178] D. Lead optimization
[0179] Potential drug candidates could be evaluated for selectivity
by incubating the candidate with the appropriate array of potential
targets. The arrays could be the entire set of genes in the
genome(s) of interest or focused subsets, e.g. GPCR's, ion
channels, enzymes, nuclear hormone receptors. The relative binding
of the drug candidate to the known target and other potential
targets could be determined. Candidates with a high degree of
non-selective binding could be abandoned or modified to reduce
non-selective binding before additional testing such as ADME
ortoxicology other tests. Potential drug candidates could be
evaluated for toxicity by incubating the candidate with the
appropriate array of targets, such as cytochrome P-450's including
pharmacogenomic variants or other variations.
[0180] Selectivity tests could also be performed on the metabolites
of a drug candidate. For instance, a radiolabeled drug could be
reacted with the appropriate biotransformation agent, such as a
liver extract, tissue culture system, or living organism such as a
rodent or dog. The radiolabeled metabolites could then be extracted
and purified and tested for binding with the array. Metabolites
with binding activity could then be characterized further by
standard methods. Two embodiments of the present method are
described in detail herein: a DNA-gelatin method, in which a
mixture comprising DNA (e.g., DNA in an expression vector, such as,
a plasmid-based or viral-based vector) and a carrier protein (e.g.,
gelatin) is used and a lipid vector-DNA method, in which a mixture
comprising DNA, such as DNA in an expression vector (e.g., a
plasmid); a carrier protein (e.g., gelatin); a sugar (e.g.,
sucrose); DNA condensation buffer; and an appropriate
lipid-containing transfection reagent is used. Any suitable gelatin
which is non-toxic, hydrated, which can immobilize the nucleic acid
mixture onto a surface and which also allows the nucleic acid
immobilized on the surface to be introduced over time into cells
plated on the surface can be used. For example, the gelatin can be
a crude protein gelatin or a more pure protein based gelatin such
as fibronectin. In addition, a hydrogel, a sugar based gelatin
(polyethylene glycol) or a synthetic or chemical based gelatin such
as acrylamide can be used.
[0181] In the first embodiment, a mixture comprising two components
(DNA such as DNA in an expression vector and a carrier protein) is
spotted onto a surface (e.g., a slide) in discrete, defined
locations or areas and allowed to dry. One example of this
embodiment is described in Example 1. After the carrier (e.g.,
gelatin)-DNA mixture has dried sufficiently that it is affixed to
the surface, transfection reagents (a lipofection mixture) and
cells to be reverse transfected are added, preferably sequentially.
The transfection mixture can be one made from available components
or can be a commercially available mixture, such as Effectene.TM.
(Qiagen), Fugene.TM. 6 (Boehringer Mannheim) or Lipofectamine.TM.
(Gibco/BRL-Life Technologies). It is added in an appropriate
quantity, which can be determined empirically, taking into
consideration the amount of DNA in each defined location. A wax
barrier can be drawn around the locations on the surface which
contain the vector-DNA mixture, prior to addition of the
transfection mixture, in order to retain the mixture or the
solution can be kept in place using a cover well. Generally, in
this embodiment, the transfection reagent is removed, such as by
vacuum suctioning, prior to addition of cells into which DNA is to
be reverse transfected. Actively growing cells are plated on top of
the locations, producing a surface that bears the DNA-containing
mixture in defined locations. The resulting product is maintained
under conditions (e.g., temperature and time) which result in entry
of DNA in the DNA spots into the growing cells. These conditions
will vary according to the types of cells and reagents used and can
be determined empirically. Temperature can be, for example, room
temperature or 37.degree. C., 25.degree. C. or any temperature
determined to be appropriate for the cells and reagents.
[0182] In the second embodiment, one example of which is described
in Example 2, a mixture comprising DNA in an expression vector; a
carrier protein (e.g., gelatin); a sugar (e.g., sucrose); DNA
condensation buffer; and a lipid-based transfection reagent is
spotted onto a surface, such as a slide, in discrete, defined
locations and allowed to dry. Actively growing cells are plated on
top of the DNA-containing locations and the resulting surface is
maintained under conditions (e.g., temperature and time) which
result in entry of DNA in the DNA spots into the growing cells
(reverse transfection). Expression of DNA in cells is detected
using known methods, as described above.
[0183] E. Optimization of plasmids
[0184] In still another embodiment, the subject method can be used
to optimize an expression system for a particular cell type.
Briefly, the transfection array can be a collection of various
permutations of a vector system. For instance, the vector library
can test various combinations and permutations of promoter and
enhance sequences, replication origins, and other components which
could effect the level of expression of a protein or the stability
of the cell line for the plasmid.
[0185] VII. Exemplary Embodiments
[0186] The present invention is illustrated by the following
examples, which are not intended to be limiting in any way.
EXAMPLE 1
Reverse Transfection: "Gelatin-DNA" Method
Materials
[0187] [DNA]: 1 .mu.g/.mu.L (eg., HA-GST pRK5, pBABE CMV GFP)
[0188] Gelatin (ICN, cat. #901771): 0.2% stock in ddH.sub.2O, all
dilutions made in PBS.sup.-0.20% gelatin=0.5g gelatin+250 mL
ddH.sub.2O
[0189] Effectene Transfection Kit (Qiagen, cat. #301425)
[0190] Plasmid-DNA: grown in 100 mL L-amp overnight from glycerol
stock, purified by standard Qiaprep Miniprep or Qiagen Plasmid
Purification Maxi protocols
[0191] Cell Type: HEK 293T cultured in DMEM/10%IFS with L-glut and
pen/strep
Diluting and Spotting DNA
[0192] Dilute DNA in 0.2% gelatin* to give final [DNA]=0.05
.mu.g/.mu.L**
[0193] Spot DNA/gelatin mix on .SIGMA. poly-L-lysine slides using
arrayer
[0194] Allow slides to dry in vacuum-dessicator overnight*** *
range of gelatin concentration that worked under the conditions
used=0.05% to 0.5% ** range of DNA concentrations that worked under
the conditions used=0.01 .mu.l/.mu.l to 0.10 .mu.g/.mu.l range of
drying time=2 hours to 1 week
Adding Tx. Reagents to Gelatin -DNA Spots
[0195] In eppendorf tube, mix 300 .mu.L DNA-condensation buffer (EC
Buffer)+16 .mu.L Enhancer
[0196] Mix by vortexing. Incubate for 5 minutes
[0197] Add 50 .mu.L Effectene and mix by pipetting
[0198] Draw a wax circular barrier on slide around spots to apply
the transfection reagent
[0199] Add 366 .mu.L mix to wax-enclosed region of spots
[0200] Incubate at room temperature for 10 to 20 minutes
[0201] Meanwhile, split cells to reverse-transfect
[0202] Vacuum-suction off reagent in hood
[0203] Place slides in dish and add cells for reverse
transfection
Splitting Cells
[0204] Split actively growing cells to [cell]=10.sup.7 cells in 25
mL
[0205] Plate cells on top of slide(s) in square
100.times.100.times.15 mm petri dish
[0206] Allow reverse transfection to proceed for 40 hours=approx. 2
cell cycles
[0207] Process slides for immunofluorescence
EXAMPLE 2
Reverse Transfection: "Lipid-DNA" Method
Materials
[0208] [DNA]: 1 .mu.g/.mu.L (eg., HA-GST pRK5, pBABE CMV GFP)
[0209] Gelatin (ICN, cat. # 901771): 0.2% stock in ddH.sub.2O, all
dilutions made in PBS.sup.-0.05% gelatin=250 .mu.L 0.2%+750 .mu.L
PBS.sup.-
[0210] Effectene Transfection Kit (Qiagen, cat. #301425):
[0211] EC Buffer in 0.4M sucrose=273.6 .mu.L 50% sucrose+726.4
.mu.L EC Buffer
[0212] Plasmid-DNA: grown in 100 mL L-amp overnight from glycerol
stock, purified by standard Qiaprep Miniprep or Qiagen Plasmid
Purification Maxi protocols
[0213] Cell Type: HEK 293T cultured in DMEM/10%IFS with L-glut and
pen/strep
Reverse Transfection Protocol with Reduced Volume
[0214] Aliquot 1.6 .mu.ug DNA in separate eppendorf tubes
[0215] Add 15 .mu.L of pre-made DNA-condensation buffer (EC Buffer)
with 0.4M sucrose* to tubes
[0216] Add 1.6 .mu.L of Enhancer solution and mix by pipetting
several times. Incubate at room temperature for 5 minutes
[0217] Add 5 .mu.L of Effectene Transfection Reagent to the
DNA-Enhancer mix and mix by pipetting. Incubate at room temperature
for 10 minutes
[0218] Add 23.2 .mu.L of 0.05% gelatin** to total transfection
reagent mix (i.e. 1:1 dilution)
[0219] Spot lipid-DNA on .SIGMA. poly-L-lysine slides mix using
arrayer
[0220] Allow slides to dry in vacuum-dessicator overnight***
[0221] Effectene.TM. kit (Qiagen) used includes Enhancer solution,
which was used according to Qiagen's instructions. * range of
sucrose that worked under the conditions used=0.1M to 0.4M ** range
of gelatin concentration that worked under the conditions
used=0.01% to 0.05% *** range of drying time=2 hours to 1 week
Splitting Cells
[0222] Split actively growing cells to [cell]=10.sup.7 cells in 25
mL
[0223] Plate cells on top of slide(s) in square
100.times.100.times.15 mm petri dish
[0224] Allow reverse transfection to proceed for 40 hours=approx. 2
cell cycles
[0225] Process slides for immunofluorescence
EXAMPLE 3
Transfected Cells Micorarrays: a Genomics Approach for the Analysis
of Gene products in mammalian cells
Lipid-DNA Method
[0226] I. Gelatin Preparation and DNA Purification
Materials
[0227] Gamma-Amino Propyl Silane (GAPS) slides (Corning catalog
#2550),
[0228] Purified cDNA,
[0229] Gelatin, Type B: 225 Bloom (Sigma, catalog #G-9391),
Methods
[0230] 0.2% Gelatin was made by incubation in a 60.degree. C. water
bath for 15 minutes. The gelatin was cooled slowly to 37.degree. C.
at which point it was filtered through 0.45 .mu.m cellular acetate
membrane (CA).
[0231] Bacterial clones with DNA plasmids were grown in a 96
Deep-Well Dish for 18 to 24 hours in 1.3 mL of terrific broth (TB)
shaking at 250 rpm at 37.degree. C. The plasmids were miniprepped
and optical density (OD) was taken. DNA purity, as indicated by
final 280 nm/260 nm absorbance ratio, was greater than 1.7.
Storage
[0232] For storage purposes, gelatin was kept at 4.degree. C. and
miniprepped DNA kept at -20.degree. C.
[0233] II. Sample Preparation and Array Printing
Materials
[0234] Effectene Transfection Reagent (Qiagen catalog #301425),
[0235] Sucrose (Life Technologies),
[0236] INTEGRID 100 mm.times.15 mm Tissue Culture Square Petri
Dishes (Becton Dickinson: Falcon catalog #35-1012),
[0237] Costar 384-well plates (VWR catalog #7402),
[0238] Stealth Micro Spotting Pins, (Telechem International, Inc.
catalog #SMP4),
[0239] PixSys 5500 Robotic Arrayer (Cartesian Technologies, Model
AD20A5),
[0240] Vacuum Dessicator with Stopcock 250 mm, NALGENE (VWR catalog
#24987-004),
[0241] DRIERITE Anhydrous Calcium Sulfate (VWR catalog
#22890-229)
[0242] Forceps to hold slides,
[0243] Human Embryonic Kidney (HEK) 293T cells,
[0244] Tissue Culture hood,
[0245] Cover Slips (50 mm.times.25 mm),
Methods
[0246] For each DNA-containing spot, 15 .mu.l of pre-made
DNA-condensation buffer (Buffer EC) with 0.2M to 0.4M sucrose was
added to 0.80 .mu.g to 1.60 .mu.g DNA in a separate eppendorf tube.
Subsequently, 1.51 .mu.l of the Enhancer solution was added to the
tube and mixed by pipetting. This was let to incubate at room
temperature for 5 minutes. 5 .mu.l Effectene transfection reagent
was added, mixed and let to incubate at room temperature for 10
minutes with the DNA-Enhancer mixture. 1.times.volume of 0.05%
gelatin was added, mixed and the appropriate amount was aliquoted
into a 384-well plate for arraying purposes.
[0247] The PixSys 5500 Robotic Arrayer was used with Telechem's
Arraylt Stealth Pins (SMP4) with each spot spaced 400 .mu.m apart
with a 50 ms to 500 ms delay time of the pin on the slide for each
spot. A 55% relative humidity environment was maintained during the
arraying. A thorough wash step was implemented between each dip
into a DNA sample in the 384-well plate to avoid clogging of the
pins that would result in missing spots in the array.
[0248] In a tissue culture hood, 10.times.10.sup.6 Human Embryonic
Kidney (HEK) 293T cells were prepared in 25 ml DME media with 10%
IFS, pen/strep and glutamine for every 3 slides that were to be
processed. After arraying, the slides were simply placed array-side
facing up on a sterile 100.times.100.times.10 mm square dish (3
slides per plate) and the cells were poured gently on the slides
while avoiding direct pouring on the arrays themselves. If the
number of slides were not a multiple of 3, dummy slides were placed
to cover the square dish.
[0249] The cells were let to grow on the arrays for approximately 2
cell cycles (.about.40 hours for 293T). Subsequently, the slides
were very gently rinsed with PBS.sup.- in a Coplin jar, and then
fixed in 3.7% paraformaldehyde/4.0% sucrose for 20 minutes in a
Coplin jar, and then transferred back to ajar with PBS.sup.-.
Storage
[0250] After arraying, slides were stored at room temperature in a
vacuum dessicator with anhydrous calcium sulfate pellets. After
fixation, slides were kept in PBS.sup.- at 4.degree. C. until
analyses were completed (maximum of 5 days).
[0251] III. Methods of Detection
[0252] Immunofluorescence
[0253] Fluorescence Microscopy
[0254] Laser Scanning
[0255] Radiolabelling and detection with sensitive film or
emulsion
[0256] If the expressed proteins to be visualized are fluorescent
proteins, they can be viewed and photographed by fluorescent
microscopy. For large expression array, slides may be scanned with
a laser scanner for data storage. If a fluorescent antibody can
detect the expressed proteins, the protocol for immunofluorescence
can be followed. If the detection is based on radioactivity, the
slides can be fixed as indicated above and radioactivity detected
by autoradiography with film or emulsion.
Immunofluorescence
[0257] After fixation, the array area was permeabilized in 0.1%
Triton X-100 in PBS.sup.- for 15 minutes. After two rinses in PBS-,
the slides were blocked for 60 minutes, probed with a primary
antibody at 1:200 to 1:500 dilution for 60 minutes, blocked for 20
minutes, probed with a fluorescent secondary antibody at 1:200
dilution for 40 minutes. The slides can be transferred to a Coplin
jar in PBS.sup.- and visualized under an upright fluorescent
microscope. After analyses, the slides can be mounted and stored in
the dark at 4.degree. C.
[0258] To create these microarrays, distinct and defined areas of a
lawn of cells were simultaneously transfected with different
plasmid DNAs (FIG. 4A). This is accomplished without the use of
individual wells to sequester the DNAs. Nanoliter volumes of
plasmid DNA in an aqueous gelatin solution are printed on a glass
slide. A robotic arrayer (PixSys 5500, Cartesian Technologies)
equipped with stealth pins (SMP4, Telechem) was used to print a
plasmid DNA/gelatin solution contained in a 384-well plate onto CMT
GAPS glass slides (Coming). The pins deposited .about.1 nl volumes
400 .mu.m apart using a 25 ms pin down slide time in a 55% relative
humidity environment. Printed slides were stored at room
temperature in a vacuum desiccator until use. Preparation of
aqueous gelatin solution is important and is as follows. 0.02%
gelatin (w/v) (Sigma G-9391) was dissolved in MilliQ water by
heating and gentle swirling in a 60.degree. C. water bath for 15
minutes. The solution was cooled slowly to room temperature and
filtered through a 0.45 um cellular acetate membrane and stored at
4.degree. C. Plasmid DNA was purified with the Plasmid Maxi or
QIAprep 96 Turbo Miniprep kits (Qiagen), and always had an
A260/A280>1.7. Concentrated solutions of DNA were diluted in the
gelatin solution so to keep the gelatin concentration >0.017%
and, unless otherwise specified, final plasmid DNA concentrations
were 0.033 .mu.g/.mu.l. To express GFP the EGFP construct in
pBABEpuro was used.
[0259] After drying, the DNA spots are briefly exposed to a lipid
transfection reagent, the slide is placed in a culture dish and
covered with adherent mammalian cells in media. The Effectene
transfection kit (301425, Qiagen) was used as follows. In a 1.5 ml
microcentrifuge tube, 16 .mu.l enhancer was added to 150 .mu.l EC
buffer, mixed, and incubated for 5 minutes at room temperature. 25
.mu.l effectene lipid was added, mixed and the entire volume
pipetted onto a 40.times.20 mm cover well (PC200, Grace Bio-Labs).
A slide with the printed side down was placed on the cover well
such that the solution covers the entire arrayed area while also
creating an airtight seal. After a 10 minute incubation, the cover
well was pried off the slide with a forceps and the transfection
reagent removed carefully by vacuum aspiration. The slide was
placed printed side up in a 100.times.100.times.10 mm square tissue
culture dish and a 1.times.10.sup.7 actively growing HEK293T cells
in 25 ml media (DMEM with 10% FBS, 50 units/ml penicillin and 50
.mu.g/ml streptomycin) were poured into the dish. Three slides can
be transfected side-by-side in this fashion. The cells grew on the
slide for 40 hours prior to fixing for 20 minutes at room
temperature in 3.7% paraformaldehyde/4.0% sucrose in PBS. Other
commonly used mammalian cells lines, such as HeLa and A549 cells,
were also tested and similar results were obtained but with
transfection efficiencies of 30-50% of those obtained with HEK293
cells. The DNA in the gelatin gel is insoluble in cell culture
media but readily enters cells growing on it to create the
transfected cell microarray.
[0260] To illustrate the method, an array with elements containing
an expression construct for the green fluorescent protein (GFP) was
printed. HEK293 cells were plated on the slide for transfection and
the fluorescence of the cells detected with a laser fluorescence
scanner. Microarrays were imaged at a resolution of 5 .mu.m with a
laser fluorescence scanner (ScanArray 5000, GSI Lumonics). GFP and
cy3 emission was measured separately after sequential excitation of
the two fluorophores. To obtain images at cellular resolution,
cells were photographed with a conventional fluorescent microscope.
All images were pseudocolored and superimposed using Photoshop 5.5
(Adobe Systems).
[0261] A low magnification scan showed a regular pattern of
fluorescent spots that matches the pattern in which the GFP
expression construct was printed (FIG. 4B). A higher magnification
image obtained via fluorescence microscopy showed that each spot is
about 150 .mu.m in diameter and consists of a cluster of 30-80
fluorescent cells (FIG. 4C). As in a conventional transfection, the
total expression level in the clusters is proportional over a
defined range to the amount of plasmid DNA used (FIG. 4D). Since it
may be useful to express two different plasmids in the same cells,
whether the technique is compatible with cotransfection was
examined. Arrays with elements containing expression constructs for
GFP, an epitope-tagged protein or both were prepared and
transfected. The cells growing on elements printed with both cDNAs
express both encoded proteins, indicating that cotransfection had
occurred (FIG. 4E).
[0262] Whether transfected cell microarrays could be used to clone
gene products based on their intrinsic properties was also
determined. As a test case, an array to identify the receptor for
FK506, a clinically important immunosuppressant whose
pharmacologically relevant target, FKBP12, is an intra-cellular
protein, was used (Kino, T., et al., J. Antiobiot., 40:1256 (1987);
Harding, M. W., et al., Nature, 26:755 (1989)). Elements containing
expression constructs for FKBP12, GFP, or both were printed on a
slide, in an easily recognizable pattern. After the transfected
cell microarray formed, radiolabeled FK506 was added to the tissue
culture media for one hour prior to processing the slide for
autoradiography and immunofluorescence. The radiolabeled FK506
bound to the array in a pattern of spots that exactly matches the
pattern of cell clusters expressing FKBP12 (FIG. 5A). Detection of
the bound FK506 with autoradiographic emulsion confirmed, at the
cellular level, colocalization between FKBP12 expression and FK506
binding (FIG. 5B). The binding is specific because the
GFP-expressing clusters and the non-transfected cells surrounding
the clusters showed only background levels of signal (FIG. 5A).
Furthermore, the prior addition of excess rapamycin, a competitive
antagonist of FK506, completely eliminated the signal. 1 .mu.M
rapamycin was added to the cell culture media 30 minutes before the
addition of radiolabeled FK506.
[0263] The utility of transfected cell microarrays for identifying
gene products that induce phenotypes of interest in mammalian cells
or have a distinct sub-cellular localization was also explored.
Arrays with a collection, enriched for signaling molecules, of 192
distinct epitope-tagged cDNAs in expression vectors were printed.
192 Genestrom expression constructs (Invitrogen) in bacteria were
cultured in two 96-well plates and plasmid DNA was purified using
the Turbo Miniprep Kit (Qiagen). Plasmid DNA was diluted with 0.02%
gelatin to a final concentration of 0.040 .mu.g/.mu.l and printed.
Cellular phosphotyrosine levels were determined by
immunofluorescence staining and scanning. Cell morphology and
subcellular localization of expressed proteins was assessed by
visual inspection via fluorescence microscopy of the cells in the
clusters after their detection with anti-V.gamma.
immunofluorescence.
[0264] After transfection, their effects on cellular
phosphotyrosine levels and morphology as well as their subcellular
localization were determined. Five cell clusters on grid 1 (A2, C7,
C9, C 11, and F6) had phosphotyrosine levels above background (FIG.
5C). The coordinates of the clusters match those of the wells of a
microtiter plate containing the source cDNAs and were used to look
up the identity of the transfected cDNAs. This revealed that four
of these clusters were transfected with known tyrosine kinases
(trkC, syk, syn, and blk) while the fifth (C11) encodes a protein
of unknown function. Simple visual examination of the morphology of
the cells in the transfected clusters revealed a diversity of
cellular phenotypes even in this small set of clones. In array 2,
cluster E8 had fragmented cells characteristic of apoptosis while
in two clusters (D10 and F7) the cells were closely attached to
each other (FIG. 5D). The presence of apoptotic cells was confirmed
by TUNEL (Terminal deoxynucleotidyl transferase mediated
dUTP-biotin nick end labeling method) staining. TUNEL staining was
performed as described (Y. Gavrieli, Y. Sherman, S. A. Ben-Sasson.
J. Cell Biol. 119, 493 (1992)).
[0265] The observed phenotypes are consistent with the presumed
functions of the cDNAs expressed in these clusters (the Table).
Subcellular localization of the expressed proteins were examined
through visual inspection the and those with distinct patterns were
noted (the Table). This revealed that several proteins that are
known transcription factors were mainly located in the cell
nucleus. This was also true for other proteins, such as phosphatase
1-beta, whose subcellular distribution has not been previously
ascertained.
1TABLE Description of selected cDNAs expressed in the transfected
cell microarray. Shown are the coordinates, the phenotype or
property detected, the Genbank accession number and the name of the
cDNA. nuc/cyto means nuclear and cytoplasmic staining was visible.
Grid: Coordinate Phenotype/property Accession number Function 2:E8
apoptosis AF016266 TRAIL receptor 2 2:D10 cell adhesion X97229 NK
receptor 2:F7 cell adhesion M98399 CD36 1:A9 nuclear U11791 CyclinH
1:B5 nuclear M60527 deoxycytidine kinase 1:B12 nuclear M60724 p70
S6 kinase kinase .alpha.1 1:C12 nuclear M90813 D-type cyclin 1:E4
mitochondrial U54645 methyhnalonyl-coA mutase 1:E10 mitochondrial
J05401 creatine kinase 1:G9 nuc/cyto U40989 tat interactive protein
1:G10 nuc/cyto U09578 MAPKAP (3pk) kinase 2:A9 nuclear X83928 TFIID
subunit TAFII28 2:A12 nuc/cyto M62831 ETR101 2:B6 nuc/cyto X06948
IgE receptor .alpha.-subunit 2:B12 nuclear X63469 TFIIE .beta.
subunit 2:C5 nuclear M76766 General transcription factor IIB 2:C7
nuc/cyto Ml 5059 CD23A 2:C12 nuclear X80910 PP1, .beta. catalytic
subunit 2:D4 nuclear AF017307 Ets-related transcription factor 2:E7
nuclear X63468 TFIIE .alpha. 2:E12 nuclear U22662 Orphan receptor
LXR-.alpha. 2:F8 nuclear L08895 MEF2C 2:F12 nuclear AF028008
SP1-like transcription factor 2:G2 nuc/cyto U37352 PP2A, regulatory
B' .alpha. 1 subunit 2:G3 nuc/cyto L14778 PP2B, catalytic .alpha.
subunit
[0266] The microarrays can be printed with the same robotic
arrayers as traditional DNA arrays, so it is feasible to achieve
densities of up 10,000-15,000 cell clusters per standard slide. At
these densities the entire set of human genes can be expressed on a
small number of slides, allowing rapid pan-genomic screens. Thus,
comprehensive collections of full-length cDNAs for all mammalian
genes can be generated (Strausberg, R. L., et al., Science, 15:455
(1999); www.hip.harvard.edu/research.html. www.guthrie.org/cDNA.)
and will be valuable tools for making such arrays.
[0267] Transfected cell microarrays have distinct advantages over
conventional expression cloning strategies using FACs or sib
selection (Simonsen, H., et al., Ttrends Pharmacol. Sci., 15:437
(1994)). First, cDNAs do not need to be isolated from the cells
exhibiting the phenotype of interest. This allows for screens using
a variety of detection methods, such as autoradiography or in situ
hybridization, and significantly accelerates the pace of expression
cloning. The experiments described herein took days to perform
instead of the weeks to months necessary with other expression
cloning strategies. Second, transfected cell microarrays can also
be used to screen living cells, allowing the detection of transient
phenotypes, such as changes in intracellular calcium
concentrations. Third, being compact and easy to handle,
transfected cell microarrays have economies of scale. The arrays
are stable for months and can be printed in large numbers, allowing
many phenotypes to be screened in parallel, with a variety of
methods, in a small number of tissue culture plates.
[0268] Described herein are arrays in which the transfected
plasmids direct gene overexpression. However, as antisense
technology improves or other methods emerge for decreasing gene
function in mammalian cells, it is likely that transfected cell
microarrays can be used to screen for phenotypes caused by loss of
gene function. Lastly, the immobilization of the plasmid DNA in a
degradable gel is the key to spatially restricting transfection
without wells.
[0269] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *
References