Note: Descriptions are shown in the official language in which they were submitted.
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PROTEIN INTERACTION AND TRANSCRIPTION FACTOR TRAP
Field of Invention
This invention relates to the use of gene trapping
methods for the identification of genes and two-hybrid
methodology for the identification of protein-protein
interactions.
Background of the Invention
The standard two-hybrid assay relies upon the fact
that many eukaryotic transcriptional regulatory systems
consist of separate domains: a DNA-binding domain (DNA-BD)
that binds to a promoter or other cis-transcriptional
regulatory element; and, an activation domain (AD) that
directs RNA polymerase II to transcribe a gene downstream
from the site on the DNA where the DNA-BD is bound. The
DNA binding domain and the activation domain may be
separate proteins but will function to activate
transcription as long as the AD is in proximity to a DNA-BD
bound to the transcriptional regulatory element. 4dhere
each of the AD and the DNA-BD is fused to members of a pair
of interacting proteins, the AD will function via the link
to the DNA-BD created by the interacting proteins. Thus,
the two-hybrid assay may be used to investigate whether
interaction occurs between two proteins (termed "bait" and
"prey") expressed as fusion products with DNA-BD and AD
peptides, respectively. A positive event is identified by
activation of a reporter gene having an upstream promoter
to which the DNA-BD binds.
The two-hybrid assay may be carried out in a variety
of eukaryotic cells including yeast (see: Fields, S. and
Song. O. 1989 A Novel Genetic System to Detect
Protein-Protein Interactions Nature 340:245-247; and
Fields, S. 1993. The Two-hybrid System to Detect
Protein-Protein Interactions. Methods: A Companion to
Meth. Enzymol. 5:116-124.) and mammalian cells (see: Luo,
Y. et a1. 1997. Mammalian Two-Hybrid System: A
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Complementary Approach to the Yeast Two-Hybrid System.
Biotechnics 22:350-52; and, Feron, E.R. et a1. 1992.
Karyoplasmic Interaction Selection Strategy: A General
Strategy to Detect Protein-Protein Interactions in
Mammalian Cells Proc. Natl. Acad. Sci. U.S.A. 89:7958-62).
Commercial yeast and mammalian two-hybrid assay kits are
available from Clontech Laboratories, Inc., 1020 East
Meadow Circle, Palo Alto, California, 94303-4230, U.S.A.
Specific protein-protein interactions are the basis
for many biological processes. Standard two-hybrid
techniques make use of specialized cDNA expression
libraries as a source of protein sequences used in
screening for specific interactions between proteins.
However, cDNA expression libraries possess some intrinsic
disadvantages. For example, cDNA libraries produce a bias
toward cloning of highly expressed genes and rare gene
transcripts are unlikely to be discovered. The source of
the mRNA for the generation of the cDNA library is critical
since many tissue restricted genes and developmentally or
temporally regulated genes are not represented by a
particular cDNA library.
Gene trap vectors target the prevalent introns of the
eukaryotic genome. These vectors may consist of either a
splice-acceptor (SA) site upstream of a reporter sequence,
or an unpaired splice-donor (SD) site downstream from a
reporter sequence. Preferably, on the latter vector
employing SD, the reporter sequence is driven by an
appropriate transcriptional regulatory element
(eg. promoter). Integration of the above-described gene
trap vectors into an intron results in production of MRNA
in which a transcript of the vector is joined to an
transcript of an adjacent exon. (see: Skarnes, W.C. et
a1. 1992. A Gene Trap Approach in Mouse Embryonic Stem
Cells: The lacX Reporter is Activated by Splicing, Reflex
Endogenous Gene Expression and is Mutagenic in Mice. Genes
Dev. 6:903-918; W.C. Skarnes 1993 The Identification of New
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Genes: Gene Trapping in Transgenic Mice. Current Opinion
in Biotechnology 4:684-89; and, United States Patent
No. 5,652,128 July 29, 1997.)
A form of gene trapping (termed "tagging") may also be
accomplished by using a vector comprising a peptide
encoding segment and both an upstream SA and a downstream
SD (see United States Patent No. 5,652,128 of Jarvik).
Features of gene trapping include:
(a) random integration into the genome;
(b) splice acceptor or splice donor containing
vectors result in a fusion of a transcript of a
reporter gene on the vector with endogenous gene
transcripts;
(c) the full repertoire of genes are represented in
the genome without a bias towards highly
expressed genes;
(d) gene trapping can provide information about
coding regions of most genes that is independent
of their transcription status; and
(e) gene trapping is independent of the source of
mRNA (therefore, rare as well as tissue specific
genes and developmental temporally regulated
genes may be trapped).
A full strategy for genome-wide functional analysis
should include a systematic strategy for identification and
characterization of gene products according to their
protein-protein interaction characteristics.
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Summary of Invention
Gene trap methodologies provide a repertoire of
protein domains encoded by exon sequences found within the
genome. Two-hybrid techniques permit identification of
protein-protein interactions. This invention makes use of
a combination of gene trap and two-hybrid methodologies for
the identification and characterization of genes according
to protein-protein interactions of the gene product or for
the identification of genes encoding transcriptional
activator domains (AD). Interaction of an exon-encoded
protein domain with a given protein, or functioning of the
exon-encoded domain as an AD, is detected by reconstituting
the activity of a transcriptional activator.
This invention also provides gene trap vectors adapted
for use in a two-hybrid assay and methodologies for
identification of genes encoding proteins capable of
interacting with a selected protein. This invention also
provides gene trap vectors and methodologies for the
selective identification of genes encoding transcription
activator domains.
This invention provides a DNA construct comprising a
DNA sequence encoding a transcriptional regulatory protein
moiety selected from the group consisting of a DNA-BD and
a AD; and, a m-RNA splice site. The term "m-RNA" splice
site is defined herein as being a splice acceptor sequence
(SA) or an unpaired splice donor sequence (SD).
This invention also provides a DNA construct
comprising a DNA sequence encoding a transcriptional
regulatory protein moiety selected from the group
consisting of a DNA-BD and an AD; and, a downstream SD.
Preferably, this construct will have a transcriptional
regulatory element operably linked to the sequence encoding
the transcriptional regulatory protein moiety.
This invention also provides a DNA construct
comprising a DNA sequence encoding a transcription
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regulatory protein moiety selected from the group
consisting of a DNA-BD and an AD, together with an upstream
SA and a downstream SD.
This invention also provides a DNA construct
comprising an SA upstream of a transcriptional regulatory
protein moiety selected from the group consisting of a
DNA-BD and an AD; and, a downstream poly-adenylation
signal.
This invention also provides a method of making the
DNA constructs of this invention comprising the step of
ligating a DNA sequence encoding a transcriptional
regulatory protein moiety as defined above with one or both
of a SA and a SD. Preferably, at least three such DNA
constructs are made in three different reading frames.
This invention also provides cells comprising the DNA
constructs of this invention obtainable by the method of
transforming eucaryotic cells with one or more DNA
constructs of this invention.
This invention also provides kits that comprise the
above-described DNA construct of this invention. The DNA
constructs may be in the form of plasmids. The kits may
also comprise host cells, two-hybrid vectors or reporter
gene constructs as described herein. The two-hybrid
vectors of the kit may be plasmids constructed (eg.
presence of suitable restriction sites) to permit insertion
of a test protein sequence to be part of a two-hybrid
vector as described herein. The kits may also comprise
materials and reagents useful for DNA insertions, reporter
gene activity assays, or sequencing of inserts (eg.
primers).
This invention also provides host cells whose genome
optionally comprises a reporter gene as described herein
and wherein the cell has incorporated into the genome, a
two-hybrid vector as described herein. The two-hybrid
vector may include a sequence encoding a test protein.
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This invention also provides a method for detecting
interaction between a protein domain encoded by an exon
sequence in the genome of a host cell and a test protein,
wherein the host cell contains a reporter gene under the
control of a transcriptional regulatory element that
expresses a detectable protein when the reporter gene is
transcribed, comprising:
(a) introducing into the host cell a first DNA
construct that is capable of being expressed in
the host cell, said first DNA construct encoding
a first hybrid protein comprising:
(i) a transcriptional regulatory protein moiety
selected from the group consisting of: a
DNA-BD that recognizes a binding site on the
transcriptional regulatory element of the
reporter gene; and, a AD functional in the
host cell; and
(ii) a test protein;
(b) introducing into the host cell a second DNA
construct that is capable of being expressed in
the host cell sequence encoding a transcriptional
regulatory protein moiety selected from the group
of said moieties defined at subparagraph (a)(i)
above to reconstitute a transcriptional
regulatory protein, and one or more m-RNA splice
sites as defined herein,
wherein integration of the second DNA construct into
the genome of the host cell results in the expression
of a second hybrid protein comprising a
transcriptional regulatory protein moiety and an
endogenous protein of the host cell; and
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(c) determining whether the reporter gene is
transcribed.
The second DNA construct described above may be
selected from the following group:
(I) A gene trap vector comprising a sequence encoding
a transcriptional regulatory protein moiety
selected from the group of said moieties defined
at subparagraph (a) (i) above to reconstitute a
transcriptional regulatory protein, followed by
a SD. Preferably, this gene trap vector will
comprise a transcriptional regulatory element
operably linked to the sequence encoding the said
moiety.
(II) A gene trap vector without a transcriptional
regulatory element and comprising SA upstream of
a sequence encoding a transcriptional regulatory
protein moiety selected from the group of said
moieties defined at subparagraph (a)(i) above to
reconstitute a transcriptional regulatory
protein. Preferably, the sequence encoding said
moiety is followed by a poly-adenylation signal.
(III) A gene trap vector comprising a sequence encoding
a transcriptional regulatory moiety selected from
the group of said moieties defined at
subparagraph (a)(i) above to reconstitute a
transcriptional regulatory protein, with an
upstream SA and a downstream SD.
Where the first DNA construct comprises a sequence
encoding a DNA-BD that recognizes a binding site on the
reporter gene, the second DNA construct will comprise a
sequence encoding the AD. Where the first DNA construct
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comprises a sequence encoding the AD, the second DNA
construct will comprise the DNA-BD. When the first DNA
construct is expressed in a cell in which the second DNA
construct is expressed resulting in a hybrid protein of
which the endogenous portion interacts with the test
protein, reconstitution of the transcriptional regulatory
protein occurs and binding of the latter protein by means
of the DNA-BD to the reporter gene results in activation of
the reporter gene.
Preferably, the second DNA construct will comprise the
AD and not the DNA-BD. This may minimize false positives
resulting from constitution of a regulatory protein by
means of the second DNA construct being expressed with an
endogenous exon that encodes a protein capable of
functioning as the AD.
This invention also provides a method for detecting
endogenous transcription activator domains of a host cell,
wherein the host cell contains a reporter gene under the
control of a transcriptional regulatory element that
expresses a detectable protein when the reporter gene is
transcribed, comprising:
(a) introducing into the host cell a DNA construct
that is capable of being expressed in the host
cell comprising a sequence encoding a DNA-BD that
recognizes a binding site on the transcriptional
regulatory element of the reporter gene and a
m-RNA splice site, wherein integration of the DNA
construct into the genome of the host cell
results in the expression of a hybrid protein
comprising the DNA-BD and an endogenous protein
of the host cell; and
(b) detecting whether the reporter gene is
transcribed.
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The DNA construct used in the method for detecting
endogenous transcription activator domains may be selected
from the following group:
(IV) A gene trap vector comprising a sequence encoding
a DNA-BD that recognizes a binding site on the
transcriptional regulatory element of the
reporter gene, followed by a SD. Preferably, a
transcriptional regulatory element is operably
linked to the sequence encoding the DNA-BD.
(V) A gene trap vector without a transcriptional
regulatory element and comprising a SA upstream
of a sequence encoding DN-BD that recognizes a
binding site in the transcriptional regulatory
element of the reporter gene. Preferably, the
sequence encoding the DNA-BD is followed by a
poly-adenylation signal.
(VI) A gene trap vector comprising a sequence encoding
a DNA-BD that recognizes a binding site in the
transcriptional regulatory element of the
reporter gene, with an upstream SA and a
downstream SD.
Detailed Description of the Invention
In the present invention, DNA constructs are
introduced into a host cell and expressed in the host cell
in sufficient quantities for a reporter gene to be
activated. The host cell may be any eukaryotic cell,
including yeast, zebrafish, c. elegans, drosophila and
mammalian cells having a genome one would like to screen
for interactive protein encoding exons or AD encoding
exons.
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The host cell contains a reporter gene having a
binding site for a DNA-BD. The reporter gene product is
detectable when the reporter gene is transcriptionally
activated. A reporter gene is one whose transcription is
detectable and or which expresses a protein which is also
detectable, either of which can be assayed. Examples of
readily detectable proteins are well-known and include:
,Q-galactosidase, green fluorescent protein, luciferase,
alkaline phosphatase, and chloramphenicol acetyl
transferase (CAT) as well as other enzymes and proteins
that are selectable markers. Other examples of detectable
proteins include cell surface markers such as CD4. In the
exemplified embodiment, the reporter gene used is the pac
gene which encodes the puromycin~resistance marker.
The reporter gene in the host cell will be driven by
a transcriptional regulatory element that is capable of
binding the DNA-BD employed in the assay and is functional
in the host cell. Many examples of suitable regulatory
elements including promoters are well-known.
The assay may make use of host cells in which the
reporter gene has been previously incorporated, or a
construct containing the reporter gene may be introduced to
the cell at the same time as other vectors used in the
assay.
Other vectors used in the assay include a gene trap
vector and a two-hybrid vector. The gene-trap vector is
employed for random insertion of a transcriptional
regulatory protein moiety into the genome of the host cell
and may comprise DNA encoding either a AD or a DNA-BD and
either: an upstream splice acceptor (SA); or, an upstream
transcriptional regulatory element (eg. a promoter) capable
of functioning in the host cell for transcription of the
downstream AD or DNA-BD which in turn is followed by an
unpaired splice donor sequence (SD). In an alternate
embodiment, the gene trap vector has both an upstream SA
and a downstream SD.
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Incorporation of the gene trap vector within an intron
will permit processing of a chimeric message comprising a
transcript of a flanking endogenous exon joined to the
transcript for the DNA-BD or AD. Use of a gene trap vector
having a downstream sD and an upstream promoter is
preferred since transcription of the chimeric message will
not be dependent upon endogenous expression of the host
cell gene.
Generally, an unpaired splice donor includes the 3'
end of an exon and the 5' end of an intron, and a splice
acceptor includes the 3' end of an intron and the 5' end of
an exon. Functionally, a splice donor is defined by its
ability to effect m-RNA splicing to a splice acceptor site,
and a splice acceptor site is defined by its ability to
effect mRNA splicing to a splice~donor site.
The two-hybrid vector will comprise an upstream
transcriptional regulatory element (eg. a promoter) capable
of a functioning in the host cell and driving transcription
of a sequence intended to reconstitute the transcriptional
regulatory protein. Thus, the two-hybrid vector will
express either a DNA-BD or a AD as the case may be,
depending upon the makeup of the gene trap vector.
Preferably, the two-hybrid vector will express DNA-BD. The
two-hybrid vector also contains a sequence under the
control of the regulatory element which encodes a selected
protein of interest (test protein) for which
protein-protein interactions are to be determined.
Expression of the two-hybrid vector in the host cell
results in the translation of a chimeric protein comprising
the transcriptional regulatory protein moiety (eg. DNA-BD)
fused with the test protein. Incorporation of the gene
trap vector into a gene encoding a protein capable of
interaction with the selected protein will result in
production in the cell of a reconstituted transcription
regulatory protein via interaction of the test protein and
the protein product of the trapped gene. Activation of the
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reporter gene occurs as a result of binding of the DNA-BD
to the reporter gene promoter.
In an alternate embodiment used for detecting exons
encoding endogenous transcription activator domains
(protein capable of functioning as an AD), the gene trap
vector comprising a DNA-BD is used without a two-hybrid
vector. When the gene trap vector integrates into a gene
containing an exon that encodes a protein capable of
functioning as an AD in the cell, the resulting gene
product is a chimeric protein that joins both the DNA-BD
coded for by the vector DNA and the AD coded for by the
endogenous exon. Thus, a transcriptional regulatory
protein is constituted, capable of activating the reporter
gene in the cell.
The DNA-BD and the AD may be derived from a single
transcriptional regulatory protein having separate
DNA-binding and transcriptional activation domains (for
example, the yeast GAL4 and GEN4 proteins). Alternatively,
the DNA-BD and AD moieties may be derived from separate
sources. For example, the DNA-BD may be derived from LexA
in E.coli. The DNA-BD may be from DNA binding proteins
other than activators (eg. repressers). The AD could be
derived from as 147-238 of GAL4. The moieties may also be
synthetic, such as the B42 activation domain. Preferably,
the DNA-BD and the AD are from different proteins. In any
case, the DNA-BD should not be capable of functioning
significantly as an activator domain on its own and the AD
should not be capable of binding to the promoter of the
reporter gene.
In the exemplified embodiment, the DNA-binding domain
is derived from the N-terminal region of the yeast GAL4
protein (eg. as 1-147) and the transcriptional activation
domain is derived from the transcriptional activator of
Herpes Simplex Virus VP16 (eg. as 411-455 of VP16) which
does not bind to DNA but functions as a transcriptional
activator.
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The reporter gene may be present in the genome of the
host cell at the time of introduction of the first and/or
second DNA constructs. Alternatively, a construct
comprising the reporter gene may be introduced into the
host cell genome at the same time as the first and/or
second DNA construct. Also, the first DNA construct may be
introduced to and made part of the host cell genome before
the second construct is introduced, or both constructs may
be introduced at the same time.
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Example I: Protein Interaction Trap
This aspect of the invention may be conveniently
practiced by modification of standard commercial two-hybrid
assay components. In the following example, the Clontech
Mammalian Matchmaker" two-hybrid assay kit is modified such
that the reporter gene is the selectable marker (pac) for
puromycin resistance; the DNA-BD is from GAL4 (as provided
in the commercial kit); and the AD is from Herpes Simplex
Virus VP16 (as provided in the kit). In this example, all
DNA constructs, including the reporter gene are introduced
into a murine R1 ES cell line host cell.
The first DNA construct (two-hybrid vector) comprises
a sequence encoding a GAL4 DNA-BD which recognizes a
binding site on the reporter gene and further comprises a
sequence encoding p53 protein (Clontech, pM-53 plasmid).
The second DNA construct (gene trap vector) comprises
a promoter capable of operation in the host cell driving a
VP16 AD upstream of a splice donor sequence. In an
alternate embodiment, the gene trap vector does not contain
a promoter and has a splice acceptor sequence upstream of
the VP16 AD followed by a poly-adenylation signal.
When the gene trap is integrated into an intron
adjacent to an exon of the host cell encoding a protein
domain capable of interaction with p53 protein, a
transcriptional regulatory protein comprising GAL4 BD and
the VP16 AD is constituted. Expression of the reporter
gene in a host cell as a result of binding by the DNA-BD is
detected by culturing the transformed cells in the presence
of puromycin. Cells in which the reporter gene has been
activated will survive. Alternatively, the reporter used
in the assay could remain as CAT and determination of
reporter gene activity may be carried out according to
standard assay procedures, for example as taught in the
Clontech kit instructions.
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Host cells are transformed~by any of the well-known
methods, selected as being suitable for the particular cell
type. Electroporation or calcium phosphate mediated
transfection are suitable for mammalian cells.
Transfection procedures as taught in the Clontech kit
instructions may be used. A preferred method known for ES
cells is electroporation.
The following plasmids are constructed and/or employed
in this example. The first (pGSPuro) is a modified version
of the GAL4 responsive CAT reporter construct from the
Clontech Matchmaker~ kit (pGSCAT) . In this example, the CAT
reporter gene is replaced by the selectable marker pac,
generating a reporter construct containing the puromycin
resistance gene under the control of the adenovirus Elb
minimal promoter used in the Clontech plasmid. Upstream,
are five copies of the 17 nucleotide consensus GAL4 binding
site (galactose upstream activating sequence: UASG).
The second plasmid is the pM-53 vector from the
Matchmaker" kit which is an expression plasmid containing
the SV40 promoter driving a GAL4 DNA-BD. The commercial
construct encodes p53 protein, but the multiple cloning
site downstream from the DNA-BD may be used to insert
different bait proteins.
A gene trap vector plasmid is constructed by inserting
an oligomer sequence encoding a consensus SD sequence in
frame into a SalI/BspMI digested pVPl6 plasmid (Clontech)
simultaneously deleting the stop codons and
poly-adenylation signal. Thus, a gene trap vector is
generated comprising an SV40 promoter driving expression of
the AD. Three versions of this vector were created
resulting in splicing in all three potential reading
frames. The following are examples of consensus SD
sequences:
AGGTAAGT
AGGTGAGT
each of which may be preceded by C or A.
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An alternate gene trap vector plasmid may be
constructed containing the VP16 AD downstream of a SA
sequence. Three constructs should be generated, each
resulting in splicing in each of three possible reading
frames. SA sequences comprise a polypyrimidine tract
followed by a nucleotide, T or C, AG, and at least G or A.
Examples are the murine En-2 splice acceptor and the splice
acceptors from human ~i-globin and rabbit b-globulin.
The following methods may be used for construction of
VP16 gene trap vectors:
(I) To construct the gene trap vector consisting of
the SV40 promoter driving the expression of VP16
fused to an unpaired splice donor sequence:
(a) Digest pVPl6 (Clontech) with SalI and BspMI;
(b) Isolate and purify the 3.0 kb fragment;
(c) Ligate the 3.0 kb pVPl6 fragment with each
of the following pairs of oligomers to
create fusions of VP16 with unpaired splice
donor sequences in all three possible
reading frames:
Pair #1: 5' tcgacaggtaagt 3'
5' tcatacttacctg 3'
Pair #2 5' tcgaccaggtaagt 3'
5' tcatacttacctgg 3'
Pair #3 5' tcgacccaggtaagt 3'
5'.tcatacttacctggg 3'
(II) To construct an alternate gene trap vector
comprising the En-2 SA sequence fused 5' of the
VP16 transcriptional activator:
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(A) (1) digest pGT4SA vector (Gossler et al.
1989 Science.244:463-465) with Xbal;
(2) fill in ends with T4 DNA polymerase to
generate blunt ends;
(3) digest with NdeI; and
(4) Isolated and purify the 2.0 kb fragment
encoding the En-2 splice acceptor
sequence.
(B) (1) digest pVPl6 (Clontech) with Nhel;
(2) fill in ends with T4 DNA polymerase to
generate blunt end;
(3) digest with NdeI; and
(4) isolated and purify the 2.8 kb fragment
encoding the VP16 transcriptional
activator sequence.
(C) Ligate 2.0 kb En-2 splice acceptor fragment
to 2.8 kb VP16 containing vector.
(D) To generate SA-VP16 in the other two
potential reading frames:
(1) digest the above vector with SexAI and
BglII;
(2) ligate the following pairs of oligomers
to generate fusions in the other two
possible reading frames:
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Pair #1 5' ccaggtcgca 3'
5' gatctgcga 3'
Pair #2' 5' ccaggtgca 3'
5' gatctgca 3'
The three forms of the gene trap vector representing
all three potential reading frames are placed in a head to
tail tandem array allowing the use of alternate promoters
to generate three hybrid mRNAs fusing the VP16 domain in
all three possible reading frames to a adjacent exon upon
integration into a gene within the host cell genome.
The following protocol may be followed:
1. Construct a reporter murine embryonic stem cell line
using standard methods by co-electroporation of linearized
pGSPuro, pM-53 and pPGKHyg into the murine R1 ES cell line.
Hygromycin resistance is used to monitor transfection
efficiency.
2. Characterize the reporter cell lines for its ability
to detect protein-protein interactions by electroporating
with pVPl6T (Clontech) as a positive control and pVPl6-CP
(Clontech) as a negative control for protein-protein
interaction. pVPl6T expresses a fusion of the VP16
activation domain to the SV40 large T antigen, which is
known to interact with p53. The pVPl6-CP negative control
plasmid expresses a fusion of the VP16 activation domain to
a viral coat protein, which does not interact with p53.
3. Upon electroporation of positive or negative control
plasmids, cells are then placed under 1.0 ug/ml puromycin
selection.
4. Select appropriate reporter cell clones that confer
puromycin resistance in the presence of VP16T but not with
pVPl6-CP (cells express pGSPuro and pM-53).
5. Electroporate gene trap vectors into reporter cell
line and select for puromycin resistance with 1.0 ug/ml
puromycin.
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6. Pick individual puromycin resistant colonies and
isolate RNA from each clone.
7. Isolate and sequence trapped exon/gene by rapid
amplification of cDNA end (RACE).PCR (eg. see: Skarnes, et
a1. 1992. Genes and Development 6:903-18). Clontech
sequencing primers for VP16 may be used.
Example II: Transcriptional Activator Domain Trap
In this example, the methods employed in the preceding
example are used in an assay employing the ES host cell ,
the same reporter gene construct (pGSCAT) employed in the
preceding example, and a gene trap vector plasmid designed
to trap genes expressing endogenous protein capable of
functioning as a transcriptional activator domain (AD) in
conjunction with the DNA-BD expressed by the gene trap
vector. Expression of chimeric proteins comprising the
DNA-BD fused to an endogenous protein capable of
functioning as a AD will result in activation of the
reporter gene which comprises a binding site for the
DNA-BD.
The gene trap vector plasmid is constructed by
inserting an oligomer sequence encoding the consensus SD
sequence in frame into the SalI/BspMI digested pM plasmid
(Clontech) resulting a vector comprising of the SV40
promoter driving the GAL4 DNA-binding domain linked to a SD
sequence. Three versions of this vector are created
resulting in splicing in each of the three potential
reading frames, respectively. A consensus splice donor
sequence domain contains the following:
Exon....AGGTAAGT...Intron
To construct the vector consisting of the SV40
promoter driving the expression of the GAL4 DNA binding
domain fused to an unpaired splice donor sequence:
(a) digest pM (Clontech) with SalI and BspMI;
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(b) isolate and purify the 3.2kb fragment; and
(c) ligate the 3.2 kb pVPl6 fragment with each of the
following pairs of oligomers to create fusions of
VP16 with SD sequences in all three possible
reading frames:
Pair #1 5' tcgacaggtaagt 3'
5' tcatacttacctg 3'
Pair #2 5' tcgaccaggtaagt 3'
5' tcatacttacctgg 3'
Pair #3 5' tcgacccaggtaagt 3'
5' tcatacttacctggg 3'
The three forms of the gene trap are then placed in a
head-to-tail tandem array allowing the use of alternative
promoters to generate three hybrid mRNAs fusing the GAL4
DNA domain in all three possible reading frames to the next
endogenous exon upon integration into a gene within the
genome.
The following protocol may be used:
1. Construct a reporter murine embryonic stem cell line
using standard methods by co-electroporation of linearized
pGSPuro, and pPGKHyg into the murine Rl ED cell line.
2. Select, and expand several clones which contain
pGSPuro.
3. Characterize the reporter cell line for ability to
express transcriptional activator domains by
electroporating with pM3-VP16 (Clontech) as a positive
control and pM-53 (Clontech) as a negative control for
transcriptional activator domains. pM3-VP16 expresses a
fusion of the VP16 activation domain to the GAL4 DNA
binding domain which is known transactivate the GAL4
responsive promoter in pGSPuro. The pm-53 negative control
plasmid expresses a fusion of the VP16 activation domain to
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p53, which does not transactivate the GAL4 responsive
promoter in pGSPuro.
4. Upon electroporation of positive or negative control
plasmids, cells are then placed under 1.0 ug/ml puromycin
selection.
5. Select appropriate reporter cell clones that confer
puromycin resistance in the presence of pM3-VP16 but not
with pM-53.
6. Electroporate gene trap vector into reporter cell line
and select puromycin resistance with 1.0 ug/ml puromycin.
7. Pick individual puromycin resistant colonies and
isolate RNA from each clone.
8. Isolate and sequence trapped exon/gene by rapid
amplification of cDNA ends (RACE-PCR).
All publications and patents cited in this
specification are incorporated herein by reference.
Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of
clarity of understanding, it will be readily apparent to
those of ordinary skill in the art in light of the
teachings of this invention that changes and modification
may be made thereto without departing from the spirit or
scope of the appended claims.
