CA2224475A1 - Protein interaction and transcription factor trap - Google Patents
Protein interaction and transcription factor trap Download PDFInfo
- Publication number
- CA2224475A1 CA2224475A1 CA 2224475 CA2224475A CA2224475A1 CA 2224475 A1 CA2224475 A1 CA 2224475A1 CA 2224475 CA2224475 CA 2224475 CA 2224475 A CA2224475 A CA 2224475A CA 2224475 A1 CA2224475 A1 CA 2224475A1
- Authority
- CA
- Canada
- Prior art keywords
- dna
- protein
- gene
- vector
- transcriptional regulatory
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/40—Systems of functionally co-operating vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/60—Vectors containing traps for, e.g. exons, promoters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor
Description
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
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
- 2 -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
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
- 3 -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.
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.
- 4 -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
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
- 5 -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.
' CA 02224475 1998-02-25
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.
' CA 02224475 1998-02-25
- 6 -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 (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 _ g _ 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.
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.
' CA 02224475 1998-02-25 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.
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 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.
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.
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.
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.
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:
(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:
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.
6. Pick individual puromycin resistant colonies and isolate RNA from each clone.
(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 (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 _ g _ 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.
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.
' CA 02224475 1998-02-25 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.
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 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.
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.
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.
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.
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:
(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:
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.
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;
(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 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.
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;
(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 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.
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.
Claims
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2224475 CA2224475A1 (en) | 1998-02-25 | 1998-02-25 | Protein interaction and transcription factor trap |
| EP99936098A EP1062368A1 (en) | 1998-02-25 | 1999-02-25 | Protein interaction and transcription factor trap |
| CA002320894A CA2320894A1 (en) | 1998-02-25 | 1999-02-25 | Protein interaction and transcription factor trap |
| PCT/CA1999/000173 WO1999043848A1 (en) | 1998-02-25 | 1999-02-25 | Protein interaction and transcription factor trap |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2224475 CA2224475A1 (en) | 1998-02-25 | 1998-02-25 | Protein interaction and transcription factor trap |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2224475A1 true CA2224475A1 (en) | 1999-08-25 |
Family
ID=4161884
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2224475 Abandoned CA2224475A1 (en) | 1998-02-25 | 1998-02-25 | Protein interaction and transcription factor trap |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP1062368A1 (en) |
| CA (1) | CA2224475A1 (en) |
| WO (1) | WO1999043848A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6713257B2 (en) | 2000-08-25 | 2004-03-30 | Rosetta Inpharmatics Llc | Gene discovery using microarrays |
| US7807447B1 (en) | 2000-08-25 | 2010-10-05 | Merck Sharp & Dohme Corp. | Compositions and methods for exon profiling |
| JP2004533245A (en) | 2001-05-04 | 2004-11-04 | ヘルス リサーチ インコーポレイテッド | High-throughput assays to identify gene expression modifiers |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5652128A (en) * | 1993-01-05 | 1997-07-29 | Jarvik; Jonathan Wallace | Method for producing tagged genes, transcripts, and proteins |
| US5525490A (en) * | 1994-03-29 | 1996-06-11 | Onyx Pharmaceuticals, Inc. | Reverse two-hybrid method |
| GB9603069D0 (en) * | 1996-02-14 | 1996-04-10 | Medical Res Council | Improvements in or relating to gene expression |
-
1998
- 1998-02-25 CA CA 2224475 patent/CA2224475A1/en not_active Abandoned
-
1999
- 1999-02-25 EP EP99936098A patent/EP1062368A1/en not_active Withdrawn
- 1999-02-25 WO PCT/CA1999/000173 patent/WO1999043848A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1999043848A1 (en) | 1999-09-02 |
| EP1062368A1 (en) | 2000-12-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6410317B1 (en) | Recombinase-based methods for producing expression vectors and compositions for use in practicing the same | |
| US7183395B2 (en) | Methods of identifying synthetic transcriptional and translational regulatory elements, and compositions relating to same | |
| AU4895096A (en) | Chimeric dna-binding proteins | |
| JP4817657B2 (en) | Inducible eukaryotic expression system | |
| JP2001506851A (en) | Prokaryotic two-hybrid system | |
| Mortensen et al. | Selection of transfected mammalian cells | |
| JP2004501601A5 (en) | ||
| CA2589191A1 (en) | Enhancer-containing gene trap vectors for random and targeted gene trapping | |
| US20030143578A1 (en) | High throughput method for identification of sequence tags | |
| CA2224475A1 (en) | Protein interaction and transcription factor trap | |
| JP2003513670A (en) | Methods and compositions for screening using diphtheria toxin constructs | |
| US5554510A (en) | Regulation of gene expression | |
| EP1358203A2 (en) | Methods and compositions for use in spliceosome mediated rna trans-splicing | |
| WO1996035777A1 (en) | Chimeric enzyme for promoting targeted integration of foreign dna into a host genome | |
| US7060433B1 (en) | Methods and compositions for screening using diphtheria toxin constructs | |
| CA2320894A1 (en) | Protein interaction and transcription factor trap | |
| US20230242922A1 (en) | Gene editing tools | |
| CN109790214B (en) | Improved methods for selecting cells producing polypeptides | |
| US20020076688A1 (en) | Compositions and methods for the discovery and selection of biological information | |
| AU2004237884B2 (en) | Spliceosome mediated RNA trans-splicing | |
| US7087382B1 (en) | Methods for detecting polypeptides regulating signal transduction pathways | |
| JP2008092911A (en) | Method for detecting bonding of protein | |
| WO2004065553A2 (en) | Method for comprehensive identification of cell lineage specific genes | |
| CA2426896A1 (en) | Compositions and methods for the discovery and selection of biological information | |
| EP1402020A2 (en) | Methods, vectors, cell lines and kits for selecting nucleic acids having a desired feature |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |