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WO2000053813A1 - Bibliotheques pangenomiques - Google Patents

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WO2000053813A1
WO2000053813A1 PCT/US2000/006301 US0006301W WO0053813A1 WO 2000053813 A1 WO2000053813 A1 WO 2000053813A1 US 0006301 W US0006301 W US 0006301W WO 0053813 A1 WO0053813 A1 WO 0053813A1
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gene
cells
nucleic acid
expression
marker polypeptide
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Zhou Songyang
David Baltimore
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Massachusetts Institute of Technology
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Massachusetts Institute of Technology
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    • 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
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
    • 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
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral 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
    • 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
    • C12N15/86—Viral vectors
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • DNA arrays provide an opportunity to analyze the differences and similarities between the expression pattern in one cell type relative to another cell type.
  • arrays are likely to contain only abundantly expressed genes derived from a particular cell source. If phenotypic differences between two cell types are correlated by the expression of novel genes not present in the particular cell source, the use of DNA arrays for comparing expression pattern may again be limited.
  • Mammalian genetic studies rely heavily on gene targeting using mouse embryonic stem (ES) cells and retrovirus-mediated mutagenesis.
  • ES mouse embryonic stem
  • retrovirus-mediated mutagenesis demand major effort and time to positionally clone the target genes because of the complexity of the mammalian genome.
  • mammalian signal pathways can be studied genetically in tissue culture cells.
  • Cultured cells can grow to large numbers within a short time, and techniques that transfer foreign genes into cultured cells have been well established.
  • One approach involves constructing expression cDNA libraries from different cell types, followed by the introduction of these genes back into the cells. Genes that regulate a pathway of interest can then be isolated because they result in phenotypic changes in the cells.
  • Genes that regulate a pathway of interest can then be isolated because they result in phenotypic changes in the cells.
  • the success of this approach is largely dependent on the message abundance in the cell source used to produce the cDNA libraries, as discussed above.
  • the invention is based on the discovery of a method for producing a pangenomic nucleic acid library (e.g., an expression library), and the use of such a method in screening for genes involved in a particular phenotype.
  • the method relies on a retroviral vector which is used to randomly insert a proviral sequence into a genome.
  • the pro virus includes an exogenous promoter, a sequence encoding a marker polypeptide, and a splice donor.
  • the exogenous promoter will drive transcription of a mRNA encoding a fusion protein formed by the linkage of the marker polypeptide and the amino acid sequence encoded by the endogenous exon.
  • a pangenomic library is a set of nucleic acid molecules in which every gene in a genome that encodes a functional protein is represented by at least one member of the library.
  • An expression library is capable of expressing an RNA corresponding to a gene represented by a member of the library.
  • the RNA can be translated in bacteria or eukaryotic cells to produce a protein encoded by the gene.
  • the invention features a method of producing a pangenomic nucleic acid library by (1) non-specifically inserting a provirus into the genomic DNA of a plurality of cells (e.g., human or mouse cells), the provirus including a long terminal repeat (LTR) having a U3 region that contains (i) a promoter (e.g., a cytomegalovirus promoter or a tetracycline-repressible promoter), (ii) a sequence encoding a marker polypeptide, the expression of the marker polypeptide being driven by the promoter, and (iii) a splice donor sequence, the provirus optionally having intervening sequences between (i) and (ii), and/or between (ii) and (iii); (2) selecting cells that express the marker polypeptide; and (3) isolating RNA having the sequence encoding the marker polypeptide from the selected cells, thereby producing a pangenomic nucleic acid library.
  • LTR long terminal repeat
  • the invention also includes a method of identifying a gene whose expression is associated with a desired phenotype by (1) non-specifically inserting a provirus into the genomic DNA of a plurality of cells, the provirus including a LTR having a U3 region that contains (i) a promoter, (ii) a sequence encoding a marker polypeptide, the expression of the marker polypeptide being driven by the promoter, and (iii) a splice donor sequence, the provirus optionally having intervening sequences between (i) and (ii), and/or between (ii) and (iii); (2) selecting cells that express the marker polypeptide; (3) screening cells that exhibit the desired phenotype; (4) isolating, from the selected and screened cells, RNA having the sequence encoding the marker polypeptide; and (5) identifying the sequence of the RNA 3' to the sequence encoding the marker polypeptide, thereby identifying the gene whose expression is associated with the desired phenotype.
  • This method is useful for investigating a gene whose expression is associated with any measurable phenotype, including immortality (e.g., by oncogene amplification), neuronal dedifferentiation or regeneration, metastasis (e.g., extracellular matrix-independent growth), multi-drug resistance (e.g., resistance to cancer drugs, such as cis-PtCl), senescence (e.g., disrupting the telomerase pathway), apoptosis, virus resistance (e.g., resistance to HIV-1 or influenza virus), pluripotency, or increased or decreased expression of any gene.
  • the screening step can be performed before or after the selecting step.
  • the RNA can be mRNA, and the methods of the invention can further include producing cDNA from the mRNA.
  • a marker polypeptide is any amino acid sequence that is detectable in a cell-free mixture or when present in or on the surface of a cell.
  • a marker polypeptide can include one or more of the following: epitope tags (e.g., an AU, HA, Flag, or c-myc tag), fluorescent proteins (e.g., green fluorescent protein), luminescent proteins (e.g., aequorin), chromogenic proteins (e.g., ⁇ -galactosidase), cellular localization signals (e.g., a type I, II, or III transmembrane or a nuclear localization signal), a myristylation signal, proteins conferring drug resistance (e.g., neo or puro) and/or protein oligomerization signals.
  • epitope tags e.g., an AU, HA, Flag, or c
  • a marker polypeptide which includes both an epitope tag and a transmembrane sequence for displaying the epitope tag on the surface of a cell is especially useful because the selecting step can be conducted by (1) contacting the plurality of cells with a solid support coated with an antibody which specifically binds the epitope tag; (2) washing the solid support; and (3) eluting the positive cells from the solid support.
  • the provirus can further include recombination sequences which allow removal of all or part of the provirus from the genome. Specific recombination sequences, such as those used for Cre/lox recombination, are described in U.S. Patent Nos. 5,658,772 and 5,654,182.
  • the invention further includes nucleic acid libraries, including pangenomic nucleic acid libraries produced by the methods of the invention.
  • the invention includes (1) a nucleic acid library whose members represent every gene within a genome; (2) a nucleic acid library, where the abundance of members of the library representing a gene is directly correlated with the genome copy number of the gene; (3) a nucleic acid library, where the abundance of members of the library representing a gene is directly correlated with the size of the gene; and (4) a nucleic acid library free of a statistically significant correlation between the abundance of members representing a gene and the expression level of the gene in a cell.
  • the invention further includes expression vector libraries (e.g., a library of DNA expression plasmids)and viral vector libraries (e.g., a library of retroviral or adeno viral vectors) that are derived from and contain a nucleic acid library of the invention.
  • expression vector libraries e.g., a library of DNA expression plasmids
  • viral vector libraries e.g., a library of retroviral or adeno viral vectors
  • retrovirus whose genomic RNA includes a 3 'LTR having a U3 region including (i) a promoter, (ii) a nucleotide sequence encoding a marker polypeptide, the expression of the marker polypeptide being driven by the promoter, and (iii) a splice donor sequence.
  • retroviruses can be used in the methods of the invention.
  • Fig. 1 is a schematic representation of a generic vector useful in the methods of the invention.
  • Fig. 2 is a schematic representation of a method of the invention using the vector shown in Fig. 1.
  • Fig. 3 is a schematic representation of a specific vector useful in the methods of the invention.
  • Fig. 4 is a schematic representation of a method of the invention using the vector shown in Fig. 3.
  • Fig. 5 is a bar graph of number of foci versus various constructs introduced into cells.
  • Figs 6 A and 6B are bar graphs of number of foci produced in the absence or presence of tetracycline.
  • Fig. 7 is a schematic representation of the sites of integration within a Raf gene.
  • Fig. 8 is a bar graph of number of live cells in the presence or absence of tetracycline.
  • the invention relates to methods of producing nucleic acid and cellular libraries in which every gene of a genome encoding a functional protein is represented by at least one member of the library.
  • These libraries which can be a library of DNA molecules, cells, or viruses, in turn are useful for screening genes affecting a particular phenotype of a cell.
  • a pangenomic library of the invention can provide a an unbiased reference expression level for any gene encoding a functional protein because the representation of each gene is not dependent on its expression level in a particular cell type.
  • the invention also includes a method of normalizing the expression of a gene in a cell by (1) comparing the level of expression of the gene in the cell with the abundance of members representing the gene in the nucleic acid libraries of the invention; and (2) expressing the level of expression as a quantity relative to the abundance of members representing the gene in the nucleic acid library.
  • the methods of the invention are useful for producing universal, pangenomic cDNA libraries, which can be screened without concern for under- or over- representation of certain transcripts or genes.
  • the nucleic acid libraries of the invention exhibit beneficial properties, including whole genome representation, consistent library member abundance regardless of cell source, and a normalized or standard level of abundance for every gene encoding a functional protein.
  • libraries can be especially useful in producing DNA microarrays whose gene coverage is far more extensive that microarrays formed from traditional cDNA libraries with the limitations discussed above.
  • the invention allows the identification of genes associated with a disease or condition, including loss of function mutations induced by subtle sequence changes such as point mutation and small deletions.
  • the libraries of the invention can be made from patient tissue samples and hybridized with control libraries. cDNA sequences that carry point mutations and deletions can then be isolated via MutS fusion proteins.
  • the universal cDNA libraries can be used to introduce a library into cells to complement a defect or phenotype exhibited by the cells.
  • the marker polypeptide includes a type II transmembrane sequence
  • a library of proteins can be displayed on the surface of mammalian cells. Such cells may be useful in raising antibodies.
  • the invention also includes a pangenomic cellular library in which member cells of the library express every possible gene encoding a functional protein on the surface of the cell.
  • the cellular libraries of the invention are especially useful for identifying which endogenous epitopes are being attacked in a patient experiencing an autoimmune disease. For this purpose, patient antibodies are used to select cells from the cellular library, and the sequence 3' to the sequence encoding the marker polypeptide is identified, thereby identifying the epitope to which the patient has developed an immune response.
  • Retroviral Vector Plasmids Referring to Fig. 1, an exogenous promoter (e.g., CMN or pgk), a sequence for expression of a marker polypeptide, and a consensus sequence for a splice donor are cloned into the U3 region of a 3' LTR of a retroviral vector. The sequence inserted into the 3 'LTR is termed a cassette sequence. In order to target endogenous genes in all three possible reading frames, three nearly identical vectors can be made.
  • CMN or pgk exogenous promoter
  • a sequence for expression of a marker polypeptide e.g., a sequence for expression of a marker polypeptide
  • a consensus sequence for a splice donor cloned into the U3 region of a 3' LTR of a retroviral vector.
  • the sequence inserted into the 3 'LTR is termed a cassette sequence.
  • three nearly identical vectors can be made.
  • these three vectors differ in that the splice junction is offset by zero, one, or two nucleotides relative to the reading frame for the marker polypeptide.
  • infecting cells with all three viral vectors should lead to the expression of any protein encoded by an endogenous exon, regardless of which reading frame is formed by ligation of the splice donor and acceptor in the R ⁇ A.
  • These sequences can be flanked by recombination sequences (e.g., those recognized by the recombinase Cre) that can be removed upon expression of the corresponding recombinase.
  • Retroviral vectors useful in the methods of the invention include various wild- type or enhancer-deleted (SIN) retroviral vectors (e.g., pBabe, MSCV, and pLSXN). Production of Live Retroviral Vectors
  • retroviral vectors described above can be transfected into retroviral packaging cells (e.g., BOSC cells).
  • Progeny viruses are harvested to infect mouse or human cells (e.g., cell lines), which optionally have been engineered to express an retroviral receptor sufficient for the retroviruses to enter the target cells.
  • the retroviral vectors should place the exogenous promoter and epitope-sequences near an endogenous exon. This will allow regulated gene expression independent of endogenous promoters. Due to the presence of the splice donor at the 3' end of the marker sequence, induced expression through the exogenous promoter will yield mRNA molecules that are marked by the sequence encoding the marker polypeptide at the 5' end.
  • retroviruses integrate into a genome randomly, high titer infection of cells using designed retroviruses can activate genome-wide genes with multiple exons, some of which may normally be silenced, depending on the cell type.
  • the resultant transcript is stable when the exogenous promoter and sequences encoding the marker polypeptide are integrated near exons and eventually spliced to join an endogenous poly- A signal. Therefore, cells that have integration events relevant to a specific phenotype can be enriched using the selection marker encoded by the cassette sequence. Genetic screens can be subsequently performed using these enriched cells. Cells that exhibit a phenotype of interest can be isolated. mRNAs are extracted form these cells and targeted genes can be rapidly cloned using RT-PCR with primers derived form the unique 5' tag sequences and universal poly(dT) primers. Uses
  • the methods and libraries of the invention allow genome- wide identification of genes critical for a variety of signaling processes. Comprehensive molecular markers for disease diagnosis and disease gene identification in humans can be generated.
  • Novel genes identified by using the methods and libraries of the invention can be used to further elucidate the signal transduction pathways which are defective in various human diseases or conditions.
  • the invention aides the generation of new reagents (e.g., probes and antibodies) to study these pathways.
  • new reagents e.g., probes and antibodies
  • the invention helps provide drug targets and therapeutics.
  • the methods of the invention generate normalized cDNA libraries that represent nearly all mRNA transcripts possible in the genome, because gene expression is controlled by an exogenous promoter, and because the integration of the promoter is random. Genes that are normally dormant in particular cell types can be activated to produce marked mRNA. These marked mRNAs can be isolated from whole cellular mRNA using biotinylated primers specific for the sequence encoding the marker polypeptide. The normalized cDNA libraries will also provide exon boundary information for mouse and human genes. Such nucleic acid libraries can be used to produce universal DNA chips for detection of differential gene expression among cell populations.
  • normalized libraries that represent close to complete mRNA transcripts of the whole genome can also be generated from human patient tissue samples using retrovirus vectors.
  • the normalized libraries can be compared with those from healthy donors and used to identify mutated genes that cause a particular disease.
  • the tagging epitope is a type II transmembrane sequence
  • the fusion protein will be expressed and displayed on the surface of cell. The invention, therefore, makes it possible to study protein-protein interactions and identify molecules that can bind to the protein of interest on the surface of mammalian cells.
  • ELM enhanced retroviral mutagen
  • ERM vector design The ERM vectors used were derived from pBabe-puro vectors (Morgenstern et al., Nucleic Acids Res. 18:3587-3596, 1990).
  • the ERM cassettes containing the tetracycline-repressible promoter, ERM Tag, and an artificial splice donor were cloned in the Nhel site of the 3' LTR. This design avoids potential complications arising from the cryptic splice acceptor sequence present in the 3' end of the 5' LTR.
  • the ERM Tag encodes the chicken c-Src myristylation signal, MGSSKSKPKDPSQR (SEQ ID NO:l), upstream of the AUl epitope DTYRYI (SEQ ID NO:2). This portion of the ERM was designated Tet-MyrAU-SD. The AUl epitope was chosen because of its small size.
  • a set of three ERM vectors were constructed in which the ERM Tags were in three possible reading frames (RF1, RF2, and RF3). Cell lines and virus production. NIH 3T3 cells were maintained in DMEM media supplemented with 10% calf serum (CS).
  • BOSC23 cells were maintained in DMEM media containing 10% fetal calf serum (FCS) as described in Pear et al., Pro. Natl. Acad. Sci. USA 90:8392-8396, 1993.
  • FCS fetal calf serum
  • the ERM vectors were used to transfect the retroviral packaging cell line BOSC23 as described in Pear et al., supra.
  • the virus supernatant was then collected, filtered through a 0.45 micron syringe filter, and used to infect NIH 3T3 cells.
  • NIH 3T3 transformation screen and transformation assay ERM viruses were generated in three possible reading frames, each frame encoding a myristylation signal, the AUl epitope, and a splice donor sequence under the control of a tetracycline-repressible promoter.
  • NIH 3T3 cells (about 12 million) were plated in 6- well plates (3 x 10 5 cells per well) and spin-infected with the ERM virus supematants in the presence of 4 mg/ml polybrene at 2500 rpm for one hour. The media were replaced with 3 ml of 10%> CS-DMEM the next day. Fresh media were added every three days, and the cells were maintained for two weeks. Individual foci (clones) were isolated, selected in puromycin (1 mg/ml), purified, and expanded to extract RNA for further analysis.
  • candidate genes were subcloned into pBabe retroviral vectors to generate viruses for subsequent infection of NIH 3T3 cells (1 x 10 5 cells per well). The cells were then maintained in 6-well plates, and transforming foci formation was determined after one week.
  • Tetracycline sensitivity assay Isolated foci were trypsinized and plated in 10%) CS-DMEM media with or without 2 mg/ml tetracycline for one week. The morphology of these cells was examined under a phase-contrast microscope. To quantitate the difference, approximately 100 transformed cells were plated with 5 x 10 5 parental NIH 3T3 cells in 6-well plates in the presence or absence of 2 mg/ml tetracycline. The number of foci formed after one week were compared.
  • RT-PCR 3' RACE, and PCR primers.
  • individual focus clones were expanded, and RNA was extracted using the RNeasy Mini Kit (QIAGEN).
  • the 5' end of RT-1 primer contained sequences for the T7 primer.
  • the cDNA was then PCR amplified with primers from the myristylation signal sequence (Myrl : ACCATGGGGAGCAGCAAGAGCAA ACCAAAAGACCCCAGCCAACGC [SEQ ID NO:4]) and the T7 primer, using Taq DNA polymerase (Gibco).
  • the PCR products were then gel purified and directly sequenced.
  • Genomic PCR for viral integration sites Genomic DNAs were purified from RF1-2 and RF1-8 clones in which the ERM retroviruses targeted near the A-Raf locus. To determine the precise integration sites, PCR reactions were performed using primer pairs Myrl/Raf-Exon-2-1 (CACTGTGC GTTGCTGTTAGGCAG [SEQ ID NO:5]) and Raf-exon-1-1 (TGGT GGTGGTAGGGTGGACAG [SEQ ID NO:6])/Raf-exon-2-l . The resulting PCR products were gel purified, cloned into the TOPO TA cloning vectors (Invitrogen), and sequenced.
  • ERM vectors have several beneficial features (Fig. 3). Sequences for expression of a mutagenesis tag (ERM Tag), and a consensus splice donor sequence were cloned into the U3 region of the 3' LTR of a retroviral vector. The designed splice donor sequences allowed splicing and subsequent fusion of the 5' ERM Tag to the endogenous exons near the integration site. Direct cloning of the retrovirally targeted genes was therefore more easily achieved by RT-PCR and 3' RACE when a fusion transcript was generated (Fig. 4).
  • ERM Tag mutagenesis tag
  • a consensus splice donor sequence allowed splicing and subsequent fusion of the 5' ERM Tag to the endogenous exons near the integration site. Direct cloning of the retrovirally targeted genes was therefore more easily achieved by RT-PCR and 3' RACE when a fusion transcript was generated (Fig. 4).
  • the vector was designed so that the expression of the ERM Tag sequences and splice donor was under the control of a promoter that could be turned off in the presence of tetracycline (tet-off) (Gossen et al., Trends Biochem. Sci. 18:471-475, 1993; Paulus et al, J. Virol. 70:62-67, 1996).
  • tet-off tetracycline
  • the tet-off system allowed one to distinguish authentic integration events from other events.
  • three sets of vectors corresponding to different reading frames were generated in order to capture target genes without regard to the position of the target reading frame.
  • ERM integration may generate a fusion transcript that contains the engineered ERM Tag sequences, the transcript being driven from the ERM tetracycline-regulated promoter.
  • ERM integration may upregulate transcription of a nearby gene locus through its endogenous promoter, due to the presence of exogenous enhancer elements in ERM (Fig. 4). Either expression of the resulting fusion molecules or enhanced expression of the endogenous gene may be sufficient for a phenotypic change.
  • the ERM Tag allows efficient mutagenesis.
  • the ERM Tag region contained sequences encoding a chicken c-Src myristylation signal upstream of an AUl epitope.
  • the mutagenesis efficiencies of three constructs were compared: a conventional vector without any ERM Tag sequences (pBabe), a vector encoding the AUl epitope alone and containing the artificial splice donor (Tet-AU-SD), and the ERM construct that carried the ERM Tag (Tet-MyrAU-SD). These constructs were transfected into the retrovirus packaging line BOSC23 (Pear et al., supra).
  • the virus titers obtained under these conditions were similar to those of conventional vectors.
  • the viral supematants were used to infect and mutagenize NIH 3T3 cells.
  • the Tet-AU-SD construct induced slightly more foci (about 2 foci/well) as compared to the conventional vector (about 1 focus/well).
  • the Tet-MyrAU-SD ERM virus generated 16 foci/well, a more than 10 fold increase in mutagenesis efficiency.
  • ERM-mediated mutagenesis is regulatable.
  • genes that might induce oncogenic transformation were identified in NIH 3T3 cells using the Tet-MyrAU-SD construct.
  • NIH 3T3 cells were infected with ERM viruses in three possible reading frames (RFl, RF2, and RF3). More than 600 foci were recovered. These foci exhibited distinct morphologies, each different from the other, suggesting that different classes of genes had been targeted by the ERM viruses.
  • ERM strategy is that the integrated retroviral mutagens can generate fusion proteins that are tagged with the AUl epitope.
  • the expression of potential fusion products was examined in 14 RFl ERM focus clones using an anti-AUl monoclonal antibody. Most of the clones examined did indeed express fusion proteins ranging in molecular weight from 40 to 150 kDa. The expression levels of these fusion proteins varied, possibly reflecting their cellular stability. A few clones did not appear to express any fusion protein; it was possible that these fusion proteins were unstable.
  • ERM could have transformed these cells, e.g., by disrupting an anti-oncogene via insertion of the ERM into an essential regulatory element of the anti-oncogene. In this scenario, the ERM fusion protein would not need to be expressed.
  • mice 7 were known mouse genes that include TRAD (Kawai et al., Gene 227:249-255, 1999), Dbs (Whitehead et al., Oncogene 10:713-721, 1995), ARL2 (Clark et al., Proc. Natl. Acad. Sci. USA 90:8952-8956, 1993), A-Raf, MEK6 (Su et al., Curr. Opin. Immunol. 8:402-411, 1996), CD30
  • GEF-T was most homologous to human Trio (49%> identity), and GEF-K was most homologous to the human KIAA0521 protein (70%> identity).
  • 3T3 cells these genes were cloned into retroviral vectors. Infection of NIH 3T3 cells with viruses encoding GEF-T, GEF-K, and OSP1 strongly induced focus formation within one week, while CD30 viruses induced moderate numbers of transformed foci (Table 2). These results demonstrated that the ERM strategy can be used to identify both known and novel genes that regulate cell signaling.
  • ERM virus integration Consistent with the idea that ERM viruses encoding different tag reading frames can activate different sets of genes, the genes isolated using RFl ERM were mostly distinct from those of RF2 ERM. Both GEF-T and GEF-K were identified from RFl as well as RF2 ERM screens. However, the gene products identified are of different length in the two reading frame screens, presumably as a result of different ERM integration events that targeted different exons of the same gene. Sequencing analyses of ERM mutant clones indicated that a number of gene loci had been targeted multiple times. For instance, the GEF-T locus was targeted 14 times (14/54) and the A-Raf locus was targeted 5 times (5/54). This may indicate the strength of the targeted genes in inducing transformation (penetrance). To investigate how identical phenotypes may have resulted from different
  • ERM integration in the same locus the integration sites were cloned from two clones (RFl-2 and RFl -8) in which the Raf locus was targeted. As shown in Fig. 7, the ERM viruses integrated at 2.3 kb and 2.6 kb upstream of the 5' end of Raf exon 2 in clones RFl-2 and RF1-8, respectively.
  • this Example validates the ERM strategy.
  • a feature of the ERM method is the use of the ERM mutagenesis tag (that can include the myristylation signal and the AUl epitope) to facilitate cloning of the targeted genes, as well as to tag these genes with different N-terminal motif sequences.
  • ERM strategy is its adaptability. Instead of myristylation signals, different mutagenesis motif sequences can be engineered in the ERM Tag region. For instance, motifs for protein dimerization can be included to identify proteins that require clustering for activation. If a transcription repression domain is included, loss-of-function genetic screens can be performed to search for transcription regulators of various pathways. With these modifications, more elaborate genetic screens similar to those performed in lower organisms such as flies should become more feasible.
  • ERM strategy One unique feature of the ERM strategy described above is that insertional activation is regulatable by tetracycline. This will be particularly helpful for genetic screens in which there may be a large number of background mutants. In addition, for screens involving cells that differentiate during the process, the tetracycline-regulated ERM approach will enable the maintenance of mutant cell populations before they differentiate and thereby facilitate gene cloning. With the completion of the mouse and human genome projects, one of the major challenges and goals will be to integrate sequence information of different genes with biological and functional data. The ERM strategy should be particularly suitable for fulfilling part of this goal. Because of the random nature of retroviral integration, the ERM vectors can activate every single gene including normally silent ones.
  • ERM can be utilized to genetically map, on a large scale, genes that regulate different signaling pathways (e.g., senescence and differentiation) in either cultured mammalian cells or whole animals. The genes identified from the genome projects can thus be cataloged based on their functions.
  • Example 2 Isolating IL-3 Survival Factors Using ERM
  • the ERM vectors described in Example 1 above were used to generate a library of cells, which was then screened for survival in the absence of IL-3 to identify
  • IL-3-dependent 32D cells were used for genetic screens and subsequent analysis of the cytokine-independent survival kinase (CISK; Baffy et al., J. Biol. Chem. 268:6511-6519, 1993).
  • the retroviral packaging cell line BOSC23 was used to generate retroviruses as previously described in Example 1 above.
  • RNAs were isolated from 32D cell clones identified by the genetic screen using the RNeasy Mini Kit (QIAGEN). These RNAs were then reverse transcribed, amplified, and sequenced as described in Example 1. Results
  • the ERM genetic screen described in Example 1 was used to search for IL-3- independent survival genes in 32D cells.
  • the ERM vectors were used to generate retroviruses and then infect 32D cells.
  • the cells were subsequently plated in the absence of IL-3 in 96-well plates. Clones of cells that escaped cell death induced by
  • IL-3 withdrawal were isolated after two weeks. These individual clones were further expanded, harvested, analyzed by RT-PCR, and sequencing to identify the gene loci that were targeted by the ERM retroviruses.
  • the ERM genetic screen can identify many known genes that mediate IL-3 survival. Approximately 100 clones were isolated during the screen. Some clones grew more slowly than others, indicating that (1) different gene loci had been targeted by the retro vimses; and/or (2) the targeted genes worked on different levels in regulating the pathways involved in IL-3-mediated survival.
  • Bcl-xL is a general programmed cell death (PCD) inhibitor that is capable of supporting cell growth in the absence of IL-3, possibly by blocking cytochrome C release in the mitochondria (Boise et al., Cell 74:597-608, 1993; Vander Heiden et al., Mol. Cell. 3:159-167, 1999; Vander Heiden et al., Cell 91:627-637, 1997).
  • PCD programmed cell death
  • PI 3 kinase and Akt are two important players in IL-3-mediated survival pathways. Akt acts downstream of PI 3 kinase and 3-phospholipid-dependent kinase 1 (PDK1) (Delcommenne et al., Proc. Natl. Acad.
  • CISK for cytokine-independent survival kinase.
  • CISK is a member of the Ser/Thr kinase family that includes Akt and SGK (serum- and glucocorticoid- regulated protein kinase). It shares 51 % homology with Aktl , and the C-terminal sequence of CISK is identical to hSGK3, a recently cloned SGK homologue (Kobayashi et al., Biochem. J. 344(Pt.l):630-634, 1999). Analysis of the CISK amino acid sequence revealed a 496 amino acid polypeptide that contains a putative amino terminal Phox homology (PX) domain and a Ser/Thr kinase domain.
  • PX putative amino terminal Phox homology
  • CISK acts downstream of PI-3 kinase and is thought to be a major player in several survival pathways (del Peso et al., supra;
  • CISK clone isolated during the screen was tested for a response to tetracycline, since the promoter present on the ERM vector was tetracycline repressible (tet-off). Compared to control cells, the addition of 2 mg/ml of tetracycline resulted in lose of IL-3 -independent survival phenotype (Fig. 8). This finding indicated that CISK was indeed the retrovirally targeted gene responsible for the CISK clone phenotype.
  • Example 2 applies the general techniques described in Example 1 to a search of genes whose expression leads to a particular phenotype of clinical importance (e.g., in cancer).

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Abstract

La présente invention concerne des bibliothèques pangénomiques d'acides nucléiques. Les éléments de chaque bibliothèque représentent chaque gène codant une protéine fonctionnelle. Cette invention concerne également des procédés de production de ces bibliothèques à l'aide de vecteurs spécifiques (voir fig. 3 représentant schématiquement les vecteurs utilisés), et d'utilisation de celles-ci dans des méthodes diagnostiques et de dépistage.
PCT/US2000/006301 1999-03-11 2000-03-10 Bibliotheques pangenomiques Ceased WO2000053813A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040412A1 (fr) * 2001-11-05 2003-05-15 California Institute Of Technology Outil non metrique de prediction de relations genetiques a partir de donnees d'expression
WO2002063037A3 (fr) * 2001-02-02 2003-10-02 Max Planck Gesellschaft Procede d'identification d'acides nucleiques fonctionnels
WO2002070684A3 (fr) * 2001-01-11 2003-10-16 Lion Bioscience Ag Banque de genes pour procedes de criblage
WO2002101393A3 (fr) * 2001-06-12 2003-11-06 Biotechnolog Forschung Gmbh Procede pour identifier des interactions entre des proteines et des fragments d'adn d'un genome

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5840525A (en) * 1991-05-24 1998-11-24 Genentech, Inc. Nucleic acids, vectors and host cells encoding heregulin
US5965440A (en) * 1995-12-07 1999-10-12 The General Hospital Corporation Controlled gene product delivery from a regulatable retroviral vector
US6025192A (en) * 1996-09-20 2000-02-15 Cold Spring Harbor Laboratory Modified retroviral vectors

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5840525A (en) * 1991-05-24 1998-11-24 Genentech, Inc. Nucleic acids, vectors and host cells encoding heregulin
US5965440A (en) * 1995-12-07 1999-10-12 The General Hospital Corporation Controlled gene product delivery from a regulatable retroviral vector
US6025192A (en) * 1996-09-20 2000-02-15 Cold Spring Harbor Laboratory Modified retroviral vectors

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002070684A3 (fr) * 2001-01-11 2003-10-16 Lion Bioscience Ag Banque de genes pour procedes de criblage
WO2002063037A3 (fr) * 2001-02-02 2003-10-02 Max Planck Gesellschaft Procede d'identification d'acides nucleiques fonctionnels
AU2002249170B2 (en) * 2001-02-02 2007-02-08 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V Method for identifying functional nucleic acids
WO2002101393A3 (fr) * 2001-06-12 2003-11-06 Biotechnolog Forschung Gmbh Procede pour identifier des interactions entre des proteines et des fragments d'adn d'un genome
WO2003040412A1 (fr) * 2001-11-05 2003-05-15 California Institute Of Technology Outil non metrique de prediction de relations genetiques a partir de donnees d'expression

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