WO1999002724A2 - Methods for identifying genes expressed in selected lineages, and a novel genes identified using the methods - Google Patents

Methods for identifying genes expressed in selected lineages, and a novel genes identified using the methods Download PDF

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Publication number
WO1999002724A2
WO1999002724A2 PCT/CA1998/000667 CA9800667W WO9902724A2 WO 1999002724 A2 WO1999002724 A2 WO 1999002724A2 CA 9800667 W CA9800667 W CA 9800667W WO 9902724 A2 WO9902724 A2 WO 9902724A2
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nucleic acid
protein
cells
seq
gene
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PCT/CA1998/000667
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French (fr)
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WO1999002724A3 (en
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William Stanford
Georgina Caruana
Michihiro Hidaka
Alan Bernstein
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Mount Sinai Hospital Corporation
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Priority to AU82035/98A priority Critical patent/AU8203598A/en
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Publication of WO1999002724A3 publication Critical patent/WO1999002724A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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

Definitions

  • the invention relates to vectors, compositions, and methods, for identifying genes pnmanly expressed in selected lineages
  • the invention also relates to novel genes pnmanly expressed in selected lineages, proteins encoded by the novel genes and truncations, analogs, homologs, and isoforms of the proteins; and, uses of the proteins and genes BACKGROUND OF THE INVENTION
  • the techniques use vectors which are randomly integrated into genes
  • the vectors typically contain a reporter gene which facilitates the identification and isolation of the vectors once they are inserted into a gene.
  • Gene trap vectors also typically contain sequences associated with eukaryotic structural genes such as splice- acceptor sites which occur at the 5' end of all exons
  • Vectors containing a splice-acceptor site integrate into introns and generate a fusion transcript containing a target endogenous gene and the reporter gene (see references 5, 10, 11 in D.P. Hill and W. Wurst, Supra)
  • the expression of the reporter gene is under the regulatory control of the endogenous gene and its expression mimics the expression pattern of the target gene (see reference 12 in D P. Hill and W.
  • the insertion of the gene trap vector can also create a mutation and disrupt the function of the target gene (see references 10 and 12 in D P. Hill and W Wurst, Supra).
  • the part of the target gene in the fusion transcript may also be cloned from the fusion tiansc ⁇ pt, or from genomic DNA upstream of the insertion site.
  • Embryonic stem (ES) cell technology offers an efficient way of introducing gene trap vectors into the mouse genome and thereby identify and mutate genes expressed during mouse development.
  • ES cells isolated from the mouse inner cell mass remain plu ⁇ potent after genetic manipulation and in vitro culture, and they contribute to all tissues of the mouse, including the germ line (see references 7 to 9 in D P. Hill and W Wurst,
  • Mutations can be transmitted through the germ line and offspring can be screened for recessive mutant phenotypes. Prescreenmg in chime ⁇ c embryos can also be carried out, and mutations resulting in interesting patterns can be transmitted through the germ line and their phenotype studied.
  • Gene trapping in ES cells is a powerful technique because it simultaneously integrates gene identification and structure, expression and functional analysis into one process.
  • gene trap screens have used one of these three types of analyses as the primary determinant to select clones for further study.
  • the first group of screens uses no pre-selection to study mutant phenotypes.
  • the present inventors have developed a gene trap strategy to identify, mutate, and characterize large numbers of genes on the basis of their cell-lineage specific expression
  • This expression trapping method complements and extends previous expression-based gene trap screens by specifically identifying integrations into genes preferentially expressed in selected cell lineages.
  • the approach simultaneously provides expression, sequence, and phenotypic information.
  • the method can be used to carry out large scale, genome-wide scans for genes of interest. Integrations with identifiable expression patterns in vitro can be catalogued to generate a biological resource of gene-trap insertions, based upon expression pattern, cDNA sequences, and mutant phenotypes.
  • the method permits identification of specific messages present in low levels that could not have been found using conventional techniques.
  • the present invention relates to a method of identifying a target nucleic acid molecule primarily expressed in selected lineages comprising:
  • transfected cells (a) integrating into a site in the genome of a host cell a gene trap vector containing a reporter gene, to form transfected cells; (b) growing the transfected cells in vitro under conditions whereby the transfected cells differentiate into embryoid bodies attached to a carrier and identifying embryoid bodies expressing the reporter gene in cells of a selected lineage, or (c) growing the transfected cells in vitro under conditions whereby the transfected cells differentiate into cells of a selected lineage, and identifying cells of the selected lineage expressing the reporter gene; wherein the target nucleic acid molecule composes sequences upstream or downstream of the site of integration of the reporter gene in the cells of the selected lineage.
  • the method may further comprise isolating nucleic acid molecules from the transfected cells, or descendents thereof expressing the reporter gene wherein the nucleic acid molecules comp ⁇ se the reporter gene and a part of the target nucleic acid molecule, or the nucleic acid molecules comprise genomic DNA upstream or downstream of the site of insertion of the gene trap vector
  • Transfected cells or descendents thereof expressing the reporter gene may be introduced into embryos to form chime ⁇ c embryos. Therefore, the present invention contemplates a chime ⁇ c embryo having integrated into its genome a gene trap vector at a site of a target nucleic acid molecule primarily expressed in cells of selected lineages. Germline transmission may be achieved by mating chime ⁇ c embryos allowed to mature to term, or mating foster recipient females having the chime ⁇ c embryos. Therefore, the invention also contemplates a transgenic non-human animal all of whose somatic cells and germ cells contain a gene trap vector at a site of a target gene primarily expressed in cells of selected lineages
  • the present inventors using the novel strategy described herein have identified novel clones expressed primarily in hematopoietic, endothe al, stromal, and/or myocyte lineages designated 17G2, K18F2,
  • K20D4, K18F2, K20D4, B2D2, GC10E10, GC11C7, and GC11E10 The invention therefore i elates to novel nucleic acid molecules isolated from these clones.
  • nucleic acid molecules of the invention permit identification of untranslated nucleic acid sequences or regulatory sequences which specifically promote expression of proteins operatively linked to the promoter regions Identification and use of such promoter sequences are particularly desirable in instances, such as gene transfer or gene therapy, which can specifically require heterologous gene expression in a limited
  • the invention therefore contemplates a nucleic acid encoding a regulatory sequence of a nucleic acid molecule of the invention, such as a promoter sequence
  • the nucleic acid molecules of the invention may be inserted into an approp ⁇ ate vector, and the vector may contain the necessary elements for the transcription and translation of the inserted coding sequence
  • vectors may be constructed which comprise a nucleic acid molecule of the invention and optionally one or more transcription and translation elements linked to the nucleic acid molecule.
  • Vectors are contemplated within the scope of the invention which comprise regulatory sequences of the invention, as well as chimenc gene constructs wherein a regulatory sequence of the invention is operably linked to a nucleic acid sequence encoding a heterologous protein, and a transcription termination signal.
  • a vector of the invention can be used to prepare transformed host cells expressing the proteins encoded by the nucleic acids of the invention, or a heterologous protein. Therefore, the invention further provides host cells containing a vector of the invention.
  • the invention also contemplates transgenic non-human mammals whose germ cells and somatic cells contain a vector comprising a nucleic acid molecule of the invention or a fragment thereof, in particular one which encodes an analog or a truncation of a pi otein of the invention.
  • the invention further provides a method for preparing novel proteins encoded by the nucleic acids of the invention utilizing the purified and isolated nucleic acid molecules of the invention.
  • a method for preparing a protein comprising (a) transferring a vector of the invention into a host cell; (b) selecting transformed host cells from untransformed host cells, (c) cultu ⁇ ng a selected ixansformed host cell under conditions which allow expression of the protein, and (d) isolating the protein.
  • a protein of the invention may be obtained as an isolate from natural cell sources, but they are preferably obtained by recombinant procedures
  • the invention further broadly contemplates an isolated protein comprising the amino acid sequence of SEQ. ID. NO 2, SEQ. ID. NO 5 , or SEQ. ID. NO. 7
  • the invention includes a truncation of a protein of the invention, an analog, an allehc or species variation thereof, or a homolog of a protein of the invention, or a truncation thereof (
  • proteins of the invention used herein includes truncations, analogs, allehc or species variations, and homologs).
  • the proteins of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins or chimenc proteins This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins
  • the invention also permits the construction of nucleotide probes which are unique to the nucleic acid molecules of the invention Therefore, the invention also relates to a probe comp ⁇ sing a sequence de ⁇ ved from a nucleic acid of the invention or encoding a protein of the invention
  • the probe may be labelled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleic acid sequence of the invention, or a nucleic acid sequence encoding a protein of the invention
  • the invention still further provides a method for identifying a substance which binds to a protein of the invention comprising reacting a protein with at least one substance which potentially can bind with the protein, under conditions which permit the formation of complexes between the substance and protein and assaying for complexes, for free substance, for non-complexed protein, or for activated protein
  • the invention provides a method for evaluating a compound for its ability to modulate the biological activity of a protein of the invention For example a substance which inhibits or enhances the interaction of the protein and a substance which binds to the protein may be evaluated
  • the method comprises providing a known concentration of a protein, with a substance which binds to the protein and a test compound under conditions which permit the formation of complexes between the substance and protein, and assaying for complexes, for free substance, for non-complexed protein, or for activated protein
  • nucleic acid or protein of the invention may also be identified using the methods of the invention by comparing the pattern and level of expression of nucleic acid or protein of the invention in tissues and cells, in the presence, and in the absence of the compounds
  • the substances and compounds identified using the methods of the invention may be used to modulate a nucleic acid or protein of the invention, and they may be used in the treatment of conditions requiring modulation of for example hematopoiesis, myocardium, the sensory nervous system, or cardiac or neural vasculature Accordingly, the substances and compounds may be formulated into compositions for administration to individuals suffering from one of these conditions Therefore, the present invention also relates to a composition comprising one or more of a protein of the invention, or a substance or compound identified using the methods of the invention, and a pharmaceutically acceptable earner, excipient or diluent
  • a method for treating or preventing a condition requi ⁇ ng modulation of hematopoiesis, the sensory nervous system, or vasculature is also provided comprising administering to a patient in need thereof, a protein of the invention or a composition of the invention
  • FIG. 2 panels A to I are photographs showing GC1 lElO-lacZ expression
  • Figure 3 panels A to F, are photographs showing Mena-lacZ (K18E2) expression DETAILED DESCRIPTION OF THE INVENTION 1.
  • Expression Trapping Method As hereinbefore mentioned, the present invention provides a method for detecting a target nucleic acid molecule primarily expressed in selected lineages In an embodiment of the invention the target nucleic acid molecule is primarily expressed in hematopoietic or endothehal cells
  • Hematopoietic cells refers to cells of the hematopoietic system including plu ⁇ potential stem cells which are capable of self-replication and of differentiation tc committed progenitor cells, progenitor cells, myeloid and lymphoid stem cells, and neutrophils, macrophages, erythroid cells, mast cells, megakaryocytes, blast cells, lymphocytes, and monocytes 'Endothehal cells” refers to a type of squamous epithelium cells that lines the interiors of cavities, spaces, and blood vessels
  • the method of the invention involves integrating into the genomes of host cells a gene trap vector containing a reporter gene, to form transfected cells
  • the gene trap vector used in the method of the invention comprises a reporter gene which allows for differentiation of cells having a gene trap vector integrated into a target nucleic acid molecule primarily expressed in selected lineages (e g hematopoietic or endothehal cells)
  • Reporter genes which are particularly useful in the method of the invention are genes encoding ⁇ -galactosidase (e g lac Z), chloramphenicol, acetyltransferase, or firefly luciferase, Transcription of the reporter gene is monitored by changes in the concentration of the protein encoded by the reporter gene such as ⁇ -galactosidase, chloramphenicol, acetyltransferase, green fluorescence protein (GFP), or firefly luciferase Transfected cells or descendents thereof showing reporter gene activity are identified using conventional methods
  • the gene trap vector may include sequences associated with eukaryotic structural genes which facilitate the insertion of the vector into a eukaryotic gene
  • the gene trap vector may include sequences associated with elimination of tron sequences from mRNA such as splicer-acceptor sequences (e.g. using an En entron), and polyadenylation signal sequences.
  • the gene trap vector may also include sequences which facilitate isolation and sequencing of the target gene.
  • the gene trap vector may contain loxp sequences before and after the lacZ sequence.
  • the loxp sequences are cleaved by ere recombinase allowing removal of the lacZ sequence
  • Preferred gene trap vectors for use in the method of the invention are PT1 which contains an En-2 intron sequence including a splice-acceptor site front of the bacterial lacZ gene and a neomycin gene d ⁇ ven by the PGK-1 promoter; PT1/ATG which is the same as PT1 with the exception that it includes a translational start signal (ATG) in the lacZ gene (Hill DP and Wurst W, Methods in Enzymology 225:664, 1993), and GTl. ⁇ geo which contains the En-2 splice acceptor site immediately upstream of a lacZ-neo vector thereby allowing neomycin resistance at a lower level of endogenous gene expression
  • the gene trap vector may be introduced into host cells by conventional methods such as transfection, pofection, precipitation, infection, electroporation, microinjection etc Methods for transfecting, etc. host cells are well known m the art (see Sambrook et al. Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, all of which is incorporated herein by reference)
  • Suitable host cells for use in the method of the invention include a wide va ⁇ ety of host cells, including stem cells, and plu ⁇ potent cells such as zygotes, embryos, and ES cells, preferably ES cells.
  • the gene trap vector stably integrates into the genome of the host cells Generally, the vector integrates randomly into the genome of the host cells and in some cells it will integrate into endogenous genes which are pnmanly expressed in hematopoietic or endothehal cells.
  • the transfected host cells containing the gene trap vector may be grown in vitro under conditions whereby the transfected cells differentiate into embryoid bodies
  • Methods for producing EB culture systems are known to the skilled artisan See for example, Bautch VL Et al, Dev. Dyn. 205:1-12, 1996
  • the embryoid bodies are grown attached to a carrier or support so that the endoderm layer is beneath the blood islands.
  • the carrier or support may be made of nitrocellulose, glass, polyacrylamide, gabbros, o - magnetite.
  • the support or earner matenal may have any possible configuration including spherical (e g bead), cylmd ⁇ cal (e.g. inside surface of a test tube or well, or the external surface of a rod), or flat (e.g. sheet, test st ⁇ p).
  • spherical e.g bead
  • cylmd ⁇ cal e.g. inside surface of a test tube or well, or the external surface of a rod
  • flat e.g. sheet, test st ⁇ p
  • the transfected host cells containing the gene trap vector may be grown in vitro under conditions selected so that the transfected cells differentiate into cells of a selected lineage, and the reporter gene is expressed in the transfected cells.
  • host cells which are embryonic stem cells may be cultured with a cell line which induces differentiation of the embryonic stem cells into hematopoietic cells such as the OP9 stromal cell line described by Nakano et al., (Science 265: 1098, 1994)
  • the methods of the invention can also be adapted to identify target nucleic acid molecules pnmanly expressed in particular cell types by adding one or more exogenous factors (e g cytokines) which induce the differentiation of specific cell types.
  • transfected host cells containing a gene trap vector may be grown on OP9 cell layers in the presence of granulocyte-macrophage colony-stimulating factor.
  • embryonic stem cells transfected with a gene trap vector containing a ⁇ -galactosidase gene and a gene conferring antibiotic resistance are seeded onto confluent OP9 cell layers on well plates at a concentration of 10 3 to 10 5 , preferably 10 4 cells per well.
  • the induced cells are trypsiruzed between day 5 and day 8, preferably day 5.
  • ⁇ -galactosidase activity is observed in the induced cells between about day 5 and day 12.
  • Nucleic acid molecules containing the reporter gene and a part of the target gene, or containing genomic DNA upstream or downstream of the site of integration of the gene trap vector may be isolated and cloned using standard methods from the transfected cells, or descendents thereof showing reporter gene activity. Cloned nucleic acid molecules may be sequenced and the predicted ammo acid sequence of the encoded protein can be determined using standard sequencing techniques, such as dideoxynucleotide chain termination, or Maxam-Gilbert chemical sequencing. The initiation codon and untranslated sequences of the protein may be determined using cunently available computer software designed for the purpose, such as
  • Transfected cells or descendents thereof expressing the reporter gene may be used to generate chimenc embryos.
  • clones showing reporter gene activity can be aggregated with diploid embryos (e.g. Nagy, A and Rossant J. In A.LJ. (ed): Gene Targeting: A practical Approach. Oxford, IRL, 1993, p. 147-
  • Chimenc mice can be mated (e.g. to CD-I mice) to provide animal lines having the mutation transmitted through the germline. Such a transgenic animal may be used to study the phenotype produced by the interruption of an endogenous gene by the gene trap vector, and to identify substances that reverse or enhance such a mutation.
  • the invention provides an isolated nucleic acid molecule having a sequence encoding a novel protein of the invention.
  • isolated refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical reactants, or other chemicals when chemically synthesized.
  • An "isolated” nucleic acid is also free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid molecule) from which the nucleic acid is denved.
  • nucleic acid is intended to include DNA and RNA and can be either double stranded or single stranded.
  • the invention specifically contemplates an isolated nucleic acid molecule which comprises: (i) a nucleic acid sequence encoding a protein having substantial sequence identity preferably at least
  • T can also be U; (n) nucleic acid sequences complementary to (I), preferably complementary to the full nucleic acid sequence of SEQ. ID. NO 1, SEQ. ID NO 3 , SEQ ID NO 4, SEQ ED. NO. 6, SEQ
  • SEQ. ID. NO. 8 SEQ. ID. NO. 9, or SEQ. ID NO 10,
  • nucleic acid capable of hybridizing to a nucleic acid of (I) and having at least 18 nucleotides; or (IV) a nucleic acid molecule differing from any of the nucleic acids of (I) to (in) in codon sequences due to the degeneracy of the genetic code.
  • a novel nucleic acid molecule designated 17G2 which is primarily expressed in vivo in hematopoietic cells, myocardium, in the cardiac and neural vasculature, and in the sensory nervous system, including the t ⁇ geminal ganglia, dorsal root ganglia, and optic nerve.
  • the nucleic acid molecule comprises the sequence of SEQ ID. No. 1
  • a novel nucleic acid molecule designated K18F2 which is pnmanly expressed in vitro by muscle cells in attached embryoid bodies, and some mesodermal cells in OP9 induction cultures, and pnmanly expressed in vivo in both tetraploid and diploid chimenc embryos exclusively in cardiac myocytes.
  • the nucleic acid molecule comprises the sequence of SEQ. ID. No. 3.
  • a novel nucleic acid molecule designated K20D4 which is expressed in vitro exclusively in vascular endothehal cells in attached embryoid bodies, and some mesodermal cells in OP9 induction.
  • the nucleic acid molecule comprises the sequence of SEQ. ID. No. 4 The sequence overlaps with EST accession No. AA239055 of clone 697718 from the Barstead mouse pooled organs cDNA library.
  • a novel nucleic acid molecule designated B2D2 which is pnmanly expressed in vitro m blood islands and vascular endothehal cells in attached EB cultures
  • expression is induced in some mesodermal cells but not in hematopoietic cells.
  • expression in the blood island may be due to endothehal cells or their precursors.
  • the nucleic acid molecule comprises the sequence of SEQ. ID. No 6. The sequence overlaps with EST accession No AA209568 of clone 676502 from the Soares NML mouse liver cDNA library.
  • GC10E10 A novel nucleic acid molecule designated GC10E10 which is highly expressed in vitro in undifferentiated embryonic cells. In attached embryoid bodies GC10E10 is expressed in blood lslands and endothehal cells. It is expressed highly in mesodermal cells and in low levels m a population of hematopoietic cells in OP9 induction cultures In vivo the gene is expressed in the forebram, midbrain, somites, notochord, otic vesicle, limb buds, branchial arches and heart in diploid chimeras.
  • the nucleic acid molecule comprises the sequence of SEQ.ID. No. 8. The sequence has 98% homology with the mu ⁇ ne Dlghl (dlgl)
  • a novel nucleic acid molecule designated GC11C7 which is primarily expressed in vitro in undifferentiated embryonic stem cells and in mesoderm and hematopoietic cells in the OP9 induction system.
  • the nucleic acid molecule comprises the sequence of SEQ.ID. No. 9. The sequence overlaps that of EST accession No. AA015451, clone 442692 from the Soares mouse placenta 4NbMP13.5 14.5 cDNA library and EST accession No. AA517189 clone 893845 from the Knowles Solter mouse embryonic stem cell cDNA library
  • a novel nucleic acid molecule designated GC11E10 which is highly expressed in vitro in undifferentiated embryonic stem cells and in blood islands and endothehal cells within attached embryoid bodies. It is also expressed in mesodermal cells and highly in hematopoietic cells in the OP-9 induction system In vivo it is expressed in endothehal and blood cells within E9.5 diploid chimeras.
  • the nucleic acid molecule comprises the sequence of SEQ.ID. No. 10.
  • the invention includes nucleic acid molecules having substantial sequence identity or similarity to the nucleic acid sequences of SEQ. ID. NO.l, SEQ. ID. NO 3 , SEQ ID. NO 4, SEQ ID. NO. 6, SEQ. ID.
  • Identity or similarity refers to sequence similarity between sequences and can be determined by comparing a position in each sequence which may be aligned l or purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are matching or have identical positions shared by the sequences.
  • the nucleic acid sequences have substantial sequence identity for example at least 75% nucleic acid identity, more preferably 80% nucleic acid identity; and most preferably at least 90 to 95% sequence identity. Isolated nucleic acid molecules having a sequence which differs from the nucleic acid sequence of
  • DNA sequence polymorphisms within the nucleotide sequence of a 17G2 protein may result in silent mutations which do not affect the amino acid sequence. Vana ⁇ ons in one or more nucleotides may exist among individuals within a population due to natural allehc variation. Any and all such nucleic acid V i ⁇ ations are within the scope of the invention. DNA sequence polymorphisms may also occur which lead to changes in the amino acid sequence of the protein. These amino acid polymorphisms are also within the scope of the present invention.
  • Another aspect of the invention provides a nucleic acid molecule which hybridizes under selective conditions, e.g. high stringency conditions, to a nucleic acid molecule of the invention. Selectivity of hybridization occurs with a certain degree of specificity rather than being random.
  • Appropriate stringency conditions which promote DNA hybndization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C may be employed.
  • the st ⁇ ngency may be selected based on the conditions used in the wash step.
  • the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be at high stringency conditions, at about 65°C.
  • the invention includes nucleic acid molecules encoding a protein of the invention including truncations, analogs and homologs of a protein of the invention as described herein.
  • fragments of a nucleic acid molecule of the invention are contemplated that are a stretch of at least about 18 nucleotides, more typically 50 to 200 nucleotides.
  • vanant forms of the nucleic acid molecules of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention.
  • An isolated nucleic acid molecule of the invention which comprises DNA can be isolated by prepanng a labelled nucleic acid probe based on all or part of a nucleic acid sequence of SEQ. ID. NO.l, SEQ. ID. NO 3., SEQ. ID. NO. 4, SEQ. ID. NO. 6, SEQ. ID. NO. 8, SEQ. ID. NO 9, or SEQ. ID. NO. 10.
  • the labelled nucleic acid probe is used to screen an appropriate DNA library (e g a cDNA or genomic DNA library).
  • a cDNA library can be used to isolate a cDNA by screening the library with the labelled probe using standard techniques.
  • a genomic DNA library can be similarly screened to isolate a genomic clone encompassing a gene of the invention.
  • Nucleic acids isolated by screening of a cDNA or genomic DNA library can be sequenced by standard techniques.
  • An isolated nucleic acid molecule of the invention which is DNA can also be isolated by selectively amplifying a nucleic acid using polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic o gonucleotide primers from the nucleotide sequence of SEQ. ID. NO.l, SEQ.
  • a nucleic acid can be amplified from cDNA or genomic DNA using these ohgonucleotide pnmers and standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropnate vector and characterized by DNA sequence analysis.
  • cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidimum-thiocyanate extraction procedure of Chirgwm et al., Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transc ⁇ ptase (for example, Moloney MLV reverse transc ⁇ ptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transc ⁇ ptase available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transc ⁇ ptase for example, Moloney MLV reverse transc ⁇ ptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transc ⁇ ptase available from Seikagaku America, Inc., St. Russia, FL).
  • An isolated nucleic acid molecule of the invention which is RNA can be isolated by cloning a nucleic acid molecule of the invention which is cDNA into an appropnate vector which allows for transcription of the cDNA to produce an RNA molecule.
  • a cDNA can be cloned downstream of a bacte ⁇ ophage promoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by conventional techniques
  • Nucleic acid molecules of the invention may be chemically synthesized using standard techmques. Methods of chemically synthesizing polydeoxynucleotides are known, including but not limited to solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers
  • Determination of whether a particular nucleic acid molecule encodes a protein of the invention can be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the expressed protein using conventional methods
  • a cDNA having the biological activity of a protein of the invention can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam- Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein
  • the initiation codon and untranslated sequences of a nucleic acid molecule of the invention may be determined using computer software designed for the purpose, such as PC/Gene (Intel Genetics Inc , Calif )
  • the mtron-exon structure and the transcnption regulatory sequences of a nucleic acid molecule or gene of the invention may be identified by using a nucleic acid molecule of the invention to probe a genomic DNA clone library Regulatory elements can be identified using standard techniques The function of the elements can be confirmed by using these elements to express a reporter gene such as the lacZ gene which is operatively linked to the elements
  • constructs may be introduced into cultured cells using conventional procedures or into non-human transgenic animal models In addition to identifying regulatory elements in DNA, such constructs may also be used to identify nuclear proteins interacting with the elements, using techniques known in the art
  • the invention contemplates polynucleotides comprising all or a portion of a nucleic acid of the invention comp ⁇ sing a regulatory sequence of a nucleic acid molecule of the invention contained in appropnate expression vectors
  • the vectors may contain sequences encoding heterologous proteins
  • the nucleic acids isolated using the methods described herein are mutant gene alleles
  • the mutant alleles may be isolated from individuals either known or proposed to have a genotype which contributes to the symptoms of a condition affecting hematopoiesis etc
  • Mutant alleles and mutant allele products may be used in therapeutic and diagnostic methods described herein
  • a cDNA of a mutant gene may be isolated using PCR as described herein, and the DNA sequence of the mutant allele may be compared to the normal allele to ascertain the mutat ⁇ on(s) responsible for the loss or alteration of function of the mutant gene product
  • a genomic library can also be constructed using DNA from an individual suspected of or known to carry a mutant allele, or a cDNA library can be constructed using RNA from tissue known, or suspected to express the mutant allele
  • a nucleic acid encoding a normal gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant allele in such libraries Clones containing mutant
  • Gene products made by the putatively mutant tissue may be expressed and screened, for example using antibodies specific for a protein of the invention as described herein
  • Library clones identified using the antibodies can be punfied and subjected to sequence analysis
  • the sequence of a nucleic acid molecule of the invention may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule
  • An antisense nucleic acid molecule may be constructed using chemical synthesis and enzymatic gation reactions using procedures known in the art 2.2 Proteins of the Invention
  • the proteins of the invention are primarily expressed in hematopoietic, endothehal, stromal, and/or myocyte lineages
  • Amino acid sequences of proteins of the invention comprise the sequences of SEQ ID NO 2, SEQ ID NO 5 , or SEQ ID NO 7
  • the proteins of the present invention include truncations of the proteins of the invention, and an -logs, and homologs of the proteins and truncations thereof as described herein
  • Truncated proteins may comprise peptides of between 3 and 275 amino acid residues, ranging in size from a tnpeptide to a 275 mer polypeptide
  • the truncated proteins may have an amino group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to hpid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the am o terminal end
  • the truncated proteins may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end
  • the proteins of the invention may also include analogs, and/or truncations thereof as descnbed herein, which may include, but are not limited to the proteins, containing one or more amino acid substitutions, insertions, and/or deletions
  • Amino acid substitutions may be of a conserved or non-conserved nature conserveed amino acid substitutions involve replacing one or more amino acids with amino acids of similar charge, size, and/or hydrophobicity characteristics When only conserved substitutions are made the resulting analog should be functionally equivalent to the native protein
  • Non-conserved substitutions involve replacing one or more amino acids with one or more amino acids which possess dissimilar charge, , ⁇ ze, and/or hydrophobicity characteristics
  • amino acid insertions may be introduced into a protein of the invention
  • Amino acid insertions may consist of single amino acid residues or sequential am o acids ranging from 2 to 15 amino acids m length
  • Deletions may consist of the removal of one or more amino acids, or discrete portions from the protein sequence
  • the deleted amino acids may or may not be contiguous
  • the lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 100 amino acids
  • allehc vanant at the protein level differs from another protein by only one, or at most, a few amino acid substitutions
  • a species vanation of a protein of the invention is a variation which is naturally occurring among different species of an organism
  • the proteins of the invention also include homologs and/or truncations thereof as descnbed herein Such homologs include proteins whose amino acid sequences are comprised of the ammo acid sequences of regions from other species that hybridize under selective hybridization conditions (see discussion of selective and in particular stringent hybridization conditions herein) with a probe used to obtain a protein of the invention These homologs will generally have the same regions which are characteristic of a piotem of the invention It is anticipated that a protein comprising an amino acid sequence which is at least 75% identical, preferably 80 to 90% identical, with an amino acid sequence of SEQ ID NO 2, SEQ ID NO 5 , or SEQ ID NO 7 will be a homolog
  • a percent amino acid sequence homology or identity is calculated as the percentage of aligned am o acids that match the reference sequence, where the sequence alignment has been determined using the alignment algorithm of Dayhoff et al, Methods in Enzymology 91 524-545 (1983)
  • the invention also contemplates isoforms of the proteins of the invention
  • An isoform contains the same number and kinds of amino acids as the protein of the invention, but the isoform has a different molecular structure
  • the isoforms contemplated by the present invention are those having the same properties a> a protein of the invention as described herein
  • the present invention also includes proteins of the invention conjugated with a selected protein, or a selectable marker protein (see below) to produce fusion proteins Additionally, immunogenic portions of a protein of the invention are within the scope of the invention
  • a protein of the invention may be prepared using recombinant DNA methods Accordingly, the nucleic acid molecules of the present invention having a sequence which encodes a protein of the invention may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the protein Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses
  • the invention therefore contemplates a vector of the invention containing a nucleic acid molecule of the invention, and optionally the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence Suitable regulatory sequences may be de ⁇ ved from a va ⁇ ety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology Methods in Enzymology 185, Academic Press, San Diego, CA (1990)
  • the invention further provides a vector comprising a DNA nucleic acid molecule of the invention cloned into the vector in an antisense orientation That is, the DNA molecule is linked to a regulatory sequence in a manner which allows for expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to a nucleic acid sequence of a nucleic acid molecule of the invention Regulatory sequences linked to the antisense nucleic acid can be chosen which direct the continuous expression of the antisense RNA molecule in a vanety of cell types, for instance a viral promoter and/or enhancer, or regulatory sequences can be chosen which direct tissue or cell type specific expression of antisense RNA
  • the expression vector of the invention may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a vector of the invention
  • selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, ⁇ -galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an lmmunoglobu n or portion thereof such as the Fc portion of an lmmunoglobuhn preferably IgG
  • the selectable markers can be introduced on a separate vector from the nucleic acid of interest
  • the vectors may also contain genes which encode a fusion moiety which provides increased expression of the recombinant protein, increased solubility of the recombinant protein, and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification
  • a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein
  • Transformant host cells include host cells which have been transformed or transfected with a vector of the invention.
  • transformed with encompass the introduction of nucleic acid (e.g a vector) into a cell by one of many standard techniques
  • nucleic acid e.g a vector
  • Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated transformation
  • Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, hpofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. (Molecular Cloning A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells
  • the proteins of the invention may be expressed in bacterial cells such as E coli, insect cells (using baculovirus), yeast cells, or mammalian cells
  • suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (199 1)
  • a host cell may also be chosen which modulates the expression of an inserted nucleic acid sequence, or modifies (e.g glycosylation or phosphorylation) and processes (e g. cleaves) the protein in a desired fashion
  • Host systems or cell lines may be selected which have specific and characteristic mechanisms for post- translational processing and modification of proteins.
  • eukaryotic host cells including CHO, VERO, BHK, HeLA, COS, MDCK, 293, 3T3, and WI38 may be used
  • eukaryotic host cells including CHO, VERO, BHK, HeLA, COS, MDCK, 293, 3T3, and WI38
  • cell lines and host systems which stably express the gene product may be engineered
  • Host cells and in particular cell lines produced using the methods described herein may be particularly useful in screening and evaluating compounds that modulate the activity of a protein of the invention.
  • the proteins of the invention may also be expressed in non-human transgenic animals including but not limited to mice, rats, rabbits, guinea pigs, micro-pigs, goats, sheep, pigs, non-human p ⁇ mates (e.g. baboons, monkeys, and chimpanzees) (see Hammer et al. (Nature 315.680-683, 1985), Palmiter et ⁇ l (Science
  • Procedures known in the art may be used to introduce a nucleic acid molecule of the invention encoding a protein of the invention into ammals to produce the founder lines of transgenic animals. Such procedures include pronuclear microinjection, retrovirus mediated gene transfer into germ lines, gene targeting in embryonic stem cells, electroporation of embryos, and sperm- mediated gene transfer
  • the present invention contemplates a transgenic animal that cames a nucleic acid molecule of the invention in all their cells, and animals which carry the transgene in some but not all their cells
  • the transgene may be integrated as a single transgene or in concatamers
  • the transgene may be selectively introduced into and activated in specific cell types (See for example, Lasko et al, 1992 Proc. Natl. Acad. Sci. USA 89. 6236)
  • the transgene may be integrated into the chromosomal site of the endogenous gene by gene targeting.
  • the transgene may be selectively introduced into a particular cell type inactivating the endogenous gene m that cell type (See Gu et al Science 265: 103-106).
  • the expression of a recombinant protein of the invention in a transgenic animal may be assayed using standard techniques. Initial screening may be conducted by Southern Blot analysis, or PCR methods to analyze whether the transgene has been integrated.
  • the level of mRNA expression in the tissues of transgenic animals may also be assessed using techniques including Northern blot analysis of tissue samples, in situ hybndization, and RT-PCR.
  • Tissue may also be evaluated lmmunocytochemically using antibodies against GNTV Protein
  • the proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Mer ⁇ field, 1964, J. Am Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol 15 I and II, Thieme, Stuttgart)
  • N-termmal or C-ter ⁇ unal fusion proteins comp ⁇ sing a protein of the invention conjugated with other molecules, such as proteins may be prepared by fusing, through recombinant techniques, the N terminal or
  • fusion proteins contain a protein of the invention fused to the selected protein or marker protein as described herein.
  • proteins which may be used to prepare fusion proteins include lmmunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.
  • nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleic acid sequences in biological materials.
  • Suitable probes include nucleic acid molecules based on nucleic acid sequences of the invention and in particular nucleic acid sequences encoding at least 6 sequential amino acids from regions of a protein of the invention (e.g SEQ. ID. NO.2, SEQ
  • a nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32 P, 3 H, 14 C or the like.
  • detectable substances include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and luminescent compounds.
  • An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization.
  • Labelled probes may be hybndized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
  • the nucleotide probes may also be useful in the diagnosis of disorders of the hematopoietic system, sensory nervous system, myocardium, or cardiac or neural vasculature, in monitoring the progression of these conditions; or monitoring a therapeutic treatment.
  • a probe may be used in hybridization techniques to detect nucleic acid molecules or genes of the invention.
  • the technique generally involves contacting and incubating nucleic acids obtained from a sample from a patient or other cellular source with a probe of the present invention under conditions favourable for the specific annealing of the probes to complementary sequences in the nucleic acids. After incubation, the non- annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected.
  • the detection of nucleic acid molecules of the invention may involve the amplification of specific gene sequences using an amplification method such as PCR, followed by the analysis of the amplified molecules using techniques known to those skilled in the art. Suitable pnmers can be routinely designed by one of skill in the art.
  • Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities in a gene or nucleic acid molecule of the invention, including point mutations, insertions, deletions, and chromosomal rearrangements.
  • direct sequencing single stranded conformational polymorphism analyses, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical mismatch cleavage, and ohgonucleotide hybridization may be utilized
  • Genotyping techniques known to one skilled in the art can be used to type polymorphisms that are in close proximity to mutations in a nucleic acid molecule or gene of the invention
  • the polymorphisms may be used to identify individuals in families that are likely to cany mutations If a polymorphism exhibits linkage disequa bnum with mutations in a gene, it can also be used to screen for individuals in the general population likely to cany mutations
  • Polymorphisms which may be used include restriction fragment length polymorphisms (RFLPs), single-base polymorphisms, and simple sequence repeat polymorphisms (SSLPs).
  • RFLPs restriction fragment length polymorphisms
  • SSLPs simple sequence repeat polymorphisms
  • a probe of the invention may be used to directly identify RFLPs
  • a probe or p ⁇ mer of the invention can additionally be used to isolate genomic clones such as YACs, B ACs, PACs, cosmids, phage or plasmids.
  • the DNA in the clones can be screened for SSLPs using hybridization or sequencing procedures.
  • Hybridization and amplification techniques described herein may be used to assay qualitative and quantitative aspects of expression of a nucleic acid molecule of the invention.
  • RNA may be isolated from a cell type or tissue known to express a gene and tested utilizing the hybridization (e.g. standard Northern analyses) or PCR techniques referred to herein
  • the techniques may be used to detect differences in transcript size which may be due to normal or abnormal alternative splicing
  • the techniques may be used to detect quantitative differences between levels of full length and/or alternatively splice transcnpts detected in normal individuals relative to those individuals exhibiting symptoms of a disease.
  • the primers and probes may be used m the above described methods in situ i.e directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections.
  • Proteins of the invention can be used to prepare antibodies specific for the proteins.
  • Antibodies can be prepared which bind a distinct epitope in an unconserved region of the protein.
  • An unconserved region of the protein is one which does not have substantial sequence homology to other proteins.
  • a region from a well- characterized region can be used to prepare an antibody to a conserved region of a protein of th ⁇ invention.
  • Antibodies having specificity for a protein of the invention may also be raised from fusion proteins created by expressing fusion proteins in bacteria as described herein.
  • the invention can employ intact monoclonal or polyclonal antibodies, and lmmunologically active fragments (e.g. a Fab or (Fab) 2 fragment), an antibody heavy chain, and antibody light chain, a genetically engineered single chain F v molecule (Ladner et al, U.S. Pat. No 4.946,778), or a chimenc antibody, for example, an antibody which contains the binding specificity of a mu ⁇ ne antibody, but in which the remaining portions are of human origin.
  • a Fab or (Fab) 2 fragment an antibody heavy chain
  • antibody light chain e.g. a genetically engineered single chain F v molecule (Ladner et al, U.S. Pat. No 4.946,778)
  • a chimenc antibody for example, an antibody which contains the binding specificity of a mu ⁇ ne antibody, but in which the remaining portions are of human origin.
  • Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art Antibodies specifically reactive with a protein of the invention, or derivatives, such as enzyme conjugates or labeled derivatives, may be used to detect the proteins in various biological materials, for example they may be used in any known lmmunoassays which rely on the binding interaction between an antigemc determinant of a protein and the antibodies.
  • radioimmunoassays examples include radioimmunoassays, enzyme lmmunoassays (e.g.ELISA), lmmunofluorescence, lmmunoprecipitation, latex agglutination, hemagglutination, and histochemical tests.
  • the antibodies may be used to detect and quantify a protein of the invention in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states.
  • the antibodies of the invention may be used in lmmuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect a protein of the invention, to localise it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a protein of the invention.
  • an antibody of the invention may be labelled with a detectable substance and a protein may be localised in tissues and cells based upon the presence of the detectable substance.
  • detectable substances include, but are not limited to, the following: radioisotopes (e.g., 3 H, 14 C, 35 S, 125 I, l31 I), fluorescent labels (e.g , FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, .beta.-galactosidase, luciferase, alkaline phosphatase, acetylchohnesterase), biotmyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calonmetnc methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucme zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached via spacer arms
  • Indirect methods may also be employed in which the pnmary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against a piotein of the invention.
  • a second antibody having specificity for the antibody reactive against a piotein of the invention.
  • the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.
  • a protein of the invention may be localized by radioautography.
  • the results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.
  • the proteins of the invention are primarily expressed in hematopoietic, endothehal stromal, and/or myocyte lineages.
  • the proteins of the invention have a role in proliferation, differentiation, activation and/or metabolism of cells of the hematopoietic, myocardium, cardiac and neural vasculature, endothehal, stromal, and/or myocyte lineages.
  • the methods described herein for detecting nucleic acid molecules can be used to monitor proliferation, differentiation, activation and/or metabolism of cells of the hematopoietic, endothehal, myocardium, cardiac and neural vasculature, stromal, and/or myocyte lineages by detecting and localizing proteins and nucleic acid molecules of the invention
  • the methods described herein may be used to study the developmental expression of a protein of the invention and, accordingly, will provide further insight into the role of the protein in the hematopoietic system, myocardium, sensory nervous system and vasculature.
  • the 17G2 protein is expressed m the myocardium, cardiac and neural vasculature, in hematopoietic cells, and in the sensory nervous system Therefore, the 17G2 protein has a role in proliferation, differentiation, activation and metabolism of cells of the hematopoietic system, myocardium, cardiac and neural vasculature, and the sensory nervous system.
  • the methods for detecting nucleic acid molecules and 17G2 proteins of the invention can be used to monitor proliferation, differentiation, activation and metabolism of hematopoietic cells, and cells of the sensory nervous system and neural and cardiac vasculature by detecting and localizing 17G2 proteins and nucleic acid molecules It would also be apparent to one skilled in the art that the above described methods may be used to study the developmental expression of 17G2 proteins and, accordingly, will provide further insight into the role of 17G2 proteins in the hematopoietic system, myocardium, neural and cardiac vasculature. and sensory nervous system
  • the nucleic acid molecules and proteins of the invention are markers for hematopoietic cells, endothehal cells, stromal cells, and/or myocytes, and accordingly the antibodies and probes described herein may be used to label these cells.
  • the 17G2 protein is a marker for early vascular endothehal cells and hematopoietic cells, and accordingly the antibodies and probes descnbed herein can be used to label early vascular endothehal cells and hematopoietic cells.
  • Substances which modulate a protein of the invention can be identified based on their ability to bind to the protein. Therefore, the invention also provides methods for identifying substances which bind to a protein of the invention. Substances identified using the methods of the inveni ion may be isolated, cloned and sequenced using conventional techniques.
  • Substances which can bind with a protein of the invention e g a 17G2 protein may be identified by reacting the protein with a substance which potentially binds to the protein, under conditions which permit the formation of substance-protein complexes and assaying for substance-protein complexes, for free substance, for non-complexed protein, or for activated protein Conditions which permit the formation of complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.
  • the substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
  • antibody against the protein or the substance, or labelled protein, or a labelled substance may be utilized.
  • the antibodies, proteins, or substances may be labelled with a detectable substance as described above
  • a protein, or the substance used in the method of the invention may be msolubihzed
  • the protein, or substance may be bound to a suitable earner such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc
  • the earner may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc
  • the insolubihzed protein or substance may be prepared by reacting the material with a suitable insoluble earner using known chemical or physical methods, for example, cyanogen bromide coupling
  • the invention also contemplates a method for evaluating a compound for its ability to modulate the biological activity of a protein of the invention, by assaying for an agonist or antagonist (l e enhancer or inhibitor) of the binding of the protein with a substance which binds with the protein
  • the enhancer or inhibitor may be an endogenous physiological compound or it may be a natural or synthetic compound
  • the agonists and antagonists l e inhibitors and enhancers that can be assayed using the methods of the invention may act on one or more of the binding sites on the protein or substance including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or alloste ⁇ c sites
  • the invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of the protein with a substance which is capable of binding to the protein
  • the invention may be used to assay for a compound that competes for the same binding site of the protein
  • kits suitable for applying the methods of the invention to evaluate compounds that modulate a protein of the invention may be packaged into convenient kits providing the necessary materials packaged into suitable containers.
  • kits may also include suitable supports useful in performing the methods of the invention
  • the substances or compounds identified by the methods described herein, antibodies, and antisense nucleic acid molecules of the invention may be used for modulating the biological activity of a protein of the invention, and they may be used m the treatment of conditions requiring modulation of cells of the hematopoietic, myocardium, cardiac and neural vasculature, endothehal, stromal, and/or myocyte lineages Accordingly, the substances, antibodies, and compounds may be formulated into pharmaceutical campositions for adminstration to subjects in a biologically compatible form suitable for administration in vivo
  • biologically compatible form suitable for administration in vivo is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects
  • the substances may be administered to living organisms including humans, and animals
  • Administration of a therapeutically active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result
  • a therapeutically active amount of a substance may vary according to factors such as the disease state
  • the active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc ), oral administration, inhalation, transdermal application, or rectal administration Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound
  • compositions described herein can be prepared by per e known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle Suitable vehicles are described, for example, in Remington s Pharmaceutical Sciences (Remington s Pharmaceutical).
  • compositions include, albeit not exclusively, solutions of the substances or compounds in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and lso-osmotic with the physiological fluids
  • the activity of the substances, compounds, antibodies, antisense nucleic acid molecules, and compositions of the invention may be confirmed in animal experimental model systems
  • the invention also provides methods for studying the function of a protein of the invention
  • Cells, tissues, and non-human animals lacking in expression or partially lacking in expression of a nucleic acid molecule or gene of the invention may be developed using recombinant expression vectors of the invention having specific deletion or insertion mutations m the gene
  • a recombinant expression vector may be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create a deficient cell, tissue or animal
  • Null alleles may be generated in cells, such as embryonic stem cells by deletion mutation
  • a recombinant gene may also be engineered to contain an insertion mutation which inactivates the gene
  • Such a construct may then be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, electroporation, injection etc
  • Cells lacking an intact gene may then be identified, for example by Southern blotting, Northern Blotting or by assaying for expression of the encoded protein using the methods described herein
  • Such cells may then be fused to embryonic stem cells to generate transgenic non-human animals deficient in a protein of the invention
  • Germ ne transmission of the mutation may be achieved, for example, by aggregating the embryonic stem cells with early stage embryos, such as 8 cell embrjos, in vitro, transfening the resulting blastocysts into recipient females and, generating germhne transmission of the resulting aggregation chimeras
  • Such a mutant animal may be used to define specific
  • PT1-ATG contains the En-2 splice acceptor site positioned immediately upstream of the lacZ reporter gene with an ATG translational start site [Hill D P , Wurst W , Methods in Enzymology 225 664-681, 1993]
  • the bacterial neomycin-resistance (neo) gene is driven by the phosphoglycerate k ⁇ nase-1 (PGK-1) promoter GT1 8geo contains the En-2 splice acceptor site immediately upstream of a lacZ-neo fusion gene [Skarnes W C et al, Proc Natl Acad Sci USA 92 6592- 6596, 1995]
  • the point mutation in the neo fragment of SA ⁇ geo is not contained in GT1 8geo vector, thereby allowing neomycin resistance at a lower level of endogenous gene expression than the SA ⁇ geo vector Generation of Trapped ES Cell Lines.
  • Rl ES cells were maintained on primary embryonic fibroblasts as previously described [Nagy A. et al., Proc. Natl. Acad. Sci. USA 90 8424-8428, 1993], After electroporation and selection in G418 for 8 days, drug-resistant colonies were transfened to 96-well plates and expanded to confluency. Clones were passaged to two 96-well plates and one set of 24-well plates. Once clones reached confluency, one 96-well plate was frozen, the second 96-well plate was assayed for ⁇ -galactosidase ( ⁇ -gal) expression, and the 24-well plates were used for attached EB differentiation cultures.
  • ⁇ -gal ⁇ -galactosidase
  • Cells were rinsed in lOOmM sodium phosphate (pH 7.5), then fixed in 0.2% glutaraldehyde, 5mM EGTA, 2mM MgCl2 and lOOmM sodium phosphate, pH 7.5 for 5 mm. The cells were washed 3 times for 5 mm. each m 2mM MgCl2, 0 02% NP-40 and lOOmM sodium phosphate, pH 7 5 The cells were stained with X-gal overnight at 37°C ⁇ -gal activity was detected m embryos as described above except the fixative included 1.5% formaldehyde and embryos were fixed for 30 mm. to 1 hour and washed 3 times for 15 min. eac i wash.
  • ES cells were allowed to differentiate into attached EBs as previously descnbed [Bautch V.L. et al., Dev. Dyn. 205:1-12, 1996] with several modifications
  • Clones were grown to confluency in 24-well plates, treated with dispase (Collaborative Research, 1:1 dilution in PBS), washed 3 times in PBS and grown in suspension in "Ultra Low Cluster" 24-well plates (COSTAR) in ES media without LIF
  • COSTAR "Ultra Low Cluster" 24-well plates
  • 5-10 embryoid bodies were transfened to 48-well tissue culture plates (Falcon). Cultures were fed every other day with fresh media, ⁇ -gal activity was determined on day 8. 12, and 16 post-dispase.
  • ES cells were allowed to differentiate on the OP9 stromal cell line as previously described [Nakano T. et al., Science 265:1098-1101, 1994] with several modifications.
  • ES clones were differentiated on OP9 stroma in replica wells of 6-well plates (10 ⁇ ES cells/well) for 5 days to generate mesodermal colonies.
  • a single cell suspension was prepared using trypsin from one well for eacli clone, and
  • 5' RACE was performed using the 5' RACE kit (Gibco/BRL), according to manufacturer's instructions with modifications previously described [Sam M. et al., Dev.
  • ES cells were aggregated with diploid embryos as described [Nay A., Rossant, J., Oxford, IRL, 1993, p 147-178], harvested at embryonic day (e) 9 5-14.5, and stained for ⁇ -gal activity. About half of the diploid embryos were allowed to mature to term for germ-line transmission. Chime ⁇ c males were bred to CDl females, and tail DNA of Fi and F2 offspring was analyzed by southern blotting and hybndization to En-2 or RACE fragment probes Results
  • ES colonies spontaneously differentiate into embryoid bodies (EBs) in suspension culture
  • the complex structure of the EB contains all three germ layers and resembles the extra-embryonic yolk sac both morphologically and transc ⁇ ptionally [Doetschmann T C et al , J Embryol Exp Morph 87 27-45, 1985], [Schmitt, R M et al , Genes & Dev 5 728-740, 1991], [Keller G et al , Mol Cell Biol 13 473-486, 1993], [Snodgrass H R et al , American Association of Blood Banks, 1993, p 65-83]
  • the mesoderm of the EB gives rise to angioblastic cords that form blood islands containing primitive hematopoietic cells sunounded by vascular endotheliumWang R et al , Development 114 303-316, 1992] Due
  • Each neo ⁇ colony represented a vector integration event If the vector integrated within an intron, a spliced fusion transcript between lacZ and the endogenous gene was generated upon transc ⁇ ptional activation of the trapped gene Because all ES cells which had an integrated PT1 vector were G418 resistant regardless of whether or not the integration occuned within a gene, genes which were not expressed in undifferentiated ES cells could be screened using this vector Five percent (37/779) of the neo ⁇ clones tested expre >sed lacZ in undifferentiated ES cells, of which 30 clones continued to be expressed in at least some cells during EB differentiation (Table 1) By comparison, 61 clones (8%) which did not express lacZ as undifferentiated ES cells demonstrated lacZ expression during EB differentiation (Table 1) Of the neo° clones that expressed lacZ as undifferentiated or differentiated ES cells, one-third (32 clones) exhibited a restricted pattern of expression (Table 1) The expression patterns of these
  • all neo ⁇ clones selected after introduction of the GTl. ⁇ geo vector represented integrations into genes which were transcnptionally active in undifferentiated ES cells. Accordingly, a much higher proportion of the GTl. ⁇ geo clones (34% versus 5% for PTl) expressed detectable levels of ⁇ -gal activity in undifferentiated ES cells (i.e.,
  • ES cells grown in co-culture with OP9 stromal cells differentiate into mesodermal colonies which when replaced differentiate into hematopoietic cells All gene trap cell lines demonstrating lacZ expression in blood islands were re-analyzed by differentiating ES cells in replicate OP9 stromal cell cultures[Nakano T. et al., Science 265- 1098-1101,
  • ES-de ⁇ ved mesodermal colonies expressing brachury were apparent by day 3 of culture. On day 5, a single cell suspension of a replicate culture was prepared and replated onto OP9 cells. Primitive erythrocytes and multipotential precursors differentiated from the mesodermal precursors within the next 2-3 days and single lineage precursors predominated the cultures by day 11. Cultures were assayed for lacZ expression at days 5, 8, and 11 The majority of blood island positive clones (70%) expressed lacZ m hematopoietic cells when cultured on an OP9 feeder layer (Table 2). Identification of Trapped Genes.
  • RNA was prepared from either differentiated or undifferentiated ES clones and used to perform 5' RACE [Frohman M.A. et al., Proc. Natl. Acad. Sci. USA 85:8998-9002, 1988].
  • the RACE products of eleven lacZ fusion transcripts were cloned and sequenced. Table 3 summarizes the lacZ expression pattern, the gene trap vector, and sequence information for each clone. Eight of the RACE product sequences conesponded to novel genes, of which four shared similanty with EST sequences.
  • Clone B2C3 encodes the munne homologue of karyopherm/importin ⁇ 3 and yeast Pselp [Yaseen N.R., Blobel G., Proc. Natl Acad. Sci. USA 94:4451-4456, 1997], proteins which are involved in the transport of proteins and mRNA across the nuclear membrane [Kutay U. et al., EMBO J. 16:1153-1163, 1997], [Seedorf M., Silver P.A., Proc. Natl. Acad
  • the RACE product suggests that a fusion protein was generated from the N- terminal 312 amino acids and lacZ. Mutational analysis of Xenopus karyophe ⁇ n- ⁇ suggests that this fusion protein will bind weakly to the nuclear pore complex and to RanGTP but not to karyophe ⁇ n- ⁇ [Kutay U. et al, EMBO J. 16 1153-1163, 1997] and may act as a weak dominate negative mutation In ES clone GC10G7, the GTl 8geo vector has integrated within the 3' coding region of the gene for guanosine 5'-monophosphate
  • GMP-synthetase catalyzes the amination of xanthosine 5'-monophosphate to form GMP in the presence of glutamme and ATP
  • GMP-synthetase is expressed in many cell types, high levels of ⁇ -gal activity were observed only in endothehal cells and a population of hematopoietic cells (Table 3) In Vitro and In Vivo Expression of Selected Clones.
  • selected ES clones were aggregated with diploid embryos to generate chimenc mice.
  • Reporter gene expression was performed first on chimenc embryos to quickly assess expression patterns and subsequently was confirmed in F ⁇ embryos, which is summarized along with sequence analysis in Table 1.
  • K17G2 Three clones conesponded to a sequence homolgous to an EST, a completely novel gene and Mena.
  • K17G2 was isolated using the PTl vector and displayed significant sequence similarity to a human EST.
  • K17G2-lacZ was expressed at low to medium levels in undifferentiated ES cells (Fig 1A), while its expression was restncted to blood islands and some endothehal cells in attached EBs (Fig IB) Differentiation on OP9 stromal cells revealed that K17G2-lacZ was expressed in some mesodermal and hematopoietic cells (Fig.
  • K17G2 ES cells were used to generate chimenc mice Analysis of F ⁇ elO 5 embryos revealed additional tissues which expressed the K17G2-lacZ fusion product (Fig IE).
  • the lacZ fusion product was expressed in the myocardium and the dorsal root ganglia (Fig. 1F&G, respectively)
  • K17G2-lacZ was expressed in some of the embryonic vasculature, including the endocardium, and circulating blood cells (Fig 1H&I).
  • K17G2-lacZ expression was observed in hematopoietic cells of the spleen and bone marrow and in the endocardium (data not shown).
  • K17G2 heterozygous httermates were mated with one another; however, these matings failed to produce viable homzygous mice indicating that K17G2 homozygous embryos die in utero (data not shown).
  • Clone GC11E10 was isolated using the GTl. ⁇ geo vector and represents a novel ORF.
  • the GC11E10- geo fusion protein was expressed at medium to high levels in undifferentiated ES cells (Fig. 2A). In attached EBs, expression appeared within blood islands and the vasculature associated with these structure s (Fig. 2B).
  • GC11E10 ES cells on OP9 stromal cells demonstrated lacZ expression within mesodermal colonies and high levels of expression within hematopoietic cell clusters (Fig 2C&D, respectively).
  • lacZ was expressed in the yolk sac, dorsal aorta, heart, the developing liver and vasculature (Fig. 2E&F)
  • Fig. 2G&H Further analysis demonstrated that lacZ expression was contained within blood cells circulating throughout the embryo and within blood islands m the yolk sac
  • the GC1 lElO-geo fusion protein was also expressed in endothehal cells throughout the embryo as demonstrated in the lntersomitic vessels (Fig. 21).
  • Clone K18E2 (a PTl clone) represents an integration into the first intron of Mena Mena is involved in actin assembly and cell motihty; therefore its ubiquitous expression in rapidly dividing cells was expected.
  • Mena-lacZ was expressed at very high levels in nearly all undifferentiated ES cells (Fig. 3A) and virtually all cells in EBs (Fig. 3B).
  • Differentiation of K18E2 on OP9 stromal cells demonstrated high levels of Mena-lacZ expression in mesodermal cells (Fig 4C) but only low level expression in a minority of hematopoietic cells (Fig. 4D).
  • the pattern and level of lacZ expression was reproduced in Fj embryos. Mena-lacZ was expressed by almost all cells in the developing embryo with the exception of hepatocytes and some hematopoietic cells (Fig. 4E&F and data not shown).
  • the present inventors developed an expression-based strategy to identify and mutate genes that are preferentially expressed in cells of the hematopoietic and vascular lineages
  • Gene trap vectors were introduced into ES cells by electroporation and sibling clones were allowed to differentiate into attached EBs to identify expression patterns Clones exhibiting reporter gene expression in blood islands were then differentiated on
  • the attached EB differentiation assay used as the primary screen enabled the identification of a large number of genes with a spatially or cell-type restncted expression for several lineages including hematopoietic, endothehal, stromal and myocyte
  • ES cells embryonic stem (ES) cells coupled with two in vitro differentiation assays was used to screen for genes involved in hematopoietic and vascular development
  • Undifferentiated ES cells were electroporated with either the pPTl-ATG vector which contains a splice acceptor site upstream of a promoterless lac Z gene and a PGK-neoR gene, or the pGTl 8 geo vector which contains a promoterless lacZ/neoR fusion gene G418 resistant clones were allowed to differentiate into attached embryoid bodies
  • EBs EBs and lacZ activity was assayed to indicate trapped gene expression in undifferentiated cells and differentiation cultures
  • Clones expressing lacZ in blood islands were also differentiated on OP9/OP9 stromal cells to confirm lacZ expression by hematopoietic cells
  • a modified attached embryoid body (EB) assay was used to screen the reporter gene expression pattern of approximately 1300 gene trapped ES cell lines for expression in hematopoietic and endothehal lineages The assay was earned out as described in V L Bautch et al , (Developmental Dynamics 205 1-12,
  • the reporter gene expression pattern of clone 17G2 demonstrated moderate expression of the trapped gene in undifferentiated ES cells and restricted expression of hematopoietic and endothehal cells in the attached
  • 17G2 EB cultures Differentiation of 17G2 on OP9 stromal cells lead to expression of the trapped gene in some mesodermal and hematopoietic cells.
  • 17G2 ES cells were aggregated with wild-type CDl embryos to generate chimeras.
  • In vivo expression analysis reveals expression of the 17G2 gene in the cardiac and neural vasculature, hematopoietic cells, myocardium, and sensory nerves including the trigeminal ganglia, dorsal root ganglia, and optic nerve. 17G2 expression is maintained in the adult heart and bone ma ⁇ ow.
  • the exon sequence upstream of the vector integration was cloned by 5' RACE, and analysis showed that the 17G2 gene encodes a novel gene (see Figure 1 for a nucleic acid sequence from the 17G2 gene).
  • the RACE product was used as a probe to screen the genotypes of F 2 litters. No homozygotes were detected out of over 200 pups. Reporter gene expression analysis of timed heterozygous matings revealed that homozygous embryos are viable at midgestation (el 1.5).
  • the 952 bp cDNA encodes a hydrophilic 317 amino acid open reading frame (ORF).
  • the ORF contains numerous Protein Kinase C (PKC) and Casein Kinase II (CK2) phosphorylation sites as well as a tyrosine phosphorylation site.
  • PKC Protein Kinase C
  • CK2 Casein Kinase II
  • a human EST, a Drosophilia EST, and a C.elegans full-length EST contiguous sequence encoding 466 amino acids were found to be 75%, 57%, and 50% identical, respectively.
  • Amino acid comparison demonstrated 62% (66% conserved), 46% (68% conserved), and 40% (56% conserved) identical between 17G2 and the human EST, the C. elegans contig. sequence, and the Drosophilia EST, respectively.
  • amino acid comparison by BLAST also demonstrated 30% and 42% identical and conserved, respectively with a yeast gene of unknown function termed yeast orfl.
  • a more sophisticated amino acid analysis comparison program called
  • Sorting nexin 1 is involved in sorting ligand-activated EGFR to endosomes. SNX1 was identified by a yeast-2-hybrid screen using the kinase domain of human EGFR as bait (Science272: 1008-1010).
  • VPS 1 is an 80kDa GTPase that associates with golgi membrane and is required for the sorting of proteins to the yeast vacuole.
  • MVP1 overexpression suppressed dominant alleles of VPS1.
  • MVP1 is a 59 kDa hydrophilic protein which was also shown to be necessary for protein sorting to yeast vacuoles.
  • FIG. 1 K17G2-lacZ expression in vitro and in vivo Overnight X-gal staining showed fusion transcript expression at medium intensity in most undifferentiated K17G2 ES cells
  • A The fusion transcript was expressed in the blood island and some of the associated vascular endothelium in attached EB culture
  • B Differentiation of clone K17G2 on op9 stromal cells demonstrated lacZ expression in mesodermal colonies (C) and hematopoietic clusters
  • D X-gal staining of an elO 5 Fj embryo demonstrated limited lacZ expression in the embryo (whole mount, E) including expression in the myocardium (F) and the dorsal root ganglia
  • G An X-gal stained el2 5 Fi embryo demonstrated lacZ expression in the endocardium (H) and vascular endothelium and circulating hematopoietic cells (I)
  • Table 1 Summary of attached EB primary gene trap screen.
  • Percentage was determined by dividing the number of clones with identifiable patterns of lacZ expression by the total number clones demonstrating ⁇ -gal activity.

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Abstract

The invention relates to vectors, compositions, and methods, for identifying genes primarily expressed in selected lineages. The invention also relates to novel genes primarily expressed in selected lineages, proteins encoded by the novel genes and truncations, analogs, homologs, and isoforms of the proteins; and, uses of the proteins and genes.

Description

Title: Methods for Identifying Genes Expressed in Selected Lineages, and Novel Genes Identified Using the Methods
FIELD OF THE INVENTION
The invention relates to vectors, compositions, and methods, for identifying genes pnmanly expressed in selected lineages The invention also relates to novel genes pnmanly expressed in selected lineages, proteins encoded by the novel genes and truncations, analogs, homologs, and isoforms of the proteins; and, uses of the proteins and genes BACKGROUND OF THE INVENTION
Gene trapping strategies have been used to identify eukaryotic genes displaying novel and familiar patterns of expression during embryogenesis (D P Hill and W Wurst, Methods in Enzymology, 225: 664,
1993). The techniques use vectors which are randomly integrated into genes The vectors typically contain a reporter gene which facilitates the identification and isolation of the vectors once they are inserted into a gene. Gene trap vectors also typically contain sequences associated with eukaryotic structural genes such as splice- acceptor sites which occur at the 5' end of all exons Vectors containing a splice-acceptor site integrate into introns and generate a fusion transcript containing a target endogenous gene and the reporter gene (see references 5, 10, 11 in D.P. Hill and W. Wurst, Supra) The expression of the reporter gene is under the regulatory control of the endogenous gene and its expression mimics the expression pattern of the target gene (see reference 12 in D P. Hill and W. Wurst, Supra) The insertion of the gene trap vector can also create a mutation and disrupt the function of the target gene (see references 10 and 12 in D P. Hill and W Wurst, Supra). The part of the target gene in the fusion transcript may also be cloned from the fusion tianscπpt, or from genomic DNA upstream of the insertion site.
Embryonic stem (ES) cell technology offers an efficient way of introducing gene trap vectors into the mouse genome and thereby identify and mutate genes expressed during mouse development. ES cells isolated from the mouse inner cell mass remain pluπpotent after genetic manipulation and in vitro culture, and they contribute to all tissues of the mouse, including the germ line (see references 7 to 9 in D P. Hill and W Wurst,
Supra).
Different approaches have been used to identify targeted genes using ES technology. Mutations can be transmitted through the germ line and offspring can be screened for recessive mutant phenotypes. Prescreenmg in chimeπc embryos can also be carried out, and mutations resulting in interesting patterns can be transmitted through the germ line and their phenotype studied.
Gene trapping in ES cells is a powerful technique because it simultaneously integrates gene identification and structure, expression and functional analysis into one process. Typically gene trap screens have used one of these three types of analyses as the primary determinant to select clones for further study. The first group of screens uses no pre-selection to study mutant phenotypes. Collectively, these studies have determined that nearly 40% of gene trap mutants result in recessive embryonic lethality [Fπedπch G, Genes
& Dev. 5:1513, 1991, Skarnes WC, INSERT1992 ;von Melchner H, Genes & Dev. 6:919, 1992; DeGregoπ J, Genes & Dev 8 265, 1994). Several sequence-based screening strategies have been developed to either rapidly isolate 5'RACE sequences (Holzschu D, Transgenic Res. 6 97, 1997; Chowdhury K, Nucleic Acid Res. 25.1531, 1997, and Townley DJ,Genome Res 7 293, 1997), isolate 3'RACE sequences (Yoshida M. et al, Trans. Res. 4.277, 1995; and Zambrowicz BP et al, Nature 392:608. 1998), or clone proviral integraton sites by plasmid rescue (Hicks GG et al Nature Genet 16:338, 1997). In addition Skarnes and colleagues modified the GTl.δgeo vector to specifically trap genes which encode secreted or transmembrane proteins (Proc. Natl
Acad. Sci. USA 92:6592, 1995) Several groups have performed screens based upon regulated expression. Each of these screens analyzed clones which contained integrations into genes which were transci lptionally active in ES cells. The expression of the fusion transcripts were either analyzed by in vivo expression (Wurst
W, Genetics 139:889, 1995), regulation by exogenous factors (Sam M et al, Dev. Dyn; Forrester L et al, Proc.
Natl. Acad. USA 93: 1677, 1996; Sam M et al, Mann. Genome 7:741, 1996), or by in vitro differentiation
(Scherer CA et al, Cell Growth & Diff. 7:1393, 1996; Shirai M et al, Zool Sci. 13 277, 1996; and Baker RK et al, Dev. Biol 185:201, 1997)
SUMMARY OF THE INVENTION
The present inventors have developed a gene trap strategy to identify, mutate, and characterize large numbers of genes on the basis of their cell-lineage specific expression This expression trapping method complements and extends previous expression-based gene trap screens by specifically identifying integrations into genes preferentially expressed in selected cell lineages. The approach simultaneously provides expression, sequence, and phenotypic information. The method can be used to carry out large scale, genome-wide scans for genes of interest. Integrations with identifiable expression patterns in vitro can be catalogued to generate a biological resource of gene-trap insertions, based upon expression pattern, cDNA sequences, and mutant phenotypes. The method permits identification of specific messages present in low levels that could not have been found using conventional techniques.
Therefore, broadly stated the present invention relates to a method of identifying a target nucleic acid molecule primarily expressed in selected lineages comprising:
(a) integrating into a site in the genome of a host cell a gene trap vector containing a reporter gene, to form transfected cells; (b) growing the transfected cells in vitro under conditions whereby the transfected cells differentiate into embryoid bodies attached to a carrier and identifying embryoid bodies expressing the reporter gene in cells of a selected lineage, or (c) growing the transfected cells in vitro under conditions whereby the transfected cells differentiate into cells of a selected lineage, and identifying cells of the selected lineage expressing the reporter gene; wherein the target nucleic acid molecule composes sequences upstream or downstream of the site of integration of the reporter gene in the cells of the selected lineage.
The method may further comprise isolating nucleic acid molecules from the transfected cells, or descendents thereof expressing the reporter gene wherein the nucleic acid molecules compπse the reporter gene and a part of the target nucleic acid molecule, or the nucleic acid molecules comprise genomic DNA upstream or downstream of the site of insertion of the gene trap vector
Transfected cells or descendents thereof expressing the reporter gene may be introduced into embryos to form chimeπc embryos. Therefore, the present invention contemplates a chimeπc embryo having integrated into its genome a gene trap vector at a site of a target nucleic acid molecule primarily expressed in cells of selected lineages. Germline transmission may be achieved by mating chimeπc embryos allowed to mature to term, or mating foster recipient females having the chimeπc embryos. Therefore, the invention also contemplates a transgenic non-human animal all of whose somatic cells and germ cells contain a gene trap vector at a site of a target gene primarily expressed in cells of selected lineages
The present inventors using the novel strategy described herein have identified novel clones expressed primarily in hematopoietic, endothe al, stromal, and/or myocyte lineages designated 17G2, K18F2,
K20D4, K18F2, K20D4, B2D2, GC10E10, GC11C7, and GC11E10 The invention therefore i elates to novel nucleic acid molecules isolated from these clones.
The nucleic acid molecules of the invention permit identification of untranslated nucleic acid sequences or regulatory sequences which specifically promote expression of proteins operatively linked to the promoter regions Identification and use of such promoter sequences are particularly desirable in instances, such as gene transfer or gene therapy, which can specifically require heterologous gene expression in a limited
(e.g. hematopoietic or vascular) environment The invention therefore contemplates a nucleic acid encoding a regulatory sequence of a nucleic acid molecule of the invention, such as a promoter sequence
The nucleic acid molecules of the invention may be inserted into an appropπate vector, and the vector may contain the necessary elements for the transcription and translation of the inserted coding sequence
Accordingly, vectors may be constructed which comprise a nucleic acid molecule of the invention and optionally one or more transcription and translation elements linked to the nucleic acid molecule.
Vectors are contemplated within the scope of the invention which comprise regulatory sequences of the invention, as well as chimenc gene constructs wherein a regulatory sequence of the invention is operably linked to a nucleic acid sequence encoding a heterologous protein, and a transcription termination signal.
A vector of the invention can be used to prepare transformed host cells expressing the proteins encoded by the nucleic acids of the invention, or a heterologous protein. Therefore, the invention further provides host cells containing a vector of the invention The invention also contemplates transgenic non-human mammals whose germ cells and somatic cells contain a vector comprising a nucleic acid molecule of the invention or a fragment thereof, in particular one which encodes an analog or a truncation of a pi otein of the invention.
The invention further provides a method for preparing novel proteins encoded by the nucleic acids of the invention utilizing the purified and isolated nucleic acid molecules of the invention. In an embodiment a method for preparing a protein is provided comprising (a) transferring a vector of the invention into a host cell; (b) selecting transformed host cells from untransformed host cells, (c) cultuπng a selected ixansformed host cell under conditions which allow expression of the protein, and (d) isolating the protein. A protein of the invention may be obtained as an isolate from natural cell sources, but they are preferably obtained by recombinant procedures
The invention further broadly contemplates an isolated protein comprising the amino acid sequence of SEQ. ID. NO 2, SEQ. ID. NO 5 , or SEQ. ID. NO. 7 The invention includes a truncation of a protein of the invention, an analog, an allehc or species variation thereof, or a homolog of a protein of the invention, or a truncation thereof ( The term "proteins of the invention" used herein includes truncations, analogs, allehc or species variations, and homologs). The proteins of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins or chimenc proteins This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins
The invention further contemplates antibodies having specificity against an epitope of a protein of the invention Antibodies may be labelled with a detectable substance and used to detect proteins of the invention in tissues and cells
The invention also permits the construction of nucleotide probes which are unique to the nucleic acid molecules of the invention Therefore, the invention also relates to a probe compπsing a sequence deπved from a nucleic acid of the invention or encoding a protein of the invention The probe may be labelled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleic acid sequence of the invention, or a nucleic acid sequence encoding a protein of the invention
The invention still further provides a method for identifying a substance which binds to a protein of the invention comprising reacting a protein with at least one substance which potentially can bind with the protein, under conditions which permit the formation of complexes between the substance and protein and assaying for complexes, for free substance, for non-complexed protein, or for activated protein
Still further the invention provides a method for evaluating a compound for its ability to modulate the biological activity of a protein of the invention For example a substance which inhibits or enhances the interaction of the protein and a substance which binds to the protein may be evaluated In an embodiment, the method comprises providing a known concentration of a protein, with a substance which binds to the protein and a test compound under conditions which permit the formation of complexes between the substance and protein, and assaying for complexes, for free substance, for non-complexed protein, or for activated protein
Compounds which modulate the biological activity of a nucleic acid or protein of the invention may also be identified using the methods of the invention by comparing the pattern and level of expression of nucleic acid or protein of the invention in tissues and cells, in the presence, and in the absence of the compounds
The substances and compounds identified using the methods of the invention may be used to modulate a nucleic acid or protein of the invention, and they may be used in the treatment of conditions requiring modulation of for example hematopoiesis, myocardium, the sensory nervous system, or cardiac or neural vasculature Accordingly, the substances and compounds may be formulated into compositions for administration to individuals suffering from one of these conditions Therefore, the present invention also relates to a composition comprising one or more of a protein of the invention, or a substance or compound identified using the methods of the invention, and a pharmaceutically acceptable earner, excipient or diluent A method for treating or preventing a condition requiπng modulation of hematopoiesis, the sensory nervous system, or vasculature is also provided comprising administering to a patient in need thereof, a protein of the invention or a composition of the invention
Other objects, features and advantages of the present invention will become apparent from the following detailed description It should be understood, however, that the detailed descπption and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since vaπous changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description
DESCRIPTION OF THE DRAWINGS
The invention will be better understood with reference to the drawings in which Figure 1, panels A to I are photographs showing K17G2-lacZ expression in vitro and in vivo,
Figure 2, panels A to I are photographs showing GC1 lElO-lacZ expression, Figure 3, panels A to F, are photographs showing Mena-lacZ (K18E2) expression DETAILED DESCRIPTION OF THE INVENTION 1. Expression Trapping Method As hereinbefore mentioned, the present invention provides a method for detecting a target nucleic acid molecule primarily expressed in selected lineages In an embodiment of the invention the target nucleic acid molecule is primarily expressed in hematopoietic or endothehal cells
The term "hematopoiesis' used herein refers to the proliferation, differentiation, and migration of hematopoietic cells in embryos and adults "Hematopoietic cells refers to cells of the hematopoietic system including pluπpotential stem cells which are capable of self-replication and of differentiation tc committed progenitor cells, progenitor cells, myeloid and lymphoid stem cells, and neutrophils, macrophages, erythroid cells, mast cells, megakaryocytes, blast cells, lymphocytes, and monocytes 'Endothehal cells" refers to a type of squamous epithelium cells that lines the interiors of cavities, spaces, and blood vessels
The method of the invention involves integrating into the genomes of host cells a gene trap vector containing a reporter gene, to form transfected cells The gene trap vector used in the method of the invention comprises a reporter gene which allows for differentiation of cells having a gene trap vector integrated into a target nucleic acid molecule primarily expressed in selected lineages (e g hematopoietic or endothehal cells) Reporter genes which are particularly useful in the method of the invention are genes encoding β-galactosidase (e g lac Z), chloramphenicol, acetyltransferase, or firefly luciferase, Transcription of the reporter gene is monitored by changes in the concentration of the protein encoded by the reporter gene such as β-galactosidase, chloramphenicol, acetyltransferase, green fluorescence protein (GFP), or firefly luciferase Transfected cells or descendents thereof showing reporter gene activity are identified using conventional methods For example, if the reporter gene encodes β-galactosidase, activity can be analyzed by staining with 5-bromo-4-chloro 3- mdolyl galactoside as described in Proc Natl Acad, Sci USA 84 156, 1987 The gene trap vector may also include a gene encoding a selectable marker which conveys a second property on transformed cells and permits the selection and/or identification of cells having the vector integrated into their genome Examples of such genes are genes which encode proteins conferring antibiotic resistance, or the ability to grow on a defined medium For example, a gene encoding neomycin (neo) phosphotransferase activity and conferring neomycin resistance may be included in the gene trap vector The differentiation and selection of cells using a reporter gene and selectable marker gene may be achieved using a single element For example, a β-geo construct which has sequences conferring both β- galactosidase and neomycin (neo) phosphotransferase activities may be incorporated into the gene trap vector The gene trap vector may include regulatory sequences such as promoter sequences which control the expression of one or both of the reporter gene and selectable marker gene The reporter gene or selectable marker gene may not be under the control of an autonomous promoter, and they may only be expressed if the gene trap vector is integrated into an actively expressed gene
The gene trap vector may include sequences associated with eukaryotic structural genes which facilitate the insertion of the vector into a eukaryotic gene For example, the gene trap vector may include sequences associated with elimination of tron sequences from mRNA such as splicer-acceptor sequences (e.g. using an En entron), and polyadenylation signal sequences.
The gene trap vector may also include sequences which facilitate isolation and sequencing of the target gene. For example, the gene trap vector may contain loxp sequences before and after the lacZ sequence. The loxp sequences are cleaved by ere recombinase allowing removal of the lacZ sequence Preferred gene trap vectors for use in the method of the invention are PT1 which contains an En-2 intron sequence including a splice-acceptor site front of the bacterial lacZ gene and a neomycin gene dπven by the PGK-1 promoter; PT1/ATG which is the same as PT1 with the exception that it includes a translational start signal (ATG) in the lacZ gene (Hill DP and Wurst W, Methods in Enzymology 225:664, 1993), and GTl.δgeo which contains the En-2 splice acceptor site immediately upstream of a lacZ-neo vector thereby allowing neomycin resistance at a lower level of endogenous gene expression than the SAβgeo vector (Skarnes
WC et al., Proc. Natl. Acad. Sci USA 92 652-6596, 1995)
The gene trap vector may be introduced into host cells by conventional methods such as transfection, pofection, precipitation, infection, electroporation, microinjection etc Methods for transfecting, etc. host cells are well known m the art (see Sambrook et al. Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, all of which is incorporated herein by reference)
Suitable host cells for use in the method of the invention include a wide vaπety of host cells, including stem cells, and pluπpotent cells such as zygotes, embryos, and ES cells, preferably ES cells The gene trap vector stably integrates into the genome of the host cells Generally, the vector integrates randomly into the genome of the host cells and in some cells it will integrate into endogenous genes which are pnmanly expressed in hematopoietic or endothehal cells.
The transfected host cells containing the gene trap vector may be grown in vitro under conditions whereby the transfected cells differentiate into embryoid bodies Methods for producing EB culture systems are known to the skilled artisan See for example, Bautch VL Et al, Dev. Dyn. 205:1-12, 1996 Preferably the embryoid bodies are grown attached to a carrier or support so that the endoderm layer is beneath the blood islands. The carrier or support may be made of nitrocellulose, glass, polyacrylamide, gabbros, o - magnetite.
The support or earner matenal may have any possible configuration including spherical (e g bead), cylmdπcal (e.g. inside surface of a test tube or well, or the external surface of a rod), or flat (e.g. sheet, test stπp).
The transfected host cells containing the gene trap vector may be grown in vitro under conditions selected so that the transfected cells differentiate into cells of a selected lineage, and the reporter gene is expressed in the transfected cells. For example, host cells which are embryonic stem cells may be cultured with a cell line which induces differentiation of the embryonic stem cells into hematopoietic cells such as the OP9 stromal cell line described by Nakano et al., (Science 265: 1098, 1994) The methods of the invention can also be adapted to identify target nucleic acid molecules pnmanly expressed in particular cell types by adding one or more exogenous factors (e g cytokines) which induce the differentiation of specific cell types. For example, to identify and isolate nucleic acid molecules associated with differentiation of macrophages-granulocytes, transfected host cells containing a gene trap vector may be grown on OP9 cell layers in the presence of granulocyte-macrophage colony-stimulating factor.
In a preferred embodiment of the invention embryonic stem cells transfected with a gene trap vector containing a β-galactosidase gene and a gene conferring antibiotic resistance are seeded onto confluent OP9 cell layers on well plates at a concentration of 103 to 105, preferably 104 cells per well. The induced cells are trypsiruzed between day 5 and day 8, preferably day 5. β-galactosidase activity is observed in the induced cells between about day 5 and day 12.
Nucleic acid molecules containing the reporter gene and a part of the target gene, or containing genomic DNA upstream or downstream of the site of integration of the gene trap vector, may be isolated and cloned using standard methods from the transfected cells, or descendents thereof showing reporter gene activity. Cloned nucleic acid molecules may be sequenced and the predicted ammo acid sequence of the encoded protein can be determined using standard sequencing techniques, such as dideoxynucleotide chain termination, or Maxam-Gilbert chemical sequencing. The initiation codon and untranslated sequences of the protein may be determined using cunently available computer software designed for the purpose, such as
PC/Gene (IntelliGenetics Inc., Calif.). The intron-exon structure and transcription regulatory sequences of a gene can be identified using conventional techniques.
Transfected cells or descendents thereof expressing the reporter gene may be used to generate chimenc embryos. For example, clones showing reporter gene activity can be aggregated with diploid embryos (e.g. Nagy, A and Rossant J. In A.LJ. (ed): Gene Targeting: A practical Approach. Oxford, IRL, 1993, p. 147-
178), and allowed to mature to term. Chimenc mice can be mated (e.g. to CD-I mice) to provide animal lines having the mutation transmitted through the germline. Such a transgenic animal may be used to study the phenotype produced by the interruption of an endogenous gene by the gene trap vector, and to identify substances that reverse or enhance such a mutation. 2. Nucleic Acid Molecules and Proteins Identified Using the Methods of the Invention
2.1 Nucleic Acid Molecules
As hereinbefore mentioned, the invention provides an isolated nucleic acid molecule having a sequence encoding a novel protein of the invention. The term "isolated" refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical reactants, or other chemicals when chemically synthesized. An "isolated" nucleic acid is also free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid molecule) from which the nucleic acid is denved. The term "nucleic acid" is intended to include DNA and RNA and can be either double stranded or single stranded.
The invention specifically contemplates an isolated nucleic acid molecule which comprises: (i) a nucleic acid sequence encoding a protein having substantial sequence identity preferably at least
75% sequence identity, with the ammo acid sequence of SEQ. ID. NO.2, SEQ. ID. NO 5., or SEQ.
ID. NO. 7; (ii) nucleic acid sequences complementary to (I);
(hi) a degenerate form of a nucleic acid sequence of (l); (ιv) a nucleic acid sequence compπsing at least 18 nucleotides and capable of hybπdizing to a nucleic acid sequence in (1), (n), or (in);
(v) a nucleic acid sequence encoding a truncation, an analog, an allehc or species variation of a protein compπsing the ammo acid sequence shown SEQ. ID NO.2, SEQ ID NO 5 , or SEQ. ID. NO. 7; or (vi) a fragment, or alle c or species variation of (l), (n) or (in).
In an embodiment of the invention a nucleic acid molecule is provided comprising:
(0 a nucleic acid sequence comprising the sequence of SEQ ID. NO.1 , SEQ. ID NO 3., SEQ.
ID. NO. 4, SEQ. ID. NO. 6, SEQ. ID. NO. 8, SEQ ID. NO. 9, or SEQ. ID. NO. 10, wherein T can also be U; (n) nucleic acid sequences complementary to (I), preferably complementary to the full nucleic acid sequence of SEQ. ID. NO 1, SEQ. ID NO 3 , SEQ ID NO 4, SEQ ED. NO. 6, SEQ
ID. NO. 8, SEQ. ID. NO. 9, or SEQ. ID NO 10,
(in) a nucleic acid capable of hybridizing to a nucleic acid of (I) and having at least 18 nucleotides; or (IV) a nucleic acid molecule differing from any of the nucleic acids of (I) to (in) in codon sequences due to the degeneracy of the genetic code.
In accordance with specific embodiments of the invention the following nucleic acid molecules or genes are provided
(a) A novel nucleic acid molecule designated 17G2 which is primarily expressed in vivo in hematopoietic cells, myocardium, in the cardiac and neural vasculature, and in the sensory nervous system, including the tπgeminal ganglia, dorsal root ganglia, and optic nerve. The nucleic acid molecule comprises the sequence of SEQ ID. No. 1
(b) A novel nucleic acid molecule designated K18F2 which is pnmanly expressed in vitro by muscle cells in attached embryoid bodies, and some mesodermal cells in OP9 induction cultures, and pnmanly expressed in vivo in both tetraploid and diploid chimenc embryos exclusively in cardiac myocytes. The nucleic acid molecule comprises the sequence of SEQ. ID. No. 3.
(c) A novel nucleic acid molecule designated K20D4 which is expressed in vitro exclusively in vascular endothehal cells in attached embryoid bodies, and some mesodermal cells in OP9 induction. The nucleic acid molecule comprises the sequence of SEQ. ID. No. 4 The sequence overlaps with EST accession No. AA239055 of clone 697718 from the Barstead mouse pooled organs cDNA library.
(d) A novel nucleic acid molecule designated B2D2 which is pnmanly expressed in vitro m blood islands and vascular endothehal cells in attached EB cultures However, on OP9 stroma, expression is induced in some mesodermal cells but not in hematopoietic cells. Thus, expression in the blood island may be due to endothehal cells or their precursors. The nucleic acid molecule comprises the sequence of SEQ. ID. No 6. The sequence overlaps with EST accession No AA209568 of clone 676502 from the Soares NML mouse liver cDNA library.
(e) A novel nucleic acid molecule designated GC10E10 which is highly expressed in vitro in undifferentiated embryonic cells. In attached embryoid bodies GC10E10 is expressed in blood lslands and endothehal cells. It is expressed highly in mesodermal cells and in low levels m a population of hematopoietic cells in OP9 induction cultures In vivo the gene is expressed in the forebram, midbrain, somites, notochord, otic vesicle, limb buds, branchial arches and heart in diploid chimeras. The nucleic acid molecule comprises the sequence of SEQ.ID. No. 8. The sequence has 98% homology with the muπne Dlghl (dlgl)
(f) A novel nucleic acid molecule designated GC11C7 which is primarily expressed in vitro in undifferentiated embryonic stem cells and in mesoderm and hematopoietic cells in the OP9 induction system. The nucleic acid molecule comprises the sequence of SEQ.ID. No. 9. The sequence overlaps that of EST accession No. AA015451, clone 442692 from the Soares mouse placenta 4NbMP13.5 14.5 cDNA library and EST accession No. AA517189 clone 893845 from the Knowles Solter mouse embryonic stem cell cDNA library
(g) A novel nucleic acid molecule designated GC11E10 which is highly expressed in vitro in undifferentiated embryonic stem cells and in blood islands and endothehal cells within attached embryoid bodies. It is also expressed in mesodermal cells and highly in hematopoietic cells in the OP-9 induction system In vivo it is expressed in endothehal and blood cells within E9.5 diploid chimeras. The nucleic acid molecule comprises the sequence of SEQ.ID. No. 10.
The invention includes nucleic acid molecules having substantial sequence identity or similarity to the nucleic acid sequences of SEQ. ID. NO.l, SEQ. ID. NO 3 , SEQ ID. NO 4, SEQ ID. NO. 6, SEQ. ID.
NO. 8, SEQ. ID. NO. 9, or SEQ. ID. NO. 10. Identity or similarity refers to sequence similarity between sequences and can be determined by comparing a position in each sequence which may be aligned l or purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are matching or have identical positions shared by the sequences. Preferably, the nucleic acid sequences have substantial sequence identity for example at least 75% nucleic acid identity, more preferably 80% nucleic acid identity; and most preferably at least 90 to 95% sequence identity. Isolated nucleic acid molecules having a sequence which differs from the nucleic acid sequence of
SEQ. ID. NO.l, SEQ. ID. NO 3., SEQ. ID. NO. 4, SEQ. ID. NO. 6, SEQ. ID NO. 8, SEQ ID. NO. 9, or SEQ. ID. NO. 10, due to degeneracy m the genetic code are also within the scope of the invention. As one example, DNA sequence polymorphisms within the nucleotide sequence of a 17G2 protein may result in silent mutations which do not affect the amino acid sequence. Vanaϋons in one or more nucleotides may exist among individuals within a population due to natural allehc variation. Any and all such nucleic acid V iπations are within the scope of the invention. DNA sequence polymorphisms may also occur which lead to changes in the amino acid sequence of the protein. These amino acid polymorphisms are also within the scope of the present invention.
Another aspect of the invention provides a nucleic acid molecule which hybridizes under selective conditions, e.g. high stringency conditions, to a nucleic acid molecule of the invention. Selectivity of hybridization occurs with a certain degree of specificity rather than being random. Appropriate stringency conditions which promote DNA hybndization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C may be employed. The stπngency may be selected based on the conditions used in the wash step. By way of example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be at high stringency conditions, at about 65°C.
It will be appreciated that the invention includes nucleic acid molecules encoding a protein of the invention including truncations, analogs and homologs of a protein of the invention as described herein. In particular, fragments of a nucleic acid molecule of the invention are contemplated that are a stretch of at least about 18 nucleotides, more typically 50 to 200 nucleotides. It will further be appreciated that vanant forms of the nucleic acid molecules of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention. An isolated nucleic acid molecule of the invention which comprises DNA can be isolated by prepanng a labelled nucleic acid probe based on all or part of a nucleic acid sequence of SEQ. ID. NO.l, SEQ. ID. NO 3., SEQ. ID. NO. 4, SEQ. ID. NO. 6, SEQ. ID. NO. 8, SEQ. ID. NO 9, or SEQ. ID. NO. 10. The labelled nucleic acid probe is used to screen an appropriate DNA library (e g a cDNA or genomic DNA library). For example, a cDNA library can be used to isolate a cDNA by screening the library with the labelled probe using standard techniques. Alternatively, a genomic DNA library can be similarly screened to isolate a genomic clone encompassing a gene of the invention. Nucleic acids isolated by screening of a cDNA or genomic DNA library can be sequenced by standard techniques.
An isolated nucleic acid molecule of the invention which is DNA can also be isolated by selectively amplifying a nucleic acid using polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic o gonucleotide primers from the nucleotide sequence of SEQ. ID. NO.l, SEQ.
ID. NO 3., SEQ. ID. NO. 4, SEQ. ID. NO. 6, SEQ. ID. NO. 8, SEQ. ID NO. 9, or SEQ. ID. NO. 10 for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using these ohgonucleotide pnmers and standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropnate vector and characterized by DNA sequence analysis. cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidimum-thiocyanate extraction procedure of Chirgwm et al., Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcπptase (for example, Moloney MLV reverse transcπptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcπptase available from Seikagaku America, Inc., St. Petersburg, FL). An isolated nucleic acid molecule of the invention which is RNA can be isolated by cloning a nucleic acid molecule of the invention which is cDNA into an appropnate vector which allows for transcription of the cDNA to produce an RNA molecule. For example, a cDNA can be cloned downstream of a bacteπophage promoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by conventional techniques
Nucleic acid molecules of the invention may be chemically synthesized using standard techmques. Methods of chemically synthesizing polydeoxynucleotides are known, including but not limited to solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers
(See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S Patent No 4,458,066, and Itakura U.S.
Patent Nos. 4,401,796 and 4,373,071). Determination of whether a particular nucleic acid molecule encodes a protein of the invention can be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the expressed protein using conventional methods A cDNA having the biological activity of a protein of the invention can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam- Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein
The initiation codon and untranslated sequences of a nucleic acid molecule of the invention may be determined using computer software designed for the purpose, such as PC/Gene (Intel Genetics Inc , Calif ) The mtron-exon structure and the transcnption regulatory sequences of a nucleic acid molecule or gene of the invention may be identified by using a nucleic acid molecule of the invention to probe a genomic DNA clone library Regulatory elements can be identified using standard techniques The function of the elements can be confirmed by using these elements to express a reporter gene such as the lacZ gene which is operatively linked to the elements These constructs may be introduced into cultured cells using conventional procedures or into non-human transgenic animal models In addition to identifying regulatory elements in DNA, such constructs may also be used to identify nuclear proteins interacting with the elements, using techniques known in the art
The invention contemplates polynucleotides comprising all or a portion of a nucleic acid of the invention compπsing a regulatory sequence of a nucleic acid molecule of the invention contained in appropnate expression vectors The vectors may contain sequences encoding heterologous proteins
In accordance with another aspect of the invention, the nucleic acids isolated using the methods described herein are mutant gene alleles For example, the mutant alleles may be isolated from individuals either known or proposed to have a genotype which contributes to the symptoms of a condition affecting hematopoiesis etc Mutant alleles and mutant allele products may be used in therapeutic and diagnostic methods described herein For example, a cDNA of a mutant gene may be isolated using PCR as described herein, and the DNA sequence of the mutant allele may be compared to the normal allele to ascertain the mutatιon(s) responsible for the loss or alteration of function of the mutant gene product A genomic library can also be constructed using DNA from an individual suspected of or known to carry a mutant allele, or a cDNA library can be constructed using RNA from tissue known, or suspected to express the mutant allele A nucleic acid encoding a normal gene or any suitable fragment thereof, may then be labeled and used as a probe to identify the corresponding mutant allele in such libraries Clones containing mutant sequences can be purified and subjected to sequence analysis In addition, an expression library can be constructed using cDN 5 from RNA isolated from a tissue of an individual known or suspected to express a mutant allele. Gene products made by the putatively mutant tissue may be expressed and screened, for example using antibodies specific for a protein of the invention as described herein Library clones identified using the antibodies can be punfied and subjected to sequence analysis The sequence of a nucleic acid molecule of the invention may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule An antisense nucleic acid molecule may be constructed using chemical synthesis and enzymatic gation reactions using procedures known in the art 2.2 Proteins of the Invention The proteins of the invention are primarily expressed in hematopoietic, endothehal, stromal, and/or myocyte lineages Amino acid sequences of proteins of the invention comprise the sequences of SEQ ID NO 2, SEQ ID NO 5 , or SEQ ID NO 7
In addition to the amino acid sequences as shown SEQ ID NO 2, SEQ ID NO 5 , or SEQ ID NO 7, the proteins of the present invention include truncations of the proteins of the invention, and an -logs, and homologs of the proteins and truncations thereof as described herein Truncated proteins may comprise peptides of between 3 and 275 amino acid residues, ranging in size from a tnpeptide to a 275 mer polypeptide
The truncated proteins may have an amino group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to hpid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the am o terminal end The truncated proteins may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end
The proteins of the invention may also include analogs, and/or truncations thereof as descnbed herein, which may include, but are not limited to the proteins, containing one or more amino acid substitutions, insertions, and/or deletions Amino acid substitutions may be of a conserved or non-conserved nature Conserved amino acid substitutions involve replacing one or more amino acids with amino acids of similar charge, size, and/or hydrophobicity characteristics When only conserved substitutions are made the resulting analog should be functionally equivalent to the native protein Non-conserved substitutions involve replacing one or more amino acids with one or more amino acids which possess dissimilar charge, ,ιze, and/or hydrophobicity characteristics
One or more amino acid insertions may be introduced into a protein of the invention Amino acid insertions may consist of single amino acid residues or sequential am o acids ranging from 2 to 15 amino acids m length Deletions may consist of the removal of one or more amino acids, or discrete portions from the protein sequence The deleted amino acids may or may not be contiguous The lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 100 amino acids
An allehc vanant at the protein level differs from another protein by only one, or at most, a few amino acid substitutions A species vanation of a protein of the invention is a variation which is naturally occurring among different species of an organism
The proteins of the invention also include homologs and/or truncations thereof as descnbed herein Such homologs include proteins whose amino acid sequences are comprised of the ammo acid sequences of regions from other species that hybridize under selective hybridization conditions (see discussion of selective and in particular stringent hybridization conditions herein) with a probe used to obtain a protein of the invention These homologs will generally have the same regions which are characteristic of a piotem of the invention It is anticipated that a protein comprising an amino acid sequence which is at least 75% identical, preferably 80 to 90% identical, with an amino acid sequence of SEQ ID NO 2, SEQ ID NO 5 , or SEQ ID NO 7 will be a homolog
A percent amino acid sequence homology or identity is calculated as the percentage of aligned am o acids that match the reference sequence, where the sequence alignment has been determined using the alignment algorithm of Dayhoff et al, Methods in Enzymology 91 524-545 (1983)
The invention also contemplates isoforms of the proteins of the invention An isoform contains the same number and kinds of amino acids as the protein of the invention, but the isoform has a different molecular structure The isoforms contemplated by the present invention are those having the same properties a> a protein of the invention as described herein
The present invention also includes proteins of the invention conjugated with a selected protein, or a selectable marker protein (see below) to produce fusion proteins Additionally, immunogenic portions of a protein of the invention are within the scope of the invention A protein of the invention may be prepared using recombinant DNA methods Accordingly, the nucleic acid molecules of the present invention having a sequence which encodes a protein of the invention may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the protein Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses
(e g replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used
The invention therefore contemplates a vector of the invention containing a nucleic acid molecule of the invention, and optionally the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence Suitable regulatory sequences may be deπved from a vaπety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology Methods in Enzymology 185, Academic Press, San Diego, CA (1990)
Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art The necessary regulatory sequences may be supplied by a native protein and/or its flanking regions
The invention further provides a vector comprising a DNA nucleic acid molecule of the invention cloned into the vector in an antisense orientation That is, the DNA molecule is linked to a regulatory sequence in a manner which allows for expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to a nucleic acid sequence of a nucleic acid molecule of the invention Regulatory sequences linked to the antisense nucleic acid can be chosen which direct the continuous expression of the antisense RNA molecule in a vanety of cell types, for instance a viral promoter and/or enhancer, or regulatory sequences can be chosen which direct tissue or cell type specific expression of antisense RNA
The expression vector of the invention may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a vector of the invention Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an lmmunoglobu n or portion thereof such as the Fc portion of an lmmunoglobuhn preferably IgG The selectable markers can be introduced on a separate vector from the nucleic acid of interest
The vectors may also contain genes which encode a fusion moiety which provides increased expression of the recombinant protein, increased solubility of the recombinant protein, and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification For example, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein
The vectors may be introduced into host cells to produce a transformant host cell "Transformant host cells" include host cells which have been transformed or transfected with a vector of the invention. The terms
"transformed with", "transfected with", "transformation" and "transfection" encompass the introduction of nucleic acid (e.g a vector) into a cell by one of many standard techniques Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated transformation
Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, hpofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. (Molecular Cloning A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks
Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells For example, the proteins of the invention may be expressed in bacterial cells such as E coli, insect cells (using baculovirus), yeast cells, or mammalian cells Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (199 1) A host cell may also be chosen which modulates the expression of an inserted nucleic acid sequence, or modifies (e.g glycosylation or phosphorylation) and processes (e g. cleaves) the protein in a desired fashion Host systems or cell lines may be selected which have specific and characteristic mechanisms for post- translational processing and modification of proteins. For example, eukaryotic host cells including CHO, VERO, BHK, HeLA, COS, MDCK, 293, 3T3, and WI38 may be used For long-term high-yield stable expression of the protein, cell lines and host systems which stably express the gene product may be engineered
Host cells and in particular cell lines produced using the methods described herein may be particularly useful in screening and evaluating compounds that modulate the activity of a protein of the invention.
The proteins of the invention may also be expressed in non-human transgenic animals including but not limited to mice, rats, rabbits, guinea pigs, micro-pigs, goats, sheep, pigs, non-human pπmates (e.g. baboons, monkeys, and chimpanzees) (see Hammer et al. (Nature 315.680-683, 1985), Palmiter et <ιl (Science
222:809-814, 1983), Bπnster et al. (Proc Natl. Acad. Sci USA 82 44384442, 1985), Palmiter and Bπnster (Cell. 41.343-345, 1985) and U S Patent No. 4,736,866) Procedures known in the art may be used to introduce a nucleic acid molecule of the invention encoding a protein of the invention into ammals to produce the founder lines of transgenic animals. Such procedures include pronuclear microinjection, retrovirus mediated gene transfer into germ lines, gene targeting in embryonic stem cells, electroporation of embryos, and sperm- mediated gene transfer
The present invention contemplates a transgenic animal that cames a nucleic acid molecule of the invention in all their cells, and animals which carry the transgene in some but not all their cells The transgene may be integrated as a single transgene or in concatamers The transgene may be selectively introduced into and activated in specific cell types (See for example, Lasko et al, 1992 Proc. Natl. Acad. Sci. USA 89. 6236)
The transgene may be integrated into the chromosomal site of the endogenous gene by gene targeting. The transgene may be selectively introduced into a particular cell type inactivating the endogenous gene m that cell type (See Gu et al Science 265: 103-106). The expression of a recombinant protein of the invention in a transgenic animal may be assayed using standard techniques. Initial screening may be conducted by Southern Blot analysis, or PCR methods to analyze whether the transgene has been integrated. The level of mRNA expression in the tissues of transgenic animals may also be assessed using techniques including Northern blot analysis of tissue samples, in situ hybndization, and RT-PCR. Tissue may also be evaluated lmmunocytochemically using antibodies against GNTV Protein The proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merπfield, 1964, J. Am Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol 15 I and II, Thieme, Stuttgart)
N-termmal or C-terπunal fusion proteins compπsing a protein of the invention conjugated with other molecules, such as proteins may be prepared by fusing, through recombinant techniques, the N terminal or
C-terminal of a protein of the invention, and the sequence of a selected protein or selectable marker protein with a desired biological function. The resultant fusion proteins contain a protein of the invention fused to the selected protein or marker protein as described herein. Examples of proteins which may be used to prepare fusion proteins include lmmunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.
2.3 Nucleotide Probes
The nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleic acid sequences in biological materials. Suitable probes include nucleic acid molecules based on nucleic acid sequences of the invention and in particular nucleic acid sequences encoding at least 6 sequential amino acids from regions of a protein of the invention (e.g SEQ. ID. NO.2, SEQ
ID. NO 5., or SEQ. ID. NO. 7) A nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32P, 3H, 14C or the like. Other detectable substances which may be used include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and luminescent compounds. An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization. Labelled probes may be hybndized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
The nucleotide probes may also be useful in the diagnosis of disorders of the hematopoietic system, sensory nervous system, myocardium, or cardiac or neural vasculature, in monitoring the progression of these conditions; or monitoring a therapeutic treatment.
A probe may be used in hybridization techniques to detect nucleic acid molecules or genes of the invention. The technique generally involves contacting and incubating nucleic acids obtained from a sample from a patient or other cellular source with a probe of the present invention under conditions favourable for the specific annealing of the probes to complementary sequences in the nucleic acids. After incubation, the non- annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected.
The detection of nucleic acid molecules of the invention may involve the amplification of specific gene sequences using an amplification method such as PCR, followed by the analysis of the amplified molecules using techniques known to those skilled in the art. Suitable pnmers can be routinely designed by one of skill in the art.
Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities in a gene or nucleic acid molecule of the invention, including point mutations, insertions, deletions, and chromosomal rearrangements. For example, direct sequencing, single stranded conformational polymorphism analyses, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical mismatch cleavage, and ohgonucleotide hybridization may be utilized
Genotyping techniques known to one skilled in the art can be used to type polymorphisms that are in close proximity to mutations in a nucleic acid molecule or gene of the invention The polymorphisms may be used to identify individuals in families that are likely to cany mutations If a polymorphism exhibits linkage disequa bnum with mutations in a gene, it can also be used to screen for individuals in the general population likely to cany mutations Polymorphisms which may be used include restriction fragment length polymorphisms (RFLPs), single-base polymorphisms, and simple sequence repeat polymorphisms (SSLPs).
A probe of the invention may be used to directly identify RFLPs A probe or pπmer of the invention can additionally be used to isolate genomic clones such as YACs, B ACs, PACs, cosmids, phage or plasmids.
The DNA in the clones can be screened for SSLPs using hybridization or sequencing procedures.
Hybridization and amplification techniques described herein may be used to assay qualitative and quantitative aspects of expression of a nucleic acid molecule of the invention. For example, RNA may be isolated from a cell type or tissue known to express a gene and tested utilizing the hybridization (e.g. standard Northern analyses) or PCR techniques referred to herein The techniques may be used to detect differences in transcript size which may be due to normal or abnormal alternative splicing The techniques may be used to detect quantitative differences between levels of full length and/or alternatively splice transcnpts detected in normal individuals relative to those individuals exhibiting symptoms of a disease.
The primers and probes may be used m the above described methods in situ i.e directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections.
2.4 Antibodies
Proteins of the invention can be used to prepare antibodies specific for the proteins. Antibodies can be prepared which bind a distinct epitope in an unconserved region of the protein. An unconserved region of the protein is one which does not have substantial sequence homology to other proteins. A region from a well- characterized region can be used to prepare an antibody to a conserved region of a protein of thϊ invention.
Antibodies having specificity for a protein of the invention may also be raised from fusion proteins created by expressing fusion proteins in bacteria as described herein.
The invention can employ intact monoclonal or polyclonal antibodies, and lmmunologically active fragments (e.g. a Fab or (Fab)2 fragment), an antibody heavy chain, and antibody light chain, a genetically engineered single chain Fv molecule (Ladner et al, U.S. Pat. No 4.946,778), or a chimenc antibody, for example, an antibody which contains the binding specificity of a muπne antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art Antibodies specifically reactive with a protein of the invention, or derivatives, such as enzyme conjugates or labeled derivatives, may be used to detect the proteins in various biological materials, for example they may be used in any known lmmunoassays which rely on the binding interaction between an antigemc determinant of a protein and the antibodies. Examples of such assays are radioimmunoassays, enzyme lmmunoassays (e.g.ELISA), lmmunofluorescence, lmmunoprecipitation, latex agglutination, hemagglutination, and histochemical tests. The antibodies may be used to detect and quantify a protein of the invention in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states.
In particular, the antibodies of the invention may be used in lmmuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect a protein of the invention, to localise it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression
Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a protein of the invention. Generally, an antibody of the invention may be labelled with a detectable substance and a protein may be localised in tissues and cells based upon the presence of the detectable substance. Examples of detectable substances include, but are not limited to, the following: radioisotopes (e.g., 3 H, 14 C, 35S, 125I, l31I), fluorescent labels (e.g , FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, .beta.-galactosidase, luciferase, alkaline phosphatase, acetylchohnesterase), biotmyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calonmetnc methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucme zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steπc hindrance. Antibodies may also be coupled to electron dense substances, such as femtin or colloidal gold, which are readily visualised by electron microscopy.
Indirect methods may also be employed in which the pnmary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against a piotein of the invention. By way of example, if the antibody having specificity against a protein of the invention is a rabbit
IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.
Where a radioactive label is used as a detectable substance, a protein of the invention may be localized by radioautography. The results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.
2.5 Applications of the Nucleic Acid Molecules and Proteins of the Invention
The proteins of the invention are primarily expressed in hematopoietic, endothehal stromal, and/or myocyte lineages. The proteins of the invention have a role in proliferation, differentiation, activation and/or metabolism of cells of the hematopoietic, myocardium, cardiac and neural vasculature, endothehal, stromal, and/or myocyte lineages. Therefore , the methods described herein for detecting nucleic acid molecules can be used to monitor proliferation, differentiation, activation and/or metabolism of cells of the hematopoietic, endothehal, myocardium, cardiac and neural vasculature, stromal, and/or myocyte lineages by detecting and localizing proteins and nucleic acid molecules of the invention The methods described herein may be used to study the developmental expression of a protein of the invention and, accordingly, will provide further insight into the role of the protein in the hematopoietic system, myocardium, sensory nervous system and vasculature.
By way of example, the 17G2 protein is expressed m the myocardium, cardiac and neural vasculature, in hematopoietic cells, and in the sensory nervous system Therefore, the 17G2 protein has a role in proliferation, differentiation, activation and metabolism of cells of the hematopoietic system, myocardium, cardiac and neural vasculature, and the sensory nervous system. Therefore, the methods for detecting nucleic acid molecules and 17G2 proteins of the invention, can be used to monitor proliferation, differentiation, activation and metabolism of hematopoietic cells, and cells of the sensory nervous system and neural and cardiac vasculature by detecting and localizing 17G2 proteins and nucleic acid molecules It would also be apparent to one skilled in the art that the above described methods may be used to study the developmental expression of 17G2 proteins and, accordingly, will provide further insight into the role of 17G2 proteins in the hematopoietic system, myocardium, neural and cardiac vasculature. and sensory nervous system
The nucleic acid molecules and proteins of the invention are markers for hematopoietic cells, endothehal cells, stromal cells, and/or myocytes, and accordingly the antibodies and probes described herein may be used to label these cells. For example, the 17G2 protein is a marker for early vascular endothehal cells and hematopoietic cells, and accordingly the antibodies and probes descnbed herein can be used to label early vascular endothehal cells and hematopoietic cells.
Substances which modulate a protein of the invention (e.g a 17G2 protein) can be identified based on their ability to bind to the protein. Therefore, the invention also provides methods for identifying substances which bind to a protein of the invention. Substances identified using the methods of the inveni ion may be isolated, cloned and sequenced using conventional techniques.
Substances which can bind with a protein of the invention e g a 17G2 protein may be identified by reacting the protein with a substance which potentially binds to the protein, under conditions which permit the formation of substance-protein complexes and assaying for substance-protein complexes, for free substance, for non-complexed protein, or for activated protein Conditions which permit the formation of complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.
The substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the assay of the components, antibody against the protein or the substance, or labelled protein, or a labelled substance may be utilized The antibodies, proteins, or substances may be labelled with a detectable substance as described above
A protein, or the substance used in the method of the invention may be msolubihzed For example, the protein, or substance may be bound to a suitable earner such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc The earner may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc The insolubihzed protein or substance may be prepared by reacting the material with a suitable insoluble earner using known chemical or physical methods, for example, cyanogen bromide coupling
The invention also contemplates a method for evaluating a compound for its ability to modulate the biological activity of a protein of the invention, by assaying for an agonist or antagonist (l e enhancer or inhibitor) of the binding of the protein with a substance which binds with the protein The enhancer or inhibitor may be an endogenous physiological compound or it may be a natural or synthetic compound It will be understood that the agonists and antagonists l e inhibitors and enhancers that can be assayed using the methods of the invention may act on one or more of the binding sites on the protein or substance including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or allosteπc sites
The invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of the protein with a substance which is capable of binding to the protein Thus, the invention may be used to assay for a compound that competes for the same binding site of the protein
The reagents suitable for applying the methods of the invention to evaluate compounds that modulate a protein of the invention may be packaged into convenient kits providing the necessary materials packaged into suitable containers The kits may also include suitable supports useful in performing the methods of the invention
The substances or compounds identified by the methods described herein, antibodies, and antisense nucleic acid molecules of the invention may be used for modulating the biological activity of a protein of the invention, and they may be used m the treatment of conditions requiring modulation of cells of the hematopoietic, myocardium, cardiac and neural vasculature, endothehal, stromal, and/or myocyte lineages Accordingly, the substances, antibodies, and compounds may be formulated into pharmaceutical campositions for adminstration to subjects in a biologically compatible form suitable for administration in vivo By "biologically compatible form suitable for administration in vivo is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects The substances may be administered to living organisms including humans, and animals Administration of a therapeutically active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result For example, a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual Dosage regima may be adjusted to provide the optimum therapeutic response For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation
The active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc ), oral administration, inhalation, transdermal application, or rectal administration Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound
The compositions described herein can be prepared by per e known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle Suitable vehicles are described, for example, in Remington s Pharmaceutical Sciences (Remington s Pharmaceutical
Sciences, Mack Publishing Company, Easton, Pa , USA 1985) On this basis, the compositions include, albeit not exclusively, solutions of the substances or compounds in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and lso-osmotic with the physiological fluids The activity of the substances, compounds, antibodies, antisense nucleic acid molecules, and compositions of the invention may be confirmed in animal experimental model systems
The invention also provides methods for studying the function of a protein of the invention Cells, tissues, and non-human animals lacking in expression or partially lacking in expression of a nucleic acid molecule or gene of the invention may be developed using recombinant expression vectors of the invention having specific deletion or insertion mutations m the gene A recombinant expression vector may be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create a deficient cell, tissue or animal
Null alleles may be generated in cells, such as embryonic stem cells by deletion mutation A recombinant gene may also be engineered to contain an insertion mutation which inactivates the gene Such a construct may then be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, electroporation, injection etc Cells lacking an intact gene may then be identified, for example by Southern blotting, Northern Blotting or by assaying for expression of the encoded protein using the methods described herein Such cells may then be fused to embryonic stem cells to generate transgenic non-human animals deficient in a protein of the invention Germ ne transmission of the mutation may be achieved, for example, by aggregating the embryonic stem cells with early stage embryos, such as 8 cell embrjos, in vitro, transfening the resulting blastocysts into recipient females and, generating germhne transmission of the resulting aggregation chimeras Such a mutant animal may be used to define specific cell populations, developmental patterns and in vivo processes, normally dependent on gene expression The following non-limiting examples are illustrative of the present invention Examples
Example 1
MATERIALS AND METHODS
Vectors Two gene trap vectors were used PT1-ATG (PT1 henceforth) contains the En-2 splice acceptor site positioned immediately upstream of the lacZ reporter gene with an ATG translational start site [Hill D P , Wurst W , Methods in Enzymology 225 664-681, 1993] The bacterial neomycin-resistance (neo) gene is driven by the phosphoglycerate kιnase-1 (PGK-1) promoter GT1 8geo contains the En-2 splice acceptor site immediately upstream of a lacZ-neo fusion gene [Skarnes W C et al, Proc Natl Acad Sci USA 92 6592- 6596, 1995] The point mutation in the neo fragment of SAβgeo is not contained in GT1 8geo vector, thereby allowing neomycin resistance at a lower level of endogenous gene expression than the SAβgeo vector Generation of Trapped ES Cell Lines. Rl ES cells were maintained on primary embryonic fibroblasts as previously described [Nagy A. et al., Proc. Natl. Acad. Sci. USA 90 8424-8428, 1993], After electroporation and selection in G418 for 8 days, drug-resistant colonies were transfened to 96-well plates and expanded to confluency. Clones were passaged to two 96-well plates and one set of 24-well plates. Once clones reached confluency, one 96-well plate was frozen, the second 96-well plate was assayed for β-galactosidase (β-gal) expression, and the 24-well plates were used for attached EB differentiation cultures. Expression of the lacZ reporter gene was carefully determined both in undifferentiated and differentiated ES cells. Clones with observable expression patterns were re-frozen and in some cases, re-analyzed. In addition, the expression patterns were photographed and cataloged. Reporter Gene Expression, β-gal activity of undifferentiated and differentiated cells was detected as follows:
Cells were rinsed in lOOmM sodium phosphate (pH 7.5), then fixed in 0.2% glutaraldehyde, 5mM EGTA, 2mM MgCl2 and lOOmM sodium phosphate, pH 7.5 for 5 mm. The cells were washed 3 times for 5 mm. each m 2mM MgCl2, 0 02% NP-40 and lOOmM sodium phosphate, pH 7 5 The cells were stained with X-gal overnight at 37°C β-gal activity was detected m embryos as described above except the fixative included 1.5% formaldehyde and embryos were fixed for 30 mm. to 1 hour and washed 3 times for 15 min. eac i wash.
Attached EB Screen. ES cells were allowed to differentiate into attached EBs as previously descnbed [Bautch V.L. et al., Dev. Dyn. 205:1-12, 1996] with several modifications Clones were grown to confluency in 24-well plates, treated with dispase (Collaborative Research, 1:1 dilution in PBS), washed 3 times in PBS and grown in suspension in "Ultra Low Cluster" 24-well plates (COSTAR) in ES media without LIF On day 3 post- dispase treatment, 5-10 embryoid bodies were transfened to 48-well tissue culture plates (Falcon). Cultures were fed every other day with fresh media, β-gal activity was determined on day 8. 12, and 16 post-dispase. OP9 Induction Assay. ES cells were allowed to differentiate on the OP9 stromal cell line as previously described [Nakano T. et al., Science 265:1098-1101, 1994] with several modifications. ES clones were differentiated on OP9 stroma in replica wells of 6-well plates (10^ ES cells/well) for 5 days to generate mesodermal colonies. A single cell suspension was prepared using trypsin from one well for eacli clone, and
105 mesodermal cells were replated onto OP9 stroma in two wells of a 6-well plate and grown for 3 days. Non- adherent hematopoietic cells were transfened from both wells to one new well for an additional 3 days, β-gal activity was determined on mesodermal cells on the duplicate day 5 OP9 plate and on adherent hematopoietic cells on days 8 and 11. 5' RACE. RNA was prepared from either undifferentiated or differentiated cells using Tπzol (Gibco/BRL) according to manufacturer's instructions. 5' RACE was performed using the 5' RACE kit (Gibco/BRL), according to manufacturer's instructions with modifications previously described [Sam M. et al., Dev. Dyn., in press]. 5' RACE products were subcloned into the CloneAmp plasmid (Gibco/BRL) and sequenced using the Sequenase kit (Pharmacia) according to manufacturers' instructions Sequences were analyzed by comparison to the non-redundant GenBank and EST of NCBI using the BLASTN program
Generation of Chimeras. ES cells were aggregated with diploid embryos as described [Nay A., Rossant, J., Oxford, IRL, 1993, p 147-178], harvested at embryonic day (e) 9 5-14.5, and stained for β-gal activity. About half of the diploid embryos were allowed to mature to term for germ-line transmission. Chimeπc males were bred to CDl females, and tail DNA of Fi and F2 offspring was analyzed by southern blotting and hybndization to En-2 or RACE fragment probes Results
Identification of Trapped Gene Expression Patterns In the absence of leukemic inhibitory factor, ES colonies spontaneously differentiate into embryoid bodies (EBs) in suspension culture The complex structure of the EB contains all three germ layers and resembles the extra-embryonic yolk sac both morphologically and transcπptionally [Doetschmann T C et al , J Embryol Exp Morph 87 27-45, 1985], [Schmitt, R M et al , Genes & Dev 5 728-740, 1991], [Keller G et al , Mol Cell Biol 13 473-486, 1993], [Snodgrass H R et al , American Association of Blood Banks, 1993, p 65-83] As in the yolk sac, the mesoderm of the EB gives rise to angioblastic cords that form blood islands containing primitive hematopoietic cells sunounded by vascular endotheliumWang R et al , Development 114 303-316, 1992] Due to the developmental potential of EBs, the differentiation of ES cells into EBs has provided an excellent model to study the effects of targeted mutations on hematopoietic, vascular and myoblast lineages [Weiss M J et al , Genes & Dev 8 1184-1197, 1994, Shalaby F et al , Cell 89 981-990, 1997, Naπta N et al , Development 122 3755-3764, 1996] However, EBs grown in suspension are difficult to manipulate in clonal cultures and the outer layer of visceral endoderm precludes the identification of small numbers of lacZ positive cells Therefore, the EB culture system was modified so that EBs grow attached to tissue culture plastic [Bautch V L et al , Dev Dyn 205 1-12, 1996] This "attached" or "flat" culture method places the endoderm layer beneath the blood islands and renders the EB more accessible to observation and experimental manipulation The PT1 gene trap vector, which contains a splice acceptor site immediately upstream of a promoterless lacZ reporter gene and the neo gene driven by PGK-1 promoter, was introduced into ES cells (clone Rl) by electroporation After G418 selection, drug-resistant colonies were transfened to 96-well plates and expanded to confluency Clones were replica plated to two 96-well plates and one set of 24-well plates Once clones reached confluency, one 96-well plate was frozen, the second 96-well plate was assayed for β- galactosidase (β-gal) expression, and the 24-well plates were used for attached EB differentiation cultures
Each neoκ colony represented a vector integration event If the vector integrated within an intron, a spliced fusion transcript between lacZ and the endogenous gene was generated upon transcπptional activation of the trapped gene Because all ES cells which had an integrated PT1 vector were G418 resistant regardless of whether or not the integration occuned within a gene, genes which were not expressed in undifferentiated ES cells could be screened using this vector Five percent (37/779) of the neo^ clones tested expre >sed lacZ in undifferentiated ES cells, of which 30 clones continued to be expressed in at least some cells during EB differentiation (Table 1) By comparison, 61 clones (8%) which did not express lacZ as undifferentiated ES cells demonstrated lacZ expression during EB differentiation (Table 1) Of the neo° clones that expressed lacZ as undifferentiated or differentiated ES cells, one-third (32 clones) exhibited a restricted pattern of expression (Table 1) The expression patterns of these clones can be grouped into seven categories (Table 2) More than a third of the clones were expressed in blood islands and/or the vasculature, in contrast, stromal and muscle cells each represented only 3% of the clones displaying restricted expression patterns In addition, 9% of the clones expressed lacZ constitutively in virtually all undifferentiated and differentiated cells The remaining clones exhibited restricted patterns of expression in other cell type(s) In a second series of experiments, the GTl.δgeo vector which contains a splice-acceptor site immediately upstream of a β-gal-neo fusion gene was used. Thus, unlike the PTl vector, all neoκ clones selected after introduction of the GTl.δgeo vector represented integrations into genes which were transcnptionally active in undifferentiated ES cells. Accordingly, a much higher proportion of the GTl.δgeo clones (34% versus 5% for PTl) expressed detectable levels of β-gal activity in undifferentiated ES cells (i.e.,
"Blue", Table 1) Of those, 159 clones continued to express lacZ in at least some cells during EB differentiation. Of the clones which were lacZ negative as undifferentiated ES cells, more than half upregulated expression of lacZ in a portion of differentiated cells in EB cultures. In total, 47 clones displayed an obvious pattern of expression (Table 1 and 2) The majority of the pattern-expressing clones expressed lacZ in the blood islands and or the endothehum (Table 2)
In contrast to EB body differentiation in which ES cells differentiate into all three germ layers which eventually give πse to many lineages including hematopoietic and vascular cells, ES cells grown in co-culture with OP9 stromal cells differentiate into mesodermal colonies which when replaced differentiate into hematopoietic cells All gene trap cell lines demonstrating lacZ expression in blood islands were re-analyzed by differentiating ES cells in replicate OP9 stromal cell cultures[Nakano T. et al., Science 265- 1098-1101,
1994], [Nakano T. et al., Science 272:722-724, 1996]. ES-deπved mesodermal colonies expressing brachury were apparent by day 3 of culture. On day 5, a single cell suspension of a replicate culture was prepared and replated onto OP9 cells. Primitive erythrocytes and multipotential precursors differentiated from the mesodermal precursors within the next 2-3 days and single lineage precursors predominated the cultures by day 11. Cultures were assayed for lacZ expression at days 5, 8, and 11 The majority of blood island positive clones (70%) expressed lacZ m hematopoietic cells when cultured on an OP9 feeder layer (Table 2). Identification of Trapped Genes. To determine the DNA sequence of the trapped genes, RNA was prepared from either differentiated or undifferentiated ES clones and used to perform 5' RACE [Frohman M.A. et al., Proc. Natl. Acad. Sci. USA 85:8998-9002, 1988]. The RACE products of eleven lacZ fusion transcripts were cloned and sequenced. Table 3 summarizes the lacZ expression pattern, the gene trap vector, and sequence information for each clone. Eight of the RACE product sequences conesponded to novel genes, of which four shared similanty with EST sequences. The sequences of three of the trapped genes conesponded to genes that encode known protein products: Mena, Karyopheπn β3, and 5'GMP synthetase. Clone K18E2 encodes Mena, the mammalian homologue of Drosophiha Enabled(ena), which was originally cloned by a genetic screen for suppressors of Abl-dependent phenotypes [Gertler F.B. et al., Genes & Dev. 9:521-533, 1995], [Gertler F.B. et al., Cell 87.227-239, 1996]. In clone K18E2, the PTl vector has integrated into the first intron of Mena, downstream of the initiation codon and, therefore, should result in a null mutation. Clone B2C3 encodes the munne homologue of karyopherm/importin β3 and yeast Pselp [Yaseen N.R., Blobel G., Proc. Natl Acad. Sci. USA 94:4451-4456, 1997], proteins which are involved in the transport of proteins and mRNA across the nuclear membrane [Kutay U. et al., EMBO J. 16:1153-1163, 1997], [Seedorf M., Silver P.A., Proc. Natl. Acad
Sci. USA 94:8590-8595, 1997] The RACE product suggests that a fusion protein was generated from the N- terminal 312 amino acids and lacZ. Mutational analysis of Xenopus karyopheπn-α suggests that this fusion protein will bind weakly to the nuclear pore complex and to RanGTP but not to karyopheπn-α [Kutay U. et al, EMBO J. 16 1153-1163, 1997] and may act as a weak dominate negative mutation In ES clone GC10G7, the GTl 8geo vector has integrated within the 3' coding region of the gene for guanosine 5'-monophosphate
(GMP) synthetase GMP-synthetase catalyzes the amination of xanthosine 5'-monophosphate to form GMP in the presence of glutamme and ATP Although GMP-synthetase is expressed in many cell types, high levels of β-gal activity were observed only in endothehal cells and a population of hematopoietic cells (Table 3) In Vitro and In Vivo Expression of Selected Clones. To determine if in vitro expression patterns conelated with in vivo expression, selected ES clones were aggregated with diploid embryos to generate chimenc mice.
Reporter gene expression was performed first on chimenc embryos to quickly assess expression patterns and subsequently was confirmed in F\ embryos, which is summarized along with sequence analysis in Table 1.
Three clones conesponded to a sequence homolgous to an EST, a completely novel gene and Mena. K17G2 was isolated using the PTl vector and displayed significant sequence similarity to a human EST. K17G2-lacZ was expressed at low to medium levels in undifferentiated ES cells (Fig 1A), while its expression was restncted to blood islands and some endothehal cells in attached EBs (Fig IB) Differentiation on OP9 stromal cells revealed that K17G2-lacZ was expressed in some mesodermal and hematopoietic cells (Fig. 1C&D, respectively) To analyze the expression pattern of K17G2-lacZ in vivo, K17G2 ES cells were used to generate chimenc mice Analysis of F\ elO 5 embryos revealed additional tissues which expressed the K17G2-lacZ fusion product (Fig IE). For example, the lacZ fusion product was expressed in the myocardium and the dorsal root ganglia (Fig. 1F&G, respectively) However, as predicted by the in vitro expression, K17G2-lacZ was expressed in some of the embryonic vasculature, including the endocardium, and circulating blood cells (Fig 1H&I). In the adult, K17G2-lacZ expression was observed in hematopoietic cells of the spleen and bone marrow and in the endocardium (data not shown). K17G2 heterozygous httermates were mated with one another; however, these matings failed to produce viable homzygous mice indicating that K17G2 homozygous embryos die in utero (data not shown).
Clone GC11E10 was isolated using the GTl.δgeo vector and represents a novel ORF. The GC11E10- geo fusion protein was expressed at medium to high levels in undifferentiated ES cells (Fig. 2A). In attached EBs, expression appeared within blood islands and the vasculature associated with these structure s (Fig. 2B).
Differentiation of GC11E10 ES cells on OP9 stromal cells demonstrated lacZ expression within mesodermal colonies and high levels of expression within hematopoietic cell clusters (Fig 2C&D, respectively). In vivo, lacZ was expressed in the yolk sac, dorsal aorta, heart, the developing liver and vasculature (Fig. 2E&F) Further analysis demonstrated that lacZ expression was contained within blood cells circulating throughout the embryo and within blood islands m the yolk sac (Fig. 2G&H) The GC1 lElO-geo fusion protein was also expressed in endothehal cells throughout the embryo as demonstrated in the lntersomitic vessels (Fig. 21).
Clone K18E2 (a PTl clone) represents an integration into the first intron of Mena Mena is involved in actin assembly and cell motihty; therefore its ubiquitous expression in rapidly dividing cells was expected. Mena-lacZ was expressed at very high levels in nearly all undifferentiated ES cells (Fig. 3A) and virtually all cells in EBs (Fig. 3B). Differentiation of K18E2 on OP9 stromal cells demonstrated high levels of Mena-lacZ expression in mesodermal cells (Fig 4C) but only low level expression in a minority of hematopoietic cells (Fig. 4D). The pattern and level of lacZ expression was reproduced in Fj embryos. Mena-lacZ was expressed by almost all cells in the developing embryo with the exception of hepatocytes and some hematopoietic cells (Fig. 4E&F and data not shown). DISCUSSION
The present inventors developed an expression-based strategy to identify and mutate genes that are preferentially expressed in cells of the hematopoietic and vascular lineages Gene trap vectors were introduced into ES cells by electroporation and sibling clones were allowed to differentiate into attached EBs to identify expression patterns Clones exhibiting reporter gene expression in blood islands were then differentiated on
OP9 stromal cells to determine if hematopoietic cells expressed the reporter gene From almost 1300 clones,
79 clones were isolated with identifiable expression patterns, of which 33 were preferentially expressed in hematopoietic and/or endothehal cells These in vitro patterns of expression, which can be analyzed relatively quickly and in large numbers, were reliable predictors of in vivo expression patterns as determined in chimenc and Fi embryos ES clones with expression patterns of interest were then used to clone and sequence the upstream coding region of the trapped gene by 5' RACE Three of the clones conesponded to known genes and eight were novel
The attached EB differentiation assay used as the primary screen enabled the identification of a large number of genes with a spatially or cell-type restncted expression for several lineages including hematopoietic, endothehal, stromal and myocyte
Example 2
Gene trapping in embryonic stem (ES) cells coupled with two in vitro differentiation assays was used to screen for genes involved in hematopoietic and vascular development Undifferentiated ES cells were electroporated with either the pPTl-ATG vector which contains a splice acceptor site upstream of a promoterless lac Z gene and a PGK-neoR gene, or the pGTl 8 geo vector which contains a promoterless lacZ/neoR fusion gene G418 resistant clones were allowed to differentiate into attached embryoid bodies
(EBs) and lacZ activity was assayed to indicate trapped gene expression in undifferentiated cells and differentiation cultures Clones expressing lacZ in blood islands were also differentiated on OP9/OP9 stromal cells to confirm lacZ expression by hematopoietic cells A modified attached embryoid body (EB) assay was used to screen the reporter gene expression pattern of approximately 1300 gene trapped ES cell lines for expression in hematopoietic and endothehal lineages The assay was earned out as described in V L Bautch et al , (Developmental Dynamics 205 1-12,
1996) with the following modifications The ES clones were grown up in 24-well plates m the presence of hf
(but without feeders) essentially as would be earned out in TC dishes The media was aspirated, each well was washed with 1 5 ml PBS and aspirate Cold diluted (1 1 IN PBS) Dispase was added to cover the well and it was allowed to sit 1-2 mm at RT The wells were filled with PBS and then pipetted up & down 2-3 times The colonies were allowed to settle and the Dispase/PBS was aspirated or pipetted off Washing was repeated with
PBS, and using 1 5 ml CEB media Clumps were transfered to 1 5 ml CEB media m wells of "Ultra Low
Cluster 24 well plate" (COSTAR cat # 3473) The plate was incubated at 37EC, 5%C02 for 3 days On the third day post-Dispase, the embryoid bodies were pipetted up & down to mix, and about 2-4 drops were transfened into about 0 8ml CEB media/ well of a 48-well plate (Falcon cat # 3078) The wells were checked to confirm that there were about 5 colonies/well The plate was then incubated at 37EC, 5% C02 and the cultures were fed every other day
The reporter gene expression pattern of clone 17G2 demonstrated moderate expression of the trapped gene in undifferentiated ES cells and restricted expression of hematopoietic and endothehal cells in the attached
EB cultures. Differentiation of 17G2 on OP9 stromal cells lead to expression of the trapped gene in some mesodermal and hematopoietic cells. 17G2 ES cells were aggregated with wild-type CDl embryos to generate chimeras. In vivo expression analysis reveals expression of the 17G2 gene in the cardiac and neural vasculature, hematopoietic cells, myocardium, and sensory nerves including the trigeminal ganglia, dorsal root ganglia, and optic nerve. 17G2 expression is maintained in the adult heart and bone maπow. The exon sequence upstream of the vector integration was cloned by 5' RACE, and analysis showed that the 17G2 gene encodes a novel gene (see Figure 1 for a nucleic acid sequence from the 17G2 gene). The RACE product was used as a probe to screen the genotypes of F2 litters. No homozygotes were detected out of over 200 pups. Reporter gene expression analysis of timed heterozygous matings revealed that homozygous embryos are viable at midgestation (el 1.5). Example 3
Analysis of 17G2 DNA sequence revealed that the cDNA sequence does not contain either the Kozak initiation sequence nor the termination and polyadenylation sequences. The 952 bp cDNA encodes a hydrophilic 317 amino acid open reading frame (ORF). The ORF contains numerous Protein Kinase C (PKC) and Casein Kinase II (CK2) phosphorylation sites as well as a tyrosine phosphorylation site. Comparison of the cDNA sequence to the non-redundant DNA databases revealed no significant matches. However, comparison of the cDNA to the EST databases using BLAST revealed six rat ESTs identified from subtractive libraries that were 97% identical to 17G2 and therefore are likely homologues to 17G2. In addition, a human EST, a Drosophilia EST, and a C.elegans full-length EST contiguous sequence encoding 466 amino acids were found to be 75%, 57%, and 50% identical, respectively. Amino acid comparison demonstrated 62% (66% conserved), 46% (68% conserved), and 40% (56% conserved) identical between 17G2 and the human EST, the C. elegans contig. sequence, and the Drosophilia EST, respectively. In addition, amino acid comparison by BLAST also demonstrated 30% and 42% identical and conserved, respectively with a yeast gene of unknown function termed yeast orfl. A more sophisticated amino acid analysis comparison program called
Psi-BLAST determined that the 17G2 orf is similar (p=e-62) to the sorting nexins. Furthermore, the rat, human, C. elegans, Drosophilia, and yeast putative homologues of 17G2 as well as the sorting nexins all share the PKC, CK2, and tyrosine phosphorylation sites with 17G2 suggesting that these proteins indeed function similarly. Sorting nexin 1 (SNX1) is involved in sorting ligand-activated EGFR to endosomes. SNX1 was identified by a yeast-2-hybrid screen using the kinase domain of human EGFR as bait (Science272: 1008-1010). The C-terminal 58 amino acids bind to the EGFR kinase domain. Overexpression of SNX1 resulted in decreased expression of EGFR by enhancing rates of constitutive and ligand-induced degradation. Originally, the only similar sequence reported in GENBANK was that of Mvpl, a yeast protein identified by a genetic screen for modifiers of VPS 1 mutants (MCB 15:1671-1678). VPS 1 is an 80kDa GTPase that associates with golgi membrane and is required for the sorting of proteins to the yeast vacuole. MVP1 overexpression suppressed dominant alleles of VPS1. MVP1 is a 59 kDa hydrophilic protein which was also shown to be necessary for protein sorting to yeast vacuoles.
Having illustrated and described the principles of the invention in a prefened embodiment, it should be appreciated to those skilled in the art that the invention can be modified in anangement and detail without departure from such principles All modifications coming within the scope of the following claims are claimed
All publications, patents and patent applications refened to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety
Detailed Figure Legends
Figure 1. K17G2-lacZ expression in vitro and in vivo Overnight X-gal staining showed fusion transcript expression at medium intensity in most undifferentiated K17G2 ES cells (A) The fusion transcript was expressed in the blood island and some of the associated vascular endothelium in attached EB culture (B) Differentiation of clone K17G2 on op9 stromal cells demonstrated lacZ expression in mesodermal colonies (C) and hematopoietic clusters (D) X-gal staining of an elO 5 Fj embryo demonstrated limited lacZ expression in the embryo (whole mount, E) including expression in the myocardium (F) and the dorsal root ganglia (G) An X-gal stained el2 5 Fi embryo demonstrated lacZ expression in the endocardium (H) and vascular endothelium and circulating hematopoietic cells (I)
Figure 2. GCl lElO-lacZ expression Overnight X-gal staining showed fusion transcript expression at medium to high levels m most undifferentiated ES cells (A) In attached EB cultures, lacZ was expressed within blood islands and the associated vascular endothelium (B) Differentiation of clone GC11E10 on op9 stromal cells demonstrated lacZ expression in mesodermal colonies (C) and a proportion of hematopoietic clusters (D) Overnight whole mount X-gal staining of an e9 5 chimenc embryo and yolk sac demonstrated lacZ expression in the dorsal aorta, heart, liver, and vasculature (E) LacZ expression in the yolk sac was confined to endothehal and hematopoietic cells (F&G) LacZ was expressed by the endocardium and circulating blood cells in the heart (H) and by the mtersomitic endothehal cells (I)
Figure 3. Mena-lacZ (K18E2) expression Overnight X-gal staining demonstrated high-level lacZ expression in undifferentiated ES cells (A) and in virtually all cells in the attached EB culture including blood islands and their associated vasculature (B) Differentiation of clone K18E2 on op9 stromal cells followed by overnight X-gal staining demonstrated high level lacZ expression in mesodermal colonies (C), whereas most hematopoietic cells did not express lacZ (thick anows) although low-level expression was observed in some isolated hematopoietic cells (thin anows, D) Mena lacZ was expressed at high levels in vivo as demonstrated by strong X-gal staining in less than 90 minutes in an elO 5 F\ embryo (E) Ovei night X-gal staining of an el3 5 Fi embryo showed strong lacZ expression in all tissues except the liver (F)
Table 1. Summary of attached EB primary gene trap screen.
VECTOR UNDIFFERENTIATED EMBRYOID BODIES NUMBER (%)
PTl BLUE1 BLUE 30 (4) GTlδ.geo 159 (31)
PTl BLUE WHITE 7 (1) GTlδ.geo 13 (3)
PTl WHΠΈ BLUE 61 (8) GT18.geo 181 (35)
PTl WHΠΈ WHITE 681 (87) GTlδ.geo 156 (31)
PTl GTl,?εeo
Total Number of Neo^ Clones 779 (100) 509 (100)
Total BLUE Clones 98 (13) 353 (69)
Identifiable Patterns Among β-gal positive Clones^ 32 (33) 47 (13)
'"BLUE" indicates detectable β-gal activity.
Percentage was determined by dividing the number of clones with identifiable patterns of lacZ expression by the total number clones demonstrating β-gal activity.
Table 2. Patterns of expression in attached EBs.
TYPE PT1-ATG GTl .8
BLOOD ISLAND* 31% 40%
ENIX)THELIAL 3% 4%
BLOOD ISLAND AND ENDOTHELIAL* 3% 19%
STROMA 3% 4%
MUSCLE 6% 0%
CONSTΠΌTΓVΈ 9% 19% UNKNOWN CELL TYPE 45% 13%
70% of clones expressing lacZ in blood islands express lacZ in hematopoieuc cells in op9 inducuon assay.
Table 3. Race product analysis.
LacZ Epression Pattern
Clone Vector In Vitro1 In Vivo2 Identity
K17B1 PT1 -ATG muscle muscie, endoderm novel ORF
K17G2 PT1-ATG hematopoieuc, vascular hematopoietic, vascular, human EST blood island nervous system, myocardium
K18E2 PT1-ATG constitutive constitutive except hepatocytes Mena
K18F3 PT1-ATG muscle myocardium novel ORF
K20D4 PT1-ATG vascular N.D. endothelial EST
B2C3 GTl.δgeo hematopoietic, vascular N.D. Karyopherin β3
B2D2 GT1.8geo blood island, vascular N.D. embryo EST
GC10A2 GT1.8gco hematopoietic, blood island N.D. novel ORF
GC10G7 GTl .δgeo vascular N.D. 5'GMP synthetase
GC11C7 GTl.δgeo hematopoietic heart, forebrain, otic and optic ES ceil and placenta vesicles, andibular ESTs
GC11E10 GTl.δgeo hematopoietic, blood island hematopoietic, vascular novel ORF vascular heart
' In vitro analysis was performed by analysis of attached EB cultures and op9 cultures.
2In vivo analysis was performed using diploid or tetraploid aggregation chimeric or Fi embryos and sacrificing between e9.5 and el4.5.

Claims

WE CLAIM
1. A method of identifying a target nucleic acid molecule primarily expressed in selected lineages comprising. (a) integrating into a site in the genome of a host cell a gene trap vector containing a reporte r gene, to form transfected cells; (b) growing the transfected cells in vitro under conditions whereby the transfected cells differentiate into embryoid bodies attached to a earner and identifying embryoid bodies expressing the reporter gene in cells of a selected lineage, or (c) growing the transfected cells in vitro under conditions whereby the transfected cells differentiate into cells of a selected lineage, and identifying cells of the selected lineage expressing the reporter gene; wherein the target nucleic acid molecule comprises sequences upstream or downstream of the site of integration of the reporter gene in the cells of the selected lineage.
2. A method as claimed in claim 1, which further comprises isolating nucleic acid molecules from the transfected cells, or descendents thereof expressing the reporter gene wherein the nucleic acid molecules comprise the reporter gene and a part of the target nucleic acid molecule, or the nucleic acid molecules compπsing genomic DNA upstream or downstream of the site of insertion of the gene trap vector.
3. A method as claimed in claim 1, which further comprises forming a chimenc embryo with cells of the selected expressing the reporter gene.
4 A method as claimed m claim 3, wherein the chimenc embryo is allowed to mature to term and mated to provide animal lines or the chimeπc embryo can be implanted in a foster recipient females and mated to provide animal lines.
5. A clone expressed primarily in hematopoietic, endothehal, stromal, and/or myocyte lineages designated 17G2, K18F2, K20D4, K18F2, K20D4, B2D2, GC10E10, GC11C7, and GC11E10.
6. An isolated nucleic acid molecule which comprises:
(i) a nucleic acid sequence encoding a protein having substantial sequence idenUty preferably at least 75% sequence identity, with the amino acid sequenceof SEQ. ID. NO.2, SEQ. ID. NO 5., or SEQ. ID. NO. 7;
(ii) nucleic acid sequences complementary to (i);
(iii) a degenerate form of a nucleic acid sequence of (i);
(iv) a nucleic acid sequence comprising at least 18 nucleotides and capable of hybridizing to a nucleic acid sequence in (I), (ii), or (iii); (v) a nucleic acid sequence encoding a truncation, an analog, an allehc or species variation of a protein comprising the amino acid sequence shown SEQ ID NO 2, SEQ ID NO 5., or SEQ. ID. NO.
7, or (vi) a fragment, or allehc or species variation of (0, (n) or (in)
A nucleic acid molecule comprising:
(l) a nucleic acid sequence compπsing the sequence of SEQ. ID. NO.l, SEQ. ID. NO 3.,
SEQ. ID. NO 4, SEQ. ID. NO. 6, SEQ. ID NO. 8, SEQ. ID. NO. 9, or SEQ. ID. NO. 10, wherein T can also be U, (u) nucleic acid sequences complementary to (i), sequenceof SEQ. ID. NO 1, SEQ. ID. NO
3., SEQ. ID. NO 4, SEQ. ID NO. 6, SEQ ID. NO. 8, SEQ. ID. NO. 9, or SEQ. ID. NO. 10, (in) a nucleic acid capable of hybridizing to a nucleic acid of (l) and having at least 18 nucleotides, or (IV) a nucleic acid molecule differing from any of the nucleic acids of (I) to (m) in codon sequences due to the degeneracy of the genetic code.
8. An isolated nucleic acid molecule which encodes a 17G2 Protein which comprises:
(0 a nucleic acid sequence encoding a protein having the amino acid sequence of SEQ. ID. NO.l,
(n) nucleic acid sequences complementary to (t), or
(iii) a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid of (l).
9. A vector compnsmg a nucleic acid molecule as claimed in claim 7 and the necessary elements for the transcription and translation of the inserted coding sequence.
10. A host cell containing a vector as claimed in claim 9
11. A method for preparing a protein compπsing (a) transfemng a vector as claimed in claim 9 into a host cell;
(b) selecting transformed host cells from untransformed host cells;
(c) cultuπng a selected transformed host cell under conditions which allow expression of the protein; and
(d) isolating the protein.
12. An isolated protein comprising the am o acid sequence of SEQ. ID. NO.2, SEQ. ID. NO 5., or SEQ.
ED. NO. 7
13. Antibodies having specificity against an epitope of a protein as claimed in claim 12.
14 A probe compπsing a sequence derived trom a nucleic acid molecule as claimed in claim 7
15. A method for identifying a substance which binds to a protein as claimed in claim 12 compnsιng reacting the piotein with at least one substance which potentially can bind with the protein, under conditions which permit the formation of complexes between the substance and protein and assaying for complexes, for free substance, for non-complexed protein, or for activated protein
16. A method for evaluating a compound for its ability to modulate the biological activity of a protein as claimed in claim 12 which comprises providing a known concentration of the protein, with a substance which binds to the protein and a test compound under conditions which permit the formation of complexes between the substance and protein, and assaying for complexes, for free substance, for non-complexed protein, or for activated protein
17. A composition comprising one or more of a protein as claimed in claim 12. or a substance or compound identified using a method as claimed in claim 16, and a pharmaceutically acceptable earner, excipient or diluent.
18. A method for treating or preventing a condition requiring modulation of hematopoiesis, the sensory nervous system, myocardium, or cardiac or neural vasculature comprising administering to a patient in need thereof, a protein as claimed in claim 12 or a composition as claimed in claim 17
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WO2022130259A3 (en) * 2020-12-16 2022-07-28 Cambridge Enterprise Limited Treatment of cancer associated with dysregulated novel open reading frame products

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