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WO1994013794A1 - Thrombospondine-4 humaine - Google Patents

Thrombospondine-4 humaine Download PDF

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WO1994013794A1
WO1994013794A1 PCT/US1993/011725 US9311725W WO9413794A1 WO 1994013794 A1 WO1994013794 A1 WO 1994013794A1 US 9311725 W US9311725 W US 9311725W WO 9413794 A1 WO9413794 A1 WO 9413794A1
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thrombospondin
type
εequence
polypeptide
εaid
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PCT/US1993/011725
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John W. Lawler
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Brigham And Women's Hospital, Inc.
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Priority to AU57374/94A priority Critical patent/AU5737494A/en
Publication of WO1994013794A1 publication Critical patent/WO1994013794A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • Platelet thrombospondin is a glycoprotein that is structurally and functionally similar to the adhesive glycoproteins found in a wide variety of cells.
  • the thrombospondin genes encode two distinct polypeptides, designated thrombospondin -1 and -2 (Bornstein et al. , . Biol. Chem. , 266:12821-12824, 1991; and 265: 16691-16698, 1991; Proc. Nat. Acad. Sci. USA 88:8636-8640 (1990); Wolf et al . , Genomics, 6 : 685-691 1990)).
  • Thrombospondin-3 is a recently discovered member of the thrombospondin gene family (Vos et al. J. Biol Chem, 267: 12192-12196 (1992)).
  • Partial or complete cDNA sequences are available for human, mouse and frog thrombospondin -1, and human, mouse and chicken thrombospondin-2 (Lawler and Hynes, J. Cell Biol ⁇ , 103 :1635-1648; (1986); Bornstein et al. , supra; Lawler et al ⁇ , J. Biol. Chem. , 266:8039-8043 (1991); Genomics, 11 : 587-600, (1991).
  • the overall molecular architecture of thrombospondin-1 and 2 are substantially the same.
  • the predicted amino acid sequences of thrombospondins-1 and -2 are very similar in their repeat sequences and their COOH-terminal domains.
  • thrombospondin The central portion of platelet thrombospondin is composed of mutiple copies of structural motifs found in other proteins (Lawler and Hynes, supra 1986). Amino acid sequences that have been shown to mediate cellular attachment are also present in the central portion of the molecule (Rich et al . , Science, 249. 1574-1577 (1990)). In addition, thrombospondin contains a region that is rich in calcium binding sites and that contains the RGD sequence that promotes adhesion of some cell types (Lawler et al. , (1988)).
  • Thrombospondin has been shown to modulate its attachment to a variety of cell types in vitro.
  • the NH2-terminal heparin-binding domain binds to proteoglycans including ⁇ yndecan and to cell surface sulfatides; (Sun et al. , J. Biol. Chem. , 264:2885-1889 (1989)).
  • Thrombospondin also interacts with CD36 or platelet glycoprotein IV (Stromski et al. , Exp. Cell Res. , 198:85-92 (1992)).
  • CD36 or platelet glycoprotein IV platelet glycoprotein IV
  • Several integrin receptors have been reported to bind thrombospondin (Lawler et al. , supra (1988)).
  • thrombospondin can modulate cell adhesion, cell migration, angiogenesis and neurite outgrowth.
  • thrombospondin refers to adhesive glycoproteins of about 420,000-dalton molecular weight that are involved in modulation of cell growth and migration. Thrombospondins are composed of three polypeptides linked by disulfide bonds. The N-terminal end binds with heparin, the C-terminal end assists in platelet aggregation.
  • thrombospondin-1 and thrombospondin-2 polypeptides Three types of internal repeating structures are found in human thrombospondin-1 and thrombospondin-2 polypeptides. These are the type 1, 2 and 3 domains ("repeats"). In addition to the three types of domains, thrombospondins 1 and 2 also contain a region of homology to procollagen, as well as amino and carboxyl-termini.
  • Type 1 domains are homologous to several of the complement factors, including C-8, C-9 and properdin. Type 1 domains are also found in two proteins produced in malaria-parasitized blood cells. These are circumsporozoite protein and the thrombospondin related anonymous protein (Robson et a . , Nature 335: 79-82, (1988)). Three copies of type 1 domains are also found in the UNC-5 gene of C. eleqans
  • Type 2 domains are similar to epidermal growth factor (EGF) in that they are framed around a characteristic spacing of six cysteines. Multiple copies of EGF repeat are commonly found in adhesive glycoproteins and cell adhesion molecules.
  • the type 2 domains extend from nucleic acid sequence 1720to 2151 on thrombospondins-1 and -2.
  • an isolated nucleotide sequence encoding a new member of the thrombospondin family, thrombospondin-4, or unique fragments of thrombospondin-4 is provided.
  • One embodiment is an isolated DNA sequence encoding a thrombospondin, that has at least four, type 2 domains.
  • the sequence encodes a thrombospondin that lacks any type 1 domains.
  • a further embodiment is a sequence encoding a thrombospondin that lacks a region of homology with procollagen.
  • Yet another embodiment is a sequence that encodes a thrombospondin that has four, type 2 domains, lacks type 1 domains and lacks a region of homology to procollagen.
  • the preferred DNA of the present invention is a human homolog of thrombospondin-4. Additionally, the invention relates to vertebrate thrombospondin-4 genes isolated from porcine, ovine, bovine, feline, avian, equine, or canine, as well as primate sources and any other species in which thrombospondin-4 structure exists.
  • recombinant cells and plasmids containing the foregoing isolated DNA preferably linked to a promoter.
  • Portions of the foregoing nucleotide sequences are also included in the invention. One such portion is contained in a vector within a host cell.
  • isolated thrombospondin protein having at least four type 2 domains.
  • Other thrombospondins lack any type 1 domains and/or lack any procollagen homology.
  • Portions of the foregoing isolated thrombospondin proteins are also included in the invention.
  • Antibodies with selective binding specificity for the thrombospondin protein of the invention also are provided.
  • Another aspect of the invention is a method for producing thrombospondin polypeptide.
  • the method includes providing an expression vector to a host, the vector containing a DNA sequence of the invention having at least four, type 2 domains; allowing the host to express the thrombospondin, and isolating the expressed thrombospondin.
  • a further aspect of the invention is a probe capable of distinguishing thrombospondin-4 from thrombospondins -1, -2, and -3.
  • the probe can include a nucleotide sequence encoding a thrombospondin-4 polypeptide with at least four, type 2 domains, that lacks any type 1 domains, and lacks a region of homology to procollagen.
  • the nucleotide sequence also can encode a thrombo ⁇ pondin-4 polypeptide having sequences unique to the polypeptide.
  • thrombospondin-4 polypeptide having a restricted range of expression in tissues.
  • the preferred polypeptide is expressed in human heart and skeletal muscle, but is not expressed in human placenta, liver or kidney.
  • a method for interfering with the activity of a thrombospondin-4 gene may be accomplished by providing a construct arranged to include a thrombospondin nucleotide sequence which, when inserted, inactivates either transcription of messenger for thrombospondin-4 and/or inactivates translation of messenger into thrombospondin-4 protein.
  • This construct further has a promotor operatively linked to the sequence.
  • the construct is introduced into a cell, and the construct is allowed to homologously recombine with complementary sequences of the cell genome. Finally, cells lacking the ability to transcribe thrombospondin-4 are selected.
  • FIG. 1 Schematic drawing of human thrombospondin-1 and thrombospondin-2.
  • FIG. 2 Schematic drawing of human thrombospondin-4 The drawing schematically depicts an actual nucleotide sequence of 3120 nucleotides, with a message of approximately 3.3 kb.
  • FIG. 3 Alignment of restriction fragments of Xenopus thrombospondin-4 clones. Restriction endonuclease sites are indicated for the two families (TSP-4A and TSP-4B) . The clones that have been isolated in the first (XF1-XF4), second (XS5-XS10) and third (XT11-XT14) rounds of screening have been grouped into their appropriate family by restriction endonuclease mapping and nucleotide sequencing.
  • Fig. 4 Photograph of a Northern blot of Xenopus stage 17 RNA probed with the XF3 clone of Fig. 3. Two micrograms of total stage 17 mRNA was electrophoresed and blotted. Positions and sizes of markers are shown on the left.
  • FIG. 5 The expression of thrombospondin-4 in adult human tissue.
  • the blot was probed with a 2.2 kb fragment of Xenopus thrombospondin-4.
  • the positions and sizes (kb) of the markers are indicated on the left.
  • Type 3 domains extend from nucleic acid 2221 to 2926 on thrombospondins -1 and -2.
  • Type 3 domains include a large number of calcium-binding sites. The consensus sequence of these type 3 domains is similar to calcium binding site sequences of calmodulin, parvalbumin and fibrinogen beta and gamma subunits. (Lawler and Hynes, supra) .
  • thrombospondins of the invention like other thrombospondins characterize to date (i.e. thrombospondins -1 -2), have an N-terminal region that is more than 200 amino acids in length. In thrombospondins -3 and -4, which lack procollagen and type 1 domains, this N-terminal region preceeds the type 2 domains. In thrombospondins -1 and -2, this N-terminal region preceeds both the procollagen and type 1 domains.
  • Thrombospondins -1 and -2 also have a region adjacent the N-terminal end that is substantially homologous to the known sequence of procollagen. This region extends from nucleotides 916 to 1209 on thrombospondins -1 and -2.
  • thrombospondin-4 The novel member of the thrombospondin family, hereinafter called "thrombospondin-4" has the schematic structure depicted in FIG. 2.
  • thrombospondin-4 In complete contrast to human thrombospondins 1 and 2, thrombospondin-4 lacks type 1 domains entirely. Thrombospondin-4 also lacks a region homologous to procollagen, in contrast to the known thrombospondins 1 and 2. The molecular architecture of much of the N-terminal end of thrombo ⁇ pondin-4 is thus distinct from that of human thrombospondins 1 or 2.
  • thrombospondin-4 has four, type 2 domains (FIG. 2) in contrast to thrombospondins -1 and -2 which have three, type 2 domains (see FIG. 1).
  • Thrombospondin-4 has the same number of calcium-binding sites located within the type 3 domains as do thrombospondins 1 and 2.
  • a thrombospondin-4 molecule is the isolated nucleotide sequence shown in SEQ ID NO.: 1.
  • isolated it i ⁇ meant a nucleic acid ⁇ equence: (i) amplified jin vitro by, for example, polymera ⁇ e chain reaction (PCR); (ii) synthesized by, for example, chemical synthe ⁇ i ⁇ ; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and gel separation.
  • isolated is also meant to include polypeptides encoded by isolated nucleic acid sequences, as well as polypeptides synthesized by, for example, chemical synthetic methods, and polypeptides separated from biological materials, and then purified using conventional" protein analytical procedures.
  • SEQ ID NO. : 1 is a thrombospondin-4 that has been isolated from the frog, Xenopus laevis.
  • An open reading frame of 889 amino acids is predicted from the Xenopus nucleotide sequence.
  • the first 216 amino acids of Xenopus thrombospondin-4 have little homology with human thrombo ⁇ pondins 1 and 2, primarily because of the lack of type 1 repeats and the lack of procollagen sequence in Xenopus thrombo ⁇ pondin-4.
  • thrombospondin-4 Four adjacent type 2 domain ⁇ can be identified in Xenopus thrombospondin-4 on the basis of the positions of the cy ⁇ teine residues.
  • the overall homology with other thrombospondins is low in this type 2 region, and the introduction of several gap ⁇ is neces ⁇ ary to optimize the alignment.
  • the ⁇ econd of the type 2 do ain ⁇ i ⁇ , however, similar to those of thrombospondins -1 and -2, in that thirteen residues are inserted between the last two cysteine residue ⁇ .
  • the amino acid sequence for the four type 2 domains of thrombospondin-4 are shown below in Table 1.
  • the type 3 domains of Xenopus thrombospondin-4 are 61.4% identical to the type 3 domains of human thrombospondins 1 and 2.
  • the consen ⁇ u ⁇ ⁇ equence and overall organization of the ⁇ even, type 3 repeat ⁇ of Xenopus thrombospondin-4 are equivalent to tho ⁇ e of thrombospondins-1 and -2, with the ⁇ econd and fourth type 3 domain ⁇ being truncated after the ⁇ econd cy ⁇ teine.
  • Thrombospondin-4 contain ⁇ 4 amino acid ⁇ (PPGP) at the end of the ⁇ ixth, type 3 domain that do not align with sequence ⁇ on thrombo ⁇ pondin ⁇ -1 and -2. Further thrombospondin-4 does not contain an RGD sequence.
  • the seven, type 3 domains of Xenopus thrombospondin-4 are shown below in Table 2.
  • DNCVYVPNSGQEDTDKDNIGDACDE DADGDGILNEQ DNCVLAANIDQKNSDQDIFGDAC
  • a particularly preferred embodiment of a thrombospondin-4 molecule has the nucleotide sequence shown in SEQ ID NO. : 3. This is a human homolog of the Xenopus ⁇ equence containing about 45 more amino acid ⁇ at the amino-terminal end than the Xenopu ⁇ ⁇ equence of SEQ ID NO.: 2. Approximately the first 10 nucleotides in SEQ ID NO. : 3 are linker ⁇ from the cloning library and are not thrombospondin-4 sequence. An open rtading frame that is about 900 amino acids long (SEQ ID NO. : 4) is predicted from the nucleotide sequence of this human homolog.
  • the methionine at the 5' end of SEQ ID NO. : 3 and 4 is the beginning of the coding region.
  • the methionine is close to the 5' end and the sequence that follows represent ⁇ a rea ⁇ onable ⁇ ignal ⁇ equence.
  • the molecular architecture of the human homolog is sub ⁇ tantially identical to that of Xenopus thrombospondin-4. That is, the human homolog nucleotide sequence has the ⁇ ame structure as Xenopus thrombospondin-4 (i.e. lacks type 1 repeats and procollagen homology, has four, type 2 repeats, and has seven, type 3 repeats).
  • the pattern of expression of thrombospondin-4 in human ti ⁇ ues is markedly different from the pattern of expression of thrombospondin 1 and 2 in human tissues.
  • Northern blots of poly A+ selected RNA from adult human tis ⁇ ue ⁇ wa ⁇ performed and probed with Xenopus thrombospondin-4 and the human homolog of Xenopus thrombo ⁇ pondin-4.
  • Thrombo ⁇ pondin-4 ⁇ howed high level ⁇ of expression in human heart and skeletal muscle (Example 3). No expres ⁇ ion wa ⁇ detected in the placenta, liver or kidney.
  • Thrombo ⁇ pondin-3 had it ⁇ ⁇ tronge ⁇ t Northern blot ⁇ ignal in the lung.
  • the human gene encoding thrombospondin-4 has been cloned, isolated and expressed.
  • a general protocol is pre ⁇ ent below.
  • Thi ⁇ protocol is intended to obtain a cDNA having a complete reading frame for the human homolog of Xenopus laevi ⁇ thrombo ⁇ pondin- .
  • This objective is achieved by generating a probe to the human homolog, ⁇ creening a human cDNA library with the probe and, finally, generating a coding ⁇ equence from the ⁇ equence identified in the library.
  • a cDNA encoding Xenopus thrombo ⁇ pondin-4 was cloned by first isolating mouse thrombospondin-l, chicken thrombospondin-2 and Xenopu ⁇ thrombospondin-l clone ⁇ by ⁇ creening libraries with existing probes for other species at low stringency.
  • the resulting sequence ⁇ for the ⁇ e thrombospondin members were aligned with human thrombospondin-l and highly conserved regions were identified.
  • degenerate oligonucleotides were ⁇ ynthe ⁇ ized and u ⁇ ed as primers for the polymerase chain reaction (PCR) (SEQ ID NO. : 5 and 6; Example 1A).
  • SEQ ID NO. : 5 depicts the sequence of the forward primers and SEQ ID NO.: 6 depicts the sequence of the reverse primers.
  • PCR Polymerase chain reaction
  • Xenopu ⁇ laevi ⁇ clones (designated XS3 and XS9 : see Example IB) were u ⁇ ed to determine the nucleotide ⁇ equence of Xenopu ⁇ thrombo ⁇ pondin-4 on both strands. Since XS9 is still 650 bp smaller than the message size predicted by Northern Blot analysi ⁇ , two approache ⁇ were u ⁇ ed to complete the ⁇ equence: (i) the Xenopu ⁇ cDNA library was rescreened; and (ii) two PCR primer ⁇ that include ⁇ equences within the 5' end have were used in conjunction with two PCR primers from the polylinker to perform PCR on the library. The PCR protocol wa ⁇ that described in Example 1A. Neither approach yielded any additional sequences.
  • the approach u ⁇ ed to ⁇ creen a DNA library for the pre ⁇ ence of a thrombo ⁇ pondin-4 coding ⁇ equence corre ⁇ ponding to a human homolog includes generating preferred probes using the polymera ⁇ e chain reaction.
  • the probe ⁇ were produced by u ⁇ ing a human heart cDNA library a ⁇ a template for primer ⁇ (SEQ ID NO. : 7 and 8). Based on the degree of codon degeneracy of the predicted amino acid sequence, primers were derived from the Xenopus thrombo ⁇ pondin-4 ⁇ equence of SEQ ID NO. : 1 and 2.
  • This preferred method required identifying tissue that expresse ⁇ thrombo ⁇ pondin-4 as a source of RNA (e.g.. human heart ti ⁇ ue).
  • ti ⁇ ue ⁇ expressing the human homolog can, however, be identified by RNA analysi ⁇ , i.e.,. Northern analy ⁇ i ⁇ under low ⁇ tringency condition ⁇ . Confirmation of a human ti ⁇ ue as an RNA source and identification of additional source ⁇ of ti ⁇ ue can be accompli ⁇ hed by preparing RNA from the ⁇ elected tissue and performing Northern Blot Analysis under low stringency condition ⁇ u ⁇ ing PCR product a ⁇ the probe(s).
  • a suitable range of such stringency conditions i ⁇ de ⁇ cribed in Krause, M.H.. and Aaronson, S.A. , 1991, Method ⁇ in Enzymology 200: 546-556.
  • genomic librarie ⁇ can be ⁇ creened for the pre ⁇ ence of the human homolog coding ⁇ equence u ⁇ ing a PCR generated probe( ⁇ ).
  • the invention al ⁇ o pertain ⁇ to a more general protocol for isolating the gene for thrombospondin-4 from vertebrates, in particular from non-human vertebrates ⁇ uch as cows, pig ⁇ , monkey ⁇ and the like.
  • total mRNA can be isolated from mammalian tis ⁇ ues or from cell lines likely to express thrombospondin-4 (e.g.. cow or chimpanzee, heart mu ⁇ cle) .
  • total RNA from the selected tissue or cell culture is isolated u ⁇ ing conventional methods. Subsequent isolation of mRNA is typically accompli ⁇ hed by oligo (dT) chromotography.
  • RNA for Northern analysis is size-fractionated by electrophoresis and the RNA tran ⁇ cripts are transferred to nitrocellulose according to conventional protocols (Sambrook, J. et al . , Molecular Cloning, Cold Spring Harbor Press, N.Y. ) .
  • a labelled PCR-generated probe capable of hybridizing with the human homolog of Xenopus thrombospondin-4 can serve to identify RNA transcripts complementary to at least a portion of the human thrombospondin-4 gene. For example, if Northern analy ⁇ i ⁇ indicate ⁇ that RNA i ⁇ olated from a cow heart muscle hybridizes with the labelled probe, then a cow heart muscle cDNA library i ⁇ a likely candidate for ⁇ creening and identification of a clone containing the coding sequence for a cow homolog of thrombospondin- .
  • Northern analysi ⁇ i ⁇ u ⁇ ed to confirm the pre ⁇ ence of mRNA fragment ⁇ which hybridize to a probe corre ⁇ ponding to all or part of thrombospondin-4.
  • Northern analy ⁇ is indicates the presence and size of the tran ⁇ cript. Thi ⁇ allows one to determine whether a given cDNA clone is long enough to encompass the entire transcript or whether it i ⁇ nece ⁇ sary to obtain further cDNA clones, i.e.,. if the length of the cDNA clone is les ⁇ than the length of RNA tran ⁇ cript ⁇ as seen by Northern analy ⁇ is.
  • cDNA is not long enough, it is necessary to perform several step ⁇ ⁇ uch as: (i) rescreen the same library with the longest probes available to identify a longer cDNA; (ii) screen a different cDNA library with the longest probe; and (iii) prepare a primer-extended cDNA library using a specific nucleotide primer corresponding to a region close to, but not at, the most 5' available region. This nucleotide sequence is used to prime reverse transcription. The primer extended library is then screened with the probe corresponding to available sequences located 5' to the primer. See for example, Rupp et al. , Neuron, 6: 811-823 (1991).
  • the preferred clone of thrombospondin-4 ha ⁇ a complete coding ⁇ equence, i.e.,. one that begins with methionine, ends with a stop codon, and preferably has another in-frame stop codon 5' to the first methionine. It is also de ⁇ irable to have a cDNA that i ⁇ "full length", i.e. includes all of the 5' and 3' untranslated sequences. To as ⁇ emble a long clone from ⁇ hort fragment ⁇ , the full-length ⁇ equence i ⁇ determined by aligning the fragment ⁇ ba ⁇ ed upon overlapping ⁇ equences. Thereafter, the full-length clone is prepared by ligating the fragments together using appropriate restriction enzymes.
  • PCR-generated probes can be used in the protocol for isolating non-human mammalian homologs to thrombospondin-4.
  • probe ⁇ to be u ⁇ ed in the general method for i ⁇ olating non-human, vertebrate thrombo ⁇ pondin-4 can now include oligonucleotide ⁇ , all of which are part of the human homolog ⁇ hown in SEQ ID NO. : 3.
  • antibodie ⁇ reactive with thi ⁇ human homolog can al ⁇ o be u ⁇ ed.
  • the above-identified probe ⁇ do not require prior i ⁇ olation of RNA from a tissue expre ⁇ ing the vertebrate homolog.
  • an oligonucleotide probe typically ha ⁇ a ⁇ equence somewhat longer than that used for the PCR primers. " A longer sequence is preferable for the probe, and it is important that codon degeneracy be minimized.
  • a representative protocol for the preparation of an oligonucleotide probe for screening a cDNA library i ⁇ de ⁇ cribed in Sambrook, J. et al. Molecular Cloning, Cold Spring Harbor Pre ⁇ , New York, 1989. In general, the probe i ⁇ labelled, e.g.. P-32, and used to screen clones of a cDNA or genomic library.
  • the library can be screened using conventional immunization techniques, such as those described in Harlowe and Lane, D. (1988), Antibodies, Cold Spring Harbor Pres ⁇ , New York.
  • Antibodie ⁇ prepared u ⁇ ing purified thrombo ⁇ pondin-4 a ⁇ an immunogen are preferably fir ⁇ t te ⁇ ted for cro ⁇ reactivity with the homolog of thrombo ⁇ pondin-4 from other ⁇ pecies.
  • Other approaches to preparing antibodies for use in screening DNA librarie ⁇ , as well as for use in diagnostic and research applications, are described below.
  • the nucleic acid sequence of the human thrombospondin-4 is depicted in SEQ ID NO: 3. This sequence, its functional equivalent, or unique fragments of this sequence may be used in accordance with the invention.
  • unique fragments refers to portion ⁇ of the thrombo ⁇ pondin-4 nucleic acid ⁇ equence that find no counterpart in the known sequences of thrombospondin ⁇ -1 and -2.
  • Sub ⁇ equences comprising hybridizable portion ⁇ of the thrombo ⁇ pondin-4 ⁇ equence have use, e.g.. , in nucleic acid hybridization assays, Southern and Northern blot analyse ⁇ , etc.
  • nucleic acid sequence depicted in SEQ ID NO: 3 can be altered by mutations such as substitution ⁇ , addition ⁇ or deletion ⁇ that provide for functionally equivalent nucleic acid sequences.
  • a nucleic acid sequence is "functionally equivalent” compared with the nucleic acid sequence depicted in SEQ ID NO: 3, if it sati ⁇ fie ⁇ at lea ⁇ t one of the following conditions: (i) the nucleic acid sequence has the ability to hybridize to thrombospondin-4, but it doe ⁇ not nece ⁇ arily hybridize to thrombo ⁇ pondin-4 with an affinity that i ⁇ the ⁇ ame as that of the natural thrombospondin-4 nucleic acid ⁇ equence; and/or (ii) the nucleic acid can ⁇ erve as a probe to distingui ⁇ h between thrombospondin-4 and the other known thrombospondins.
  • a probe that can "di ⁇ tingui ⁇ h” between thrombo ⁇ pondin-4 and the other thrombospondins refers to a probe that will hybridize to a thrombospondin nucleic acid ⁇ equence that encode ⁇ for a polypeptide having has at least four, type 2 domains; that lacks any type 1 domains and/or that lacks a region of procollagen homology.
  • the term "probe” refers to a ligand of known qualities that can bind selectively to a target.
  • the term “probe” refers to a strand of nucleic acid having a base sequence complementary to a target strand.
  • nucleic acid ⁇ equence of thrombo ⁇ pondin-4 is now known, tho ⁇ e of ordinary ⁇ kill in the art can readily determine tho ⁇ e nucleic acid ⁇ equences of thrombospondin-4 that are not homologous to any other nucleic acid sequence, including the other thrombospondin sequence ⁇ . These non-homologous sequences, and peptides encoded by them, are referred to a ⁇ "unique" fragment ⁇ and are meant to be included within the ⁇ cope of the pre ⁇ ent invention.
  • nucleic acid sequences may be used in the practice of the present invention. These include, but are not limited to, sequence ⁇ compri ⁇ ing all or portion ⁇ of the thrombo ⁇ pondin-4 gene ⁇ depicted in SEQ ID NO: 1 and 3 which are altered by the sub ⁇ titution of different codon ⁇ that encode the ⁇ ame amino acid residue within the sequence, thu ⁇ producing a silent change. Such altered sequences are regarded as equivalents of the specifically claimed sequence ⁇ .
  • Thrombospondin-4 proteins or unique fragments or derivatives thereof include, but are not limited to, those containing a ⁇ a primary amino acid sequence all, or unique parts of the amino acid residue ⁇ ⁇ ub ⁇ tantially a ⁇ depicted in SEQ ID NO.: 2 and SEQ ID NO.: 4, including altered sequences in which functionally equivalent amino acid residue ⁇ are ⁇ ub ⁇ tituted for re ⁇ idue ⁇ within the ⁇ equence, re ⁇ ulting in a ⁇ ilent change.
  • an amino acid is "functionally equivalent" compared with the sequence ⁇ depicted in SEQ ID NOS.
  • amino acid sequence contains one or more amino acid residues within the sequence which can be ⁇ ub ⁇ tituted by another amino acid of a ⁇ imilar polarity which act ⁇ a ⁇ a functional equivalent.
  • Sub ⁇ titutes for an amino acid within the sequence may be ⁇ elected from other member ⁇ of the cla ⁇ to which the amino acid belong ⁇ .
  • the non-polar (hydrophobic) amino acid ⁇ include alanine, leucine, i ⁇ oleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, ⁇ erine, threonine, cy ⁇ teine, tyro ⁇ ine, a ⁇ paragine, and glutamine.
  • the po ⁇ itively charged (ba ⁇ ic) amino acid ⁇ include arginine, ly ⁇ ine and hi ⁇ tidine.
  • the negatively charged (acidic) amino acids include asparatic acid and glutamic acid.
  • thrombospondin-4 protein ⁇ or unique fragment ⁇ or derivative ⁇ thereof which are differentially modified during or after tran ⁇ lation, e.g.. , by phosphorylation, glycosylation, cro ⁇ slinking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand, (Ferguson et al. , 1988, Ann. Rev. Biochem. 57:285-320) .
  • the recombinant thrombo ⁇ pondin-4- encoding nucleic acid ⁇ equence ⁇ of the invention may be engineered ⁇ o as to modify processing or expres ⁇ ion of thrombo ⁇ pondin-4.
  • the thrombo ⁇ pondin-4 gene may be combined with a promoter ⁇ equence and/or a ribo ⁇ ome binding ⁇ ite u ⁇ ing well characterized method ⁇ , and thereby facilitate harve ⁇ ting or bioavailability.
  • a given thrombospondin-4 can be mutated in vitro or in vivo, to create variations in coding regions and/or form new restriction endonuclease site ⁇ or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be u ⁇ ed including, but not limited to, m vitro ⁇ ite-directed mutagene ⁇ i ⁇ (Hutchin ⁇ on, et al. , 1978, J. Biol . Chem. 253:6551), u ⁇ e of TAB® linkers (Pharmacia), PCR-directed mutagenesis, and the like.
  • the thrombospondin-4 of the invention also includes non-human homologs of the amino acid sequence of SEQ ID NO: 4.
  • the thrombospondin-4 peptide ⁇ of the invention may be prepared by recombinant nucleic acid expre ⁇ ion techniques or by chemical synthesis using ⁇ tandard peptide synthesis techniques.
  • Nucleic acid sequences complementary to DNA or RNA sequences encoding thrombospondin-4 or a functionally active portion thereof are al ⁇ o provided.
  • animal ⁇ particularly transgenic animals, RNA tran ⁇ cripts of a de ⁇ ired gene or gene ⁇ may be translated into polypeptide products having a host of phenotypic actions.
  • antisense thrombospondin-4 oligonucleotides can be synthe ⁇ ized.
  • the ⁇ e oligonucleotide ⁇ may have activity in their own right, such as antisense reagents which block translation or inhibit RNA function.
  • the DNA sequence can be in an inverted orientation which gives rise to a negative sense (“antisense”) RNA on transcription.
  • antisense RNA on transcription.
  • This antisense RNA is not capable of being translated to the desired thrombospondin-4 product, a ⁇ it i ⁇ in the wrong orientation and would give a nonsensical product if translated.
  • the present invention also permits the expre ⁇ sion, isolation, and purification of the thrombo ⁇ pondin-4 polypeptide.
  • a thrombospondin-4 gene may be cloned or subcloned u ⁇ ing any method known in the art.
  • a large number of vector-host ⁇ y ⁇ tem ⁇ known in the art may be u ⁇ ed.
  • Po ⁇ ible vector ⁇ include, but are not limited to, co ⁇ mid ⁇ , pla ⁇ mid ⁇ or modified viru ⁇ es, but the vector system must be compatible with the host cell used.
  • Viral vectors include, but are not limited to, vaccinia viru ⁇ , or lambda derivatives.
  • Plasmid ⁇ include, but are not limited to, pBR322, pUC, or Blue ⁇ cript® (Stratagene) pla ⁇ mid derivative ⁇ .
  • Recombinant thrombo ⁇ pondin-4 molecule ⁇ can be introduced into ho ⁇ t cell ⁇ via tran ⁇ formation, tran ⁇ fection, infection, electroporation, etc..
  • introduction of thrombospondin-4 molecules into a ho ⁇ t is accomplished using a vector containing thrombospondin DNA under control by regulatory regions of the DNA that function in the ho ⁇ t cell.
  • the cDNA that corre ⁇ pond ⁇ to the entire coding region of human thrombo ⁇ pondin-4, con ⁇ tructed from two overlapping clone ⁇ , wa ⁇ moved to the mammalian expre ⁇ sion vector, pLEN-PT (See Example 4).
  • the details of the experimental approach for transfection, selection and characterization of the expres ⁇ ed thrombo ⁇ pondin-4 protein were similar to those that have been u ⁇ ed previou ⁇ ly for human thrombospondin-l (see Biochemistry, 31: 1173-1180 (1992)), the entire content ⁇ of which are incorporated herein by reference.
  • thrombo ⁇ pondin-4 protein may be i ⁇ olated and purified by ⁇ tandard method ⁇ including chromatography (e.g., ion exchange, affinity, and ⁇ izing column chromatography) , centrifugation, differential ⁇ olubility, or by any other ⁇ tandard technique for the purification of protein ⁇ .
  • thrombo ⁇ pondin-4 protein may be i ⁇ olated by binding to an affinity column compri ⁇ ing antibodie ⁇ to thrombospondin-4 bound to a stationary ⁇ upport.
  • antibodies is meant to include monoclonal a.itibodies, polyclonal antibodies and antibodies prepared by recombinant nucleic acid techniques that are selectively reactive with thrombospondin-4.
  • selectively reactive refers to those antibodies that react with thrombospondin-4, and do not react with the other thrombo ⁇ pondin ⁇ .
  • Antibodie ⁇ include antibodie ⁇ rai ⁇ ed against Xenopus thrombospondin-4 polypeptide (SEQ ID NO. : 2) and intended to cross-react with the human homolog. These antibodies are useful for diagnostic application ⁇ .
  • antibodie ⁇ include antibodies raised against Xenopus thrombospondin-4, which antibodie ⁇ are generally u ⁇ ed for re ⁇ earch purpo ⁇ es.
  • the ⁇ e antibodie ⁇ include tho ⁇ e rai ⁇ ed against short, synthetic peptides of the Xenopu ⁇ thrombo ⁇ pondin-4 ⁇ equence.
  • a peptide immunogen is first attached to a carrier to enhance the immunogenic response.
  • the peptide immunogen can correspond to any portion of the amino acid sequence of the human thrombospondin-4 protein or to variants of the sequence, such as the amino acid sequence ⁇ corresponding to the primers and probes described, certain peptides are more likely than others to provoke an immediate respon ⁇ e. For example, a peptide including the C-terminal amino acid is more likely to generate an antibody respon ⁇ e.
  • preparing antibodie ⁇ reactive with the human homolog include: immunizing an animal with a protein expre ⁇ ed by a bacterial or eucaryotic cell, which cell include ⁇ the coding ⁇ equence for: (i) all or part of the human homolog; or (ii) the coding ⁇ equence for all or part of the Xenopu ⁇ thrornbo ⁇ pondin-4 protein.
  • Antibodie ⁇ can al ⁇ o be prepared by immunizing an animal with whole cell ⁇ that are expre ⁇ sing all or a part of a cDNA encoding the thrombospondin-4 protein.
  • the amino acid sequence of thrombospondin-4 may be analyzed in order to identify portions of the molecule which may be associated with increased immunogenicity.
  • the amino acid sequence may be subjected to computer analysis to identify surface epitopes which pre ⁇ ent computer-generated plot ⁇ of antigenic index, an amphiphilic helix, amphiphilic ⁇ heet, hydrophilicity, and the like.
  • the deduced amino acid ⁇ equence ⁇ of thrombo ⁇ pondin-4 from different species could be compared, and relatively non-homologous region ⁇ identified. These non-homologous regions would be more likely to be immunogenic across various species.
  • any technique which provides for the production of antibody molecules by continuous cell lines and culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (Nature, 256: 495-497), a ⁇ well a ⁇ the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , Immunology Today, 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodie ⁇ , and the like, are within the scope of the present invention.
  • SCA single-chain antibody
  • the monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other specie ⁇ ) monoclonal antibodie ⁇ .
  • the pre ⁇ ent invention provide ⁇ for antibody molecule ⁇ a ⁇ well as fragments of such antibody molecules.
  • the pre ⁇ ent invention provide ⁇ for a ⁇ ay system ⁇ in which activity or activitie ⁇ re ⁇ ulting from expo ⁇ ure to a peptide or non-peptide compound may be detected by mea ⁇ uring a phy ⁇ iological response to the compound in a cell or cell line which expresses the thrombo ⁇ pondin-4 molecules of the invention.
  • a "physiological response” may comprise any biological response, including but not limited to transcriptional activation of certain nucleic acid sequences (e.g.. promoter/enhancer elements as well as structural genes), translation, or phosphorylation, the induction of secondary processes, and morphological changes, such as neurite sprouting.
  • the present invention thus provides for the development of novel as ⁇ ay sy ⁇ tems which may be utilized in the screening of compound ⁇ .
  • Target cells expres ⁇ ing thrombo ⁇ pondin-4, which bind to the compound ⁇ may be produced by tran ⁇ fection with thrombo ⁇ pondin-4-encoding nucleic acid.
  • target cell line ⁇ Once target cell line ⁇ are produced or identified, it may be de ⁇ irable to ⁇ elect for cells which are exceptionally sensitive to a particular compound.
  • Such target cells may express large amounts of thrombospondin-4; target cells expressing a relative abundance of thrombospondin-4 could be identified by selecting target cells which bind to high levels of the compound, for example cells which, when incubated with a compound/tag and subjected to immunofluorescence assay, produce a relatively higher degree of fluorescence.
  • cell lines which are exceptionally sensitive to a compound may exhibit a relatively strong biological response, such as a sharp increase in immediate early gene products such as c-fos or c-jun, in re ⁇ pon ⁇ e to thrombospondin-4 binding.
  • the present invention provide ⁇ for methods of screening for low levels of thrombospondin-4 activity.
  • the present invention provides for thrombospondin-4 target cell ⁇ which are engineered to be highly ⁇ en ⁇ itive to thrombospondin-4 binding compounds.
  • the thrombospondin-4 gene cloned according to the methods set forth above, may be inserted into cells which naturally express thrombospondin-4 ⁇ uch that the recombinant thrombo ⁇ pondin-4 gene i ⁇ expre ⁇ ed at high levels .
  • thrombospondin ⁇ generally bind large amount ⁇ of calcium
  • cell ⁇ expressing thrombospondin-4 may find u ⁇ ed in calcium bioa ⁇ ay method ⁇ , particularly in clinical setting ⁇ where elevated blood calcium may be indicative of parathyroid or bone dy ⁇ function.
  • the pre ⁇ ent invention al ⁇ o provide ⁇ for experimental model ⁇ y ⁇ tem ⁇ for ⁇ tudying the phy ⁇ iological role of the native thrombo ⁇ pondin-4.
  • thrombospondin-4 protein, peptide fragment, or a derivation thereof may be either supplied to the system or produced within the system.
  • Such model systems could be used to study the effects of thrombospondin-4 excess or depletion.
  • the experimental model systems may be used to study the effects of increased or decreased respon ⁇ e to ligand in cell or tissue cultures, in whole animals, or in particular cells or tis ⁇ ue ⁇ within whole animal ⁇ or tissue culture sy ⁇ tem ⁇ , or over ⁇ pecified time interval ⁇ (including during embryogenesis) .
  • a recombinant thrombo ⁇ pondin-4 gene may be u ⁇ ed to inactivate the endogenou ⁇ gene by homologou ⁇ recombination, and thereby create a thrombospondin-4 deficient cell, tissue, or animal.
  • a recombinant thrombospondin-4 gene may be engineered to contain an insertional mutation (e.g.. the neo gene) which, when inserted, inactivates transcription of thrombospondin-4.
  • Such a construct under the control of a suitable promoter operatively linked to the thrombospondin-4 gene, may be introduced into a cell by a technique such a ⁇ tran ⁇ fection, tran ⁇ duction, injection, etc..
  • stem cells lacking an intact thrombospondin-4 gene may generate transgenic animal ⁇ deficient in thrombo ⁇ pondin-4.
  • the endogenous thrombo ⁇ pondin-4 gene of a cell may be inactivated by homologou ⁇ recombination with a mutant thrombo ⁇ pondin-4 gene to form a transgenic animal lacking the ability to express thrombospondin-4.
  • a con ⁇ truct can be provided that, upon transcription, produces an "anti-sense" nucleic acid sequence which, upon tran ⁇ lation, will not produce the required thrombo ⁇ pondin-4 protein.
  • a "transgenic animal” is an animal having cells that contain DNA which has been artificially in ⁇ erted into a cell, which DNA become ⁇ part of the genome of the animal which develop ⁇ from that cell.
  • the preferred DNA encodes for thrombospondin-4 and may be entirely foreign to the tran ⁇ genic animal or may be homologous to the natural thrombospondin-4 of the transgenic animal, but which is inserted into the animal's genome at a location which differs from that of the natural homolog.
  • thrombospondin-4 expre ⁇ ion may be reduced by providing thrombospondin-4 expres ⁇ ing cell ⁇ , preferably in a tran ⁇ genic animal, with an amount of thrombo ⁇ pondin-4 anti- ⁇ en ⁇ e RNA or DNA effective to reduce expres ⁇ ion of thrombo ⁇ pondin-4 protein.
  • a tran ⁇ genic animal (preferably a non-human mammal) can al ⁇ o be provided with a thrombospondin-4 DNA ⁇ equence that also encodes a repressor protein (e.g. , the E.coli lac repres ⁇ or) .
  • the repressor protein can bind to a specific DNA sequence of thrombospondin-4, thereby reducing (“repre ⁇ ing") the level of tran ⁇ cription of thrombo ⁇ pondin-4.
  • Tran ⁇ genic animal ⁇ of the invention which have attenuated level ⁇ of thrombo ⁇ pondin-4 expre ⁇ ion have general applicability to the field of transgenic animal generation, as they permit control of the level of expres ⁇ ion of genes.
  • thrombo ⁇ pondin-4 probe ⁇ may be used to identify cell ⁇ and tissues of transgenic animals which lack the ability to transcribe thrombospondin-4.
  • Thrombospondin-4 expre ⁇ ion may be evidenced by tran ⁇ cription of thrombo ⁇ pondin-4 mRNA or production of thrombo ⁇ pondin-4 protein, detected u ⁇ ing probes which can distingui ⁇ h thrombo ⁇ pondin-4 from thrombo ⁇ pondins -1 and -2, as de ⁇ cribed above.
  • One variety of probe which may be used to detect thrombospondin-4 expres ⁇ ion is a nucleic acid probe, containing a sequence encoding for at least four, type 2 domains.
  • the probe can contain a thrombospondin sequence of the invention lacking type 1 domains or procollagen homology. Detection of thrombospondin-4-encoding mRNA may be easily accomplished by any method known in the art, including, but not limited to, in situ hybridization, Northern blot analysis, or PCR related techniques.
  • Another variety of probe which may be used is anti-thrombospondin-4 antibody.
  • the above-mentioned probes may be used experimentally to identify cells or tissues which hitherto had not been shown to express thrombospondin-4. Furthermore, these methods may be u ⁇ ed to identify the expre ⁇ ion of thrombospondin-4 by aberrant tissues, such as malignancies.
  • EXAMPLE 1 Cloning the Xenopus thrombospondin-4 gene
  • the reaction mixture was heated to 95°C for 5 minutes and then equilibrated to the annealing temperature (37-48°C) .
  • TAQ polymerase 2.5 units was added and the sample was heated to 72°C for 3 minutes.
  • the amplification cycles were (1) incubate at 94°C for l minute and 20 second ⁇ , (2) incubate at 48°C for 2 minutes, (3) ramp to 72°C over 2 minutes, and (4) incubate at 72°C for 3 minutes. This cycle was repeated 30-40 times; finally the sample was incubated at 72°C for 7 minutes.
  • the PCR products were separated by agaro ⁇ e gel electrophoresis and the appropriately sized products were ⁇ ubcloned into pBlue ⁇ cript KS or SK (Stratagene, LaJolla, CA) .
  • the Xenopus laevis stage 45 library was screened with the PCR product as the probe.
  • the probe was labeled with digoxigenin-dUTP, and hybridization performed using the Genius Kit® following the supplier ' ⁇ protocol ⁇ (Boehringer Mannheim, Indianapoli ⁇ , IN) .
  • Po ⁇ itive plaque ⁇ were taken through ⁇ ucce ⁇ ive round ⁇ of screening with the same probe at progressively lower plague densities.
  • the purified plaques were amplified to yield high titre plate ⁇ tock ⁇ .
  • Becau ⁇ e the Xenopus laevi ⁇ library can be constructed in the ⁇ ZAPII vector pBluescript II SK, the in ⁇ ert ⁇ are exci ⁇ ed with helper phage and grown up directly following the ⁇ upplier' ⁇ protocol ⁇ (Stratagene). BamHI and EcoRI fragments were subcloned into pBlue ⁇ cript II SK and KS. All sequencing was done by the chain termination method of Sanger et al . (1977) with Sequenase reagents (U.S.. Biochemical Corp., Cleveland, OH) .
  • the largest clone that we obtained from screening the Xenopus library was 2.8 kb.
  • two oligonucleotides that corresponded to the bottom strand sequence near the 5' end were ⁇ ynthe ⁇ ized.
  • the oligonucleotide ⁇ and the pBluescript SK and primers (Stratagene, LaJolla, CA) were u ⁇ ed as PCR primer ⁇ with the library as the template.
  • Degenerate PCR using the Xenopus laevis stage 45 library has produced four distinct sequences that are related to the thrombospondins.
  • Two of the four sequences correspond to the two copie ⁇ of the thrombospondin-l gene that are pre ⁇ ent in tne Xenopus genome (Urry et al. , supra 1991) . In ⁇ ome cases, both copies of the gene are expressed (e.g., J. Biol Chem. 263: 5333-5340, DeSimone and Hynes, 1988). To date, the thrombospondin-l sequences represent the majority of the products that we have obtained. However, two PCR products comprise sequence ⁇ that are related to, but clearly di ⁇ tinct from thrombospondin-l . The sequences of these two PCR products (labeled TSP-4A and TSP-4B in FIG. 3, below) are very similar to each other suggesting that they represent the two copies of a newly identified gene in the Xenopus genome.
  • a probe was prepared from the PCR product and used to screen the library.
  • a screen of 120,000 plaques produced four positive clones that range in size from 1.7 kb to 2.3 kb (FIG. 3, XF1-XF4).
  • FIG. 3 the re ⁇ triction map ⁇ of the clones indicate that two distinct gene product ⁇ can be identified.
  • the longe ⁇ t clone for each gene ha ⁇ been ⁇ equenced on both ⁇ trand ⁇ .
  • the ⁇ equence of the PCR products is included in the ⁇ equence ⁇ of the ⁇ e clones.
  • Clone XS9 is approximately 469 nucleotide smaller than the message and the reading frame is open at the 5' end of the predicted amino acid ⁇ equence.
  • the library was rescreened in a third round of screening with the EcoRI to BamHI fragment of XS9.
  • Four additional clones have been isolated (XT11-XT14) however, they did not contain additional nucleotide sequence.
  • a Xenopus laevis ⁇ tage 22 library (a gift of Dr. Douglas Melton) wa ⁇ ⁇ creened. Restriction endonuclease mapping indicated that one of the clones (XM15; not shown in Fig. 3) contained additional 5' end sequence for the TSP-4B family.
  • Example 2 I ⁇ olating the human homolog of Xenopus thrombospondin-4
  • Example 3A Northern blot analysis of eight adult human tis ⁇ ue ⁇ indicated that thrombospondin-4 is expressed in high levels in the heart and skeletal muscle (Example 3A) .
  • a heart cDNA library (the generou ⁇ gift of Dr. Paul Allen) ha ⁇ been u ⁇ ed a ⁇ the template for polymera ⁇ e chain reaction (PCR) with the primers 89PCR (SEQ ID NO.: 7) and 90PCR (SEQ ID NO.: 8).
  • the product of the PCR reaction has been cloned into pBluescript vectors (Stratagene) .
  • the library ha ⁇ been ⁇ creened with the PCR product a ⁇ the probe.
  • Clones have been isolated and characterized in terms of the ⁇ ite ⁇ for endonuclease and nucleotide sequence. The longest clone is approximately 2kb.
  • Computer-a ⁇ i ⁇ ted progre ⁇ ive ⁇ equence alignment has been u ⁇ ed to con ⁇ truct a phylogenetic tree of the thrombo ⁇ pondin ⁇ equences. The results of this analysis are con ⁇ istent with the hypothesis that the clones that have been isolated from the human heart library represent the human homolog of Xenopus thrombospondin-4.
  • the Xenopus thrombospondin-4 clone XF3 was digested with EcoRI and Xhol and the insert purified. A variety of probes were used in the Northern analy ⁇ i ⁇ .
  • a human thrombo ⁇ pondin-l probe wa ⁇ the human full-length cDNA (Lawler et al. , 1992).
  • a human thrombo ⁇ pondin-3 probe was developed a ⁇ follows: A genomic clone GPEM-2 containing human thrombospondin-3 wa ⁇ kindly provided by Dr.Sandra Gendler (Imperial Cancer Re ⁇ earch Fund, London; Lancaster et al . , Biochem. Biophy ⁇ . Re ⁇ . Comm. , 173: 1019-1-29 1990). BamHI fragment ⁇ of GPEM-2 were ⁇ ubcloned into pBlue ⁇ cript KS and the end ⁇ of each clone were sequenced.
  • One of the ⁇ e clones contained sequence ⁇ that were homologou ⁇ to the 3' end of thrombospondin-l, 2 and 4. Based on this homology, the position of the 5' end of the last exon was determined. The 3' end of this exon was taken to be the polyadenylation signal. Oligonucleotide ⁇ that primed at the 5' and 3' end ⁇ of the la ⁇ t exon were u ⁇ ed to amplify and clone a 293 bp DNA segment that correspond ⁇ to the la ⁇ t exon of human thrombospondin-3.
  • a third probe was a ⁇ -actin probe (Clontech, Palo Alto, CA) .
  • the PCR product for the last exon of thrombospondin-3 and the actin probe were radiolabeled directly with the Multiprime DNA Labelling System (Amersham, Arlington Heights, IL).
  • a Northern blot that was prepared with Poly A+ RNA from adult human heart, brain, placenta, lung, liver, ⁇ keletal muscle, kidney and pancreas was obtained from Clontech. The blot was prehybridized and hybridized as de ⁇ cribed previou ⁇ ly (Lawler and Hynes, ⁇ upra 1986).
  • the lanes are represented a ⁇ : (a) adult human heart; (b) adult human brain; (c) adult human placenta; (d) adult human lung; (e) adult human liver; (f) adult human ⁇ keletal muscle; (g) adult human kidney; and (h) adult human pancrea ⁇ .
  • Thrombo ⁇ pondin-4 (TSP-4) ⁇ howed a re ⁇ tricted pattern of expre ⁇ ion a ⁇ thi ⁇ expre ⁇ ion i ⁇ vi ⁇ ualized u ⁇ ing a 2.2kb fragment of Xenopus thrombospondin-4.
  • the po ⁇ ition ⁇ and ⁇ ize ⁇ of the markers are indicated on the left.
  • Varying levels of thrombo ⁇ pondin-1 were ob ⁇ erved in all of the ti ⁇ ues on the blot. In this case, the principal me ⁇ age was 6.0 kb with faint bands at 4.5 and 3.6 kb.
  • the new clone ⁇ provided additional sequence ⁇ o that the total ⁇ equence i ⁇ now 3074 bp.
  • the 5' end include ⁇ a methionine re ⁇ idue that is followed by a 21 amino acid sequence that could repre ⁇ ent a ⁇ ignal ⁇ equence.
  • Two human thrombospondin-4 clones were used to construct a full-length coding region cDNA.
  • An EcoRV fragment of D9892 tt9 containing DNA (corresponding to nucleotides 1639 to 3074 of SEQ ID NO. 3) was cloned into EcoRv cut D9892 #11 containing DNA (corresponding to nucleotides 1 to 1638 of SEQ ID NO. 3).
  • DNA was made from tran ⁇ formant ⁇ and wa ⁇ cut with EcoRI to determine the orientation of the in ⁇ erted DNA.
  • the DNA was cut with XmnI followed by EcoRI to purify a full-length cDNA for thrombospondin-4 that was cloned into the EcoRI site of pLEN-PT.
  • each construct is moved from M13mp8 to the mammalian expres ⁇ ion vector pLEN-PT u ⁇ ing Xbal ⁇ ite ⁇ .
  • the cell ⁇ are grown in 100-mm di ⁇ hes until they are approximately 50% confluent.
  • the cells are washed once with 3 mL of OptiMEMI reduced serum medium (Gibco Laboratories, Gaither ⁇ burg, M.D.) containing no ⁇ erum, and then 3 mL of the ⁇ ame medium i ⁇ placed in the dish.
  • the DNA-Lipofectin mixture is added to the dishes with continuous swirling. After 24 h, the medium is changed to DME containing 10% FBS. After 48 h, the cells were trypsinized and replated in DME containing 10% FBS and 1 mg/mL Geneticin (G418, Gibco Laboratories).
  • G418-resi ⁇ tant colonie ⁇ are ⁇ ubcloned, or the cells allowed to grow and handled as pools of G418-resistant clones.
  • the cells are grown to confluence in four T75 flasks. Fresh medium i ⁇ placed on the cell ⁇ , and the cell ⁇ are grown for 48 h. The conditioned medium i ⁇ removed, and DFP added to 1 mM and PMSF added to 5 mM. After ⁇ everal hour ⁇ at 0°C, the culture ⁇ upernatant ⁇ are frozen and stored at -20°C.
  • the ⁇ pecific methodology for construction of the fusion proteins varies depending upon the availability of restriction endonuclease ⁇ ites. In general, endonuclease sites are chosen in close proximity to the region of cDNA of interest.
  • the insert i ⁇ purified by preparative agaro ⁇ e gel electrophore ⁇ i ⁇ .
  • the insert is blunted and the appropriate EcoRI linker is added ⁇ o that the reading frame of the insert is the same as that of the ⁇ -galacto ⁇ ida ⁇ e gene.
  • the in ⁇ ert i ⁇ cut with EcoRI and ligated into ⁇ gtll by procedure ⁇ recommended by the supplier (Promega Biotec., Protoclone ⁇ gtll System) .
  • Ly ⁇ ogen ⁇ of the Y1089 ⁇ train are ⁇ elected by their ability to grow at 30°C but not at 42°C.
  • fusion protein To prepare fusion protein, an overnight grow at 30°C is diluted 1:10 (v/v) and grown for an additional hour at 30°C. The culture is incubated at 45°C for 15 minutes and 10 ⁇ g/ml of isopropyl ⁇ -D-thiogalactopyrano ⁇ ide i ⁇ added. The cultures are incubated for l to 2 hours at 37°C. The cell ⁇ are pelleted by centrifugation and resuspended in 100 mM Tris (pH 8.0), 0.25 M NaCl and 0.2 mg/ml lysozyme (Sigma). After 30 minutes at 0°C, the sample i ⁇ rapidly frozen and thawed twice and then ⁇ onicated to di ⁇ rupt the cell ⁇ .
  • the sample is centrifuged and the ⁇ upernatant i ⁇ applied to an anti-beta-galacto ⁇ ida ⁇ e antibody affinity column (Promega Biotec, Proto ⁇ orb, lacZ Immuno Affinity Ad ⁇ orbent).
  • the bound fu ⁇ ion protein i ⁇ eluted with 0.1 M NaHCO_/Na 2 CO_ (pH 10.8) and dialyzed to neutral pH.
  • a glutathione S-tran ⁇ fera ⁇ e fusion protein is used as an antigen to rai ⁇ e a polyclonal rabbit anti-Xenopu ⁇ laevis thrombospondin-4 antibody.
  • the fusion protein is expressed and purified according to establi ⁇ hed procedures (Current Protocols in Molecular Biology, John Wiley and Sons). The fu ⁇ ion protein is still bound to glutathione-agarose bead ⁇ when it i ⁇ used as an antigen.
  • the antibody to human thrombospondin-4 can be produced by preparing a peptide fragment of human thrombo ⁇ pondin-4 believed to be immunogenic.
  • a preferred sequence i ⁇ the ⁇ equence of the la ⁇ t 14 amino acid ⁇ that is predicted from the cDNA sequence of SEQ ID NO. : 10 (FQEFQTQNFDRFDN) .
  • the rabbit ⁇ will receive a ⁇ ubcutaneou ⁇ boo ⁇ ter injection after 4-6 week ⁇ of purified antigen emul ⁇ ified in Freund' ⁇ incomplete adjuvant and are boosted once each month until a good titre of antibody is obtained. Rabbits are bled 10 days after boosting.
  • DNA can be injected into the pronucleu ⁇ of a fertilized egg before fu ⁇ ion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo) following the initiation of cell division (Brinster et al., Proc. Nat. Acad. Sci. USA, 82: 4438-4442 (1985)).
  • Embryos can be infected with viruse ⁇ , e ⁇ pecially retroviru ⁇ es, modified to bear thrombospondin-4 genes of the invention.
  • Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate thrombospondin-4 genes of the invention.
  • a transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term.
  • mice suitable for tran ⁇ genic experiment ⁇ can be obtained from standard commercial source ⁇ ⁇ uch as Charles River (Wilmington, MA), Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapoli ⁇ , IN), etc. Swi ⁇ Web ⁇ ter female mice are preferred for embryo retrieval and transfer. B6D2F-. males can be used for mating and vasectomized Swi ⁇ s Webster stud ⁇ can be u ⁇ ed to ⁇ timulate p ⁇ eudopregnancy. Va ⁇ ectomized mice and rat ⁇ can be obtained from the ⁇ upplier.
  • mice ⁇ ix week ⁇ of age are induced to superovulate with a 5 IU injection (0.1 cc, ip) of pregnant mare ⁇ erum gonadotropin (PMSG; Sigma) followed 48 hours later by a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG; Sigma) .
  • Females are placed with male ⁇ immediately after hCG injection.
  • Randomly cycling adult female mice are paired with va ⁇ ectomized male ⁇ . Swi ⁇ Web ⁇ ter or other comparable ⁇ trains can be used for thi ⁇ purpo ⁇ e.
  • Recipient females are mated at the same time a ⁇ donor female ⁇ .
  • the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight.
  • the oviducts are expo ⁇ ed by a ⁇ ingle midline dorsal incision. An incision is then made through the body wall directly over the oviduct.
  • the ovarian bursa is then torn with watchmakers forceps.
  • Embryos to be tran ⁇ ferred are placed in DPBS and in the tip of a tran ⁇ fer pipet (about 10-12 e bryo ⁇ ) .
  • the pipet tip i ⁇ in ⁇ erted into the infundibulum and the embryo ⁇ tran ⁇ ferred. After the tran ⁇ fer, the incision is closed by two sutures.
  • transgenic rats The procedure for generating transgenic rats is similar to that of mice See Hammer et al. , Cell, 63:1099-1112 (1990).
  • Thirty day-old female rat ⁇ are given a ⁇ ubcutaneou ⁇ injection of 20 IU of PMSG (0.1 cc) and 48 hours later each female placed with a proven male.
  • 40-80 day old female ⁇ are placed in cage ⁇ with va ⁇ ectomized males. These will provide the foster mother ⁇ for embryo transfer.
  • the next morning females are checked for vaginal plugs .
  • Females who have mated with va ⁇ ectomized male ⁇ are held aside until the time of transfer.
  • Donor female ⁇ that have mated are ⁇ acrificed (C0 2 a ⁇ phyxiation) and their oviducts removed, placed in DPSS with 0.5% BSA and the embryo ⁇ collected. Cumulu ⁇ cells ⁇ urrounding the embryo ⁇ are removed with hyaluronida ⁇ e (1 mg/ml). The embryos are then washed and placed in EBSS (Earle's balanced ⁇ alt ⁇ olution) containing 0.5% BSA in a 37.5°C incubator until the time of microinjection.
  • EBSS Earle's balanced ⁇ alt ⁇ olution
  • the live embryo ⁇ are moved to DPBS for tran ⁇ fer into fo ⁇ ter mother ⁇ .
  • the foster mothers are anesthetized with ketamine (40 mg/kg, ip) and xylazine (5 mg/kg, ip) .
  • a dor ⁇ al midline inci ⁇ ion is made through the skin and the ovary and oviduct are exposed by an inci ⁇ ion tarough the mu ⁇ cle layer directly over the ovary.
  • the ovarian bursa is torn, the embryos are picked up into the transfer pipet, and the tip of the transfer pipet is inserted into the infundibulum.
  • Approximately 10-12 embryos are transferred into each rat oviduct through the infundibulum.
  • the incision i ⁇ then clo ⁇ ed with sutures, and the foster mothers are housed singly.
  • a clone containing the thrombospondin-4 gene of the invention i ⁇ co-tran ⁇ fected with a gene encoding neomycin re ⁇ i ⁇ tance.
  • Transfection is carried out by any one of several methods well known to those of ordinary skill in the art (E.J. Robertson, supra) . Calcium phosphate/DNA precipitation, direct injection, and electroporation are the preferred methods.
  • cell ⁇ are fed with ⁇ election medium containing 10% fetal bovine ⁇ erum in DMEM ⁇ upplemented with G418 (between 200 and 500 ⁇ g/ml biological weight) .
  • Colonies of cells resi ⁇ tant to G418 are i ⁇ olated using cloning rings and expanded.
  • DNA is extracted from drug resi ⁇ tant clone ⁇ and Southern blotting experiment ⁇ u ⁇ ing a tran ⁇ gene- ⁇ pecific DNA probe are u ⁇ ed to identify tho ⁇ e clone ⁇ carrying the thrombo ⁇ pondin-4 ⁇ equence ⁇ .
  • PCR method ⁇ are used to identify the clones of interest.
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the proces ⁇ of homologou ⁇ recombination. Copecchi, Science, 244: 1288-1292 (1989). Direct injection re ⁇ ult ⁇ in a high efficiency of integration. De ⁇ ired clones are identified through PCR of DNA prepared from pools of injected ES cell ⁇ . Positive cell ⁇ within the pool ⁇ are identified by PCR ⁇ ub ⁇ equent to cell cloning. DNA introduction by electroporation is le ⁇ efficient and requires a ⁇ election step. Methods for positive selection of the recombination event (i.e. , neo resi ⁇ tance) and dual po ⁇ itive-negative ⁇ election (i.e.
  • Naturally cycling or ⁇ uperovulated female mice mated with male ⁇ are u ⁇ ed to harve ⁇ t embryo ⁇ for the implantation of ES cells. It is desirable to use the C57BL165 strain for thi ⁇ purpose when using mice. Embryos of the appropriate age are recovered approximately 3.5 day ⁇ after ⁇ ucce ⁇ ful mating. Mated females are sacrificed by C0 2 asphyxiation or cervical dislocation and embryo ⁇ are flu ⁇ hed from exci ⁇ ed uterine horns and placed in Dulbecco's modified e ⁇ ential medium plus 10% calf serum for injection with ES cells. Approximately 10-20 ES cells are injected into blastocysts using a glas ⁇ microneedle with an internal diameter of approximately 20 ⁇ m.
  • Tran ⁇ fer of Embryo ⁇ to Receptive Female ⁇ Randomly cycling adult female mice are paired with vasectomized males. Mouse strains such as Swiss Webster, ICR or others can be used for this purpose. Recipient females are mated such that they will be at 2.5 to 3.5 days post-mating when required for implantation with blastocy ⁇ t ⁇ containing ES cells. At the time of embryo transfer, the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight. The ovaries are exposed by making an inci ⁇ ion in the body wall directly over the oviduct and the ovary and uterus are externalized.
  • a hole is made in the uterine horn with a 25 gauge needle through which the blastocy ⁇ t ⁇ are tran ⁇ ferred. After the transfer, the ovary and uterus are pushed back into the body and the incision is closed by two suture ⁇ . This procedure is repeated on the oppo ⁇ ite ⁇ ide if additional tran ⁇ fer ⁇ are to be made.
  • Tail ⁇ ample ⁇ (1-2 cm) are removed from three week old animal ⁇ .
  • DNA i ⁇ prepared and analyzed by Southern blot or PCR to detect transgenic founder (F Q ) animals and their progeny (F.. and F ).
  • F Q transgenic founder
  • F.. and F progeny
  • thi ⁇ animals that have become transgenic for the de ⁇ ired thrombospondin-4 genes are identified. Because not every transgenic animal expre ⁇ es the thrombo ⁇ pondin-4 gene, and not all of tho ⁇ e that do will have the expre ⁇ ion pattern anticipated by the experimenter, it is nece ⁇ ary to characterize each line of tran ⁇ genic animal ⁇ with regard to expression of the thrombospondin-4 in different ti ⁇ sues.
  • An organism is identified as a potential tran ⁇ genic by taking a sample of the organism for DNA extraction and hybridization analy ⁇ i ⁇ with a probe complementary to the thrombospondin-4 gene of interest.
  • DNA extracted from the organism can be subjected to PCR analysi ⁇ using PCR primers complementary to the thrombospondin-4 gene of intere ⁇ t.
  • Mouse genomic clones are isolated by screening a genomic library from the D3 strain of mouse with a Xenopus thrombospondin-4 probe. Duplicate lift ⁇ are hybridized with a radiolabeled probe by e ⁇ tablished protocols (Sambrook, J. et al. , The Cloning Manual, Cold Spring Harbor Pre ⁇ , N.Y.). Plaque ⁇ that corre ⁇ pond to po ⁇ itive ⁇ ignal on both lifts are isolated and purified by succe ⁇ ive ⁇ creening round ⁇ at decrea ⁇ ing plaque density. The validity of the isolated clones is confirmed by nucleotide sequencing.
  • the genomic clones are used to prepare a gene targeting vector for the deletion of thrombospondin-4 in embryonic ⁇ tem cell ⁇ by homologous recombination.
  • a neomycin resistance gene (neo) with its tran ⁇ criptional and tran ⁇ lational ⁇ ignal ⁇ , i ⁇ cloned into convenient ⁇ ite ⁇ that are near the 5' end of the gene. This will di ⁇ rupt the coding ⁇ equence of thrombo ⁇ pondin-4 and allow for ⁇ election by the drug Geneticin (G418) by embryonic ⁇ tem (ES) cell ⁇ transfected with the vector.
  • HSV-tk Herpe ⁇ ⁇ implex virus thymidine kinase
  • Homologous recombination will also take place between homologou ⁇ DNA ⁇ equence ⁇ of the ES thrombo ⁇ pondin-4 genome and the targeting vector. Thi ⁇ u ⁇ ually re ⁇ ult ⁇ in the excision of the HSV-tk gene because it is not homologous with the thrombo ⁇ pondin-4 gene.
  • the cell lines in which homologous recombination has occurred will be highly enriched.
  • the ⁇ e cell ⁇ will contain a di ⁇ rupted coding ⁇ equence of thrombo ⁇ pondin-4.
  • Individual clones are isolated and grown up to produce enough cell ⁇ for frozen ⁇ tock ⁇ and for preparation of DNA.
  • Clone ⁇ in which the thro ⁇ rtbo ⁇ pondin-4 gene ha ⁇ been ⁇ ucce ⁇ fully targeted are identified by Southern blot analy ⁇ i ⁇ .
  • the re ⁇ ulting chimeric animal ⁇ are bred and the off ⁇ pring are analyzed by Southern blotting to identify individual ⁇ that carry the mutated form of the gene in the germ line.
  • the ⁇ e animal ⁇ will be mated to determine the effect of thrombo ⁇ pondin-4 deficiency on murine development and phy ⁇ iology.
  • ADDRESSEE Wolf, Greenfield, & Sacks, P.C.
  • MOLECULE TYPE Other nucleic acid
  • A DESCRIPTION: primer
  • MOLECULE TYPE Other nucleic acid
  • A DESCRIPTION: primer
  • MOLECULE TYPE Other nucleic acid
  • A DESCRIPTION: primer
  • MOLECULE TYPE Other nucleic acid
  • A DESCRIPTION: primer

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Abstract

Nouveau membre de la famille génétique des thrombospondines, la thrombospondine-4 a été clonée et séquencée. L'invention porte sur un ADN de thrombospondine-4 de la grenouille et sur l'homologue chez les mammifères de ce premier ADN. L'invention porte sur des vecteurs et des cellules de recombinaison, des méthodes d'obtention d'ADN et de séquences polypeptides de thrombospondine-4 isolée ainsi que sur des méthodes d'obtention d'animaux transgéniques présentant ou ne présentant pas ce gène de thrombospondine-4.
PCT/US1993/011725 1992-12-04 1993-12-03 Thrombospondine-4 humaine WO1994013794A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001040294A1 (fr) * 1999-11-29 2001-06-07 Bioroad Gene Development Ltd. Shanghai Nouveau polypeptide, thrombospondine humaine 30, et polynucleotide codant pour ce polypeptide
WO2001061011A3 (fr) * 2000-02-17 2002-03-14 Lexicon Genetics Inc Nouvelles proteines humaines contenant des sequences repetees de thrombospondine et nouveaux polynucleotides codant celles-ci
WO2001090179A3 (fr) * 2000-05-23 2002-06-13 Lexicon Genetics Inc Proteines ressemblant a la thrombospondine humaine et polynucleotides les codant
US7511131B2 (en) * 2002-11-13 2009-03-31 Genzyme Corporation Antisense modulation of apolipoprotein B expression
US8735364B2 (en) 2001-08-01 2014-05-27 Genzyme Corporation Antisense modulation of apolipoprotein B expression
US8916694B2 (en) 2004-05-05 2014-12-23 Genzyme Corporation SNPs of apolipoprotein B and modulation of their expression
US9107933B2 (en) 2009-03-16 2015-08-18 Isis Pharmaceuticals, Inc. Compositions and methods of targeting apolipoprotein B for the reduction of apolipoprotein C-III
US9347061B2 (en) 2007-03-24 2016-05-24 Genzyme Corporation Administering antisense oligonucleotides complementary to human apolipoprotein B
CN114732896A (zh) * 2022-06-09 2022-07-12 广东省科学院微生物研究所(广东省微生物分析检测中心) 骨骼肌分泌因子Thbs4在制备改善系统糖脂代谢药物中的应用

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Title
H.L. VOS ET AL.: "Thrombospondin 3 (Thbs3) a new member of the thrombospondin gene family", J. BIOL. CHEM., vol. 267, no. 17, 15 June 1992 (1992-06-15), AM. SOC. MOL. BIOL., INC.,BALTIMORE, US;, pages 12192 - 12196 *
J. LAWLER ET AL.: "Identification and characterization of thrombospondin-4, a new member of the thrombospondin gene family", J. CELL BIOLOGY, vol. 120, no. 4, February 1993 (1993-02-01), ROCKEFELLER UNIV. PRESS, N.Y. , US;, pages 1059 - 1067 *
J. LAWLER ET AL.: "The evolution of the thrombospondin gene family", J. MOL. EVOLUTION, vol. 36, no. 6, June 1993 (1993-06-01), SPRINGER VERLAG, NEW YORK, US, pages 509 - 516 *
J.ADAMS AND J. LAWLER: "The thrombospondin family", CURRENT BIOLOGY, vol. 3, no. 3, March 1993 (1993-03-01), LONDON,GB;, pages 188 - 190 *
P. BORNSTEIN ET AL.: "A second, expressed thrombospndin gene (Thbs2) exist in the mouse genome", J. BIOL. CHEM., vol. 266, no. 20, 15 July 1991 (1991-07-15), AM. SOC. MOL. BIOL., INC.,BALTIMORE, US;, pages 12821 - 12824 *
P. BORNSTEIN ET AL.: "Characterization of the mouse thrombospondin gene and evaluation of the role of the first intron in human gene expression", J. BIOL. CHEM., vol. 265, no. 27, 25 September 1990 (1990-09-25), AM. SOC. MOL. BIOL., INC.,BALTIMORE, US;, pages 16691 - 16698 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001040294A1 (fr) * 1999-11-29 2001-06-07 Bioroad Gene Development Ltd. Shanghai Nouveau polypeptide, thrombospondine humaine 30, et polynucleotide codant pour ce polypeptide
WO2001061011A3 (fr) * 2000-02-17 2002-03-14 Lexicon Genetics Inc Nouvelles proteines humaines contenant des sequences repetees de thrombospondine et nouveaux polynucleotides codant celles-ci
US6720412B2 (en) 2000-02-17 2004-04-13 Lexicon Genetics Corporation Human thrombospondin repeat proteins and polynucleotides encoding the same
WO2001090179A3 (fr) * 2000-05-23 2002-06-13 Lexicon Genetics Inc Proteines ressemblant a la thrombospondine humaine et polynucleotides les codant
US8735364B2 (en) 2001-08-01 2014-05-27 Genzyme Corporation Antisense modulation of apolipoprotein B expression
USRE44760E1 (en) 2002-11-13 2014-02-11 Genzyme Corporation Antisense modulation of apolipoprotein B-expression
US7803930B2 (en) 2002-11-13 2010-09-28 Isis Pharmaceuticals, Inc. Antisense modulation of apolipoprotein B-expression
US7511131B2 (en) * 2002-11-13 2009-03-31 Genzyme Corporation Antisense modulation of apolipoprotein B expression
US8916694B2 (en) 2004-05-05 2014-12-23 Genzyme Corporation SNPs of apolipoprotein B and modulation of their expression
US9347061B2 (en) 2007-03-24 2016-05-24 Genzyme Corporation Administering antisense oligonucleotides complementary to human apolipoprotein B
US9107933B2 (en) 2009-03-16 2015-08-18 Isis Pharmaceuticals, Inc. Compositions and methods of targeting apolipoprotein B for the reduction of apolipoprotein C-III
CN114732896A (zh) * 2022-06-09 2022-07-12 广东省科学院微生物研究所(广东省微生物分析检测中心) 骨骼肌分泌因子Thbs4在制备改善系统糖脂代谢药物中的应用
CN114732896B (zh) * 2022-06-09 2022-09-02 广东省科学院微生物研究所(广东省微生物分析检测中心) 骨骼肌分泌因子Thbs4在制备改善系统糖脂代谢药物中的应用

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