WO1992009616A1 - Augmentation de la musculature chez les animaux - Google Patents

Augmentation de la musculature chez les animaux Download PDF

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Publication number
WO1992009616A1
WO1992009616A1 PCT/US1991/008881 US9108881W WO9209616A1 WO 1992009616 A1 WO1992009616 A1 WO 1992009616A1 US 9108881 W US9108881 W US 9108881W WO 9209616 A1 WO9209616 A1 WO 9209616A1
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ski
protein
dna
muscle
construct
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PCT/US1991/008881
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Stephen H. Hughes
Pramod Sutrave
Vernon G. Pursel
Robert J. Wall
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THE UNITED OF AMERICA, represented by THE SECRETARY, UNITED STATES DEPARTMENT OF COMMERCE
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Priority to JP4509921A priority Critical patent/JPH06502078A/ja
Publication of WO1992009616A1 publication Critical patent/WO1992009616A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • 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
    • A01K2267/02—Animal zootechnically ameliorated
    • 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
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to the c-ski gene.
  • the present invention relates to DNA segments encoding chicken c-ski protein, to DNA constructs comprising the DNA segments and to cells transformed therewith.
  • the present invention further relates to animals having increased muscle size and/or reduced amounts of fat.
  • Viruses that contain the v-ski oncogene are not only capable of causing morphological transformation in vitro, but also can induce myogenic differentiation (Stavnezer et al., 1981, J. Virol. 39, 920-934; Li et al., 1986, J. Virol. 57, 1065-1072; Stavnezer et al., 1986, J. Virol. 57, 1073-1083; Colmenares and Stavnezer, 1989, Cell 59, 293-303) . Viruses that carry and express c-ski cDNAs also induce foci and myogenic differentiation (Sutrave et al., 1990, Mol. Cell. Biol.. 10, 3137-3144).
  • ski oncogene is bifunc- tional since the two known functions of ski, transfor ⁇ mation and differentiation, would appear to be contra ⁇ dictory properties.
  • genomic clones for c-ski have been isolated and partially sequenced (Stavnezer et al., 1989, Mol. Cell. Biol.. 9, 4038-4045).
  • Comparisons of the properties of two forms of c-ski that are related by alternative splicing, and of several v-ski and c-ski deletion mutants have shown that the portions of ski required for transformation and differentiation are quite similar.
  • the present invention relates to a DNA segment encoding a chicken c-ski protein or a DNA fragment complementary to said segment.
  • the present invention relates to a DNA construct comprising a DNA segment encoding a chicken c-ski protein and a vector.
  • the present invention relates to a DNA construct comprising a DNA segment encoding a truncated chicken c-ski protein having the function of c-ski and a vector.
  • the present invention also relates to host cells stably transformed with either one of the two DNA con ⁇ structs described above, in a manner allowing expression of the protein encoded in the construct.
  • the present invention relates to a animal having increased muscle size, all of whose cells contain a DNA construct comprising a DNA segment encoding a ski protein and a vector, introduced into the animal, or an ancestor of the animal.
  • the DNA segment may encode the entire protein or a truncated version thereof.
  • the present invention relates to an animal having increased muscle size and/or reduced fat, all of whose cells contain a DNA construct comprising a DNA segment encoding a truncated ski protein having the function of ski and a vector, introduced into the animal, or an ancestor of the animal.
  • the present invention relates to a method of stimulating muscle growth or preventing muscle degeneration comprising delivering a DNA construct of the present invention to the muscle under conditions such that the protein of the construct is expressed and muscle growth induced.
  • the present invention relates to a method of treating a muscle degenerative disease comprising delivering a DNA construct of the present invention to the effected muscle under conditions such that the protein of the construct is expressed and treatment effected.
  • Figure 1 shows the Structure of the c-ski cDNA clones.
  • the lengths of the cDNAs are drawn to scale and the restriction sites indicated. V-ski is shown for comparison.
  • the dotted boxes in v-ski represent the gag region of the gag-ski fusion in the acutely transforming virus SKV.
  • A, B, C and D represent the regions used for generating single-strand probes for SI nuclease protection analysis.
  • Figure 2 shows the complete coding sequence and the potential coding region of a cDNA of the FB29 type. This assumes that the 5' end sequences of the FB29 are similar to the 5' ends of the FB28 and CEL clones.
  • t indicates the site where FB28 and CEL diverge. The 25 bases found only in the CEL clones are not shown. 4* indicates the boundaries of v-ski.
  • exon boundaries are numbered and the alternately spliced exons are boxed.
  • the single base and a ino acid change between c-ski and v- ski is also boxed with a dashed line.
  • the translation termination codon is boxed in thick liens.
  • the potential polyadenylation signals are densely underlined.
  • the AT rich region containing ATTTA sequences that might be involved in mRNA stability is underlined with dashed lines.
  • Figure 3 shows a diagrammatic illustration of the alternate mRNAs generated for c-ski locus as deduced from the cDNA sequence analysis. The exons are not drawn to scale.
  • the c-ski mRNAs are shown in relation to v-ski.
  • FIGS. 4A-4E show SI nuclease protection analysis of total RNA. Uniformly labeled single-strand probes used for hybridization are shown schematically below each picture, the thick lines represent the cDNA sequence while the thin lines represent M13 sequence. The overall length of probes and expected lengths of protected fragments are also shown.
  • the RNA hybridization is indicated at the top of each lane. The numbers (8, 10, 12, 15 or 17) above the lane indicate the age of the embryo from which the RNA was prepared.
  • Probe A contains a Kpnl-Hindlll fragment of the FB27/29 type. This probe produced a fragment of 645 base pairs (bp) and two smaller fragments of 262 and 272 bp, shown by arrows.
  • Probe B contains a Kpnl-Hindlll fragment of the FB28 type. This probe produced a fragment of 534 bp as shown by the arrows. Smaller fragments were not detected.
  • Probe C (see Figure 1) contains a 497-bp Hindlll fragment of the FB27 type linked to M13 sequences. The probe yielded a 497-bp fragment and two smaller fragments of 243/254. Only the 479-bp fragment is marked by an arrow.
  • Probe D (see Figure 1) is 1116 bp in length containing a Hindlll fragment of FB28/29 type. Probe D produced a 799-bp fragment which is marked by an arrow.
  • Figure 5 shows sequence homology between c-ski and the pl9 region of gag from avian leukosis virus.
  • the c- ski sequences are from positions 218 to 242 and the pl9 sequence of gag region are from positions 633 to 658.
  • the homologous regions are boxed.
  • Figure 6 shows the c-ski expression cassette.
  • the Pvul to Nrul segment shown in the drawing was isolated by gel electrophoresis following double digestion of the plasmid.
  • the linear DNA was used to create the transgenic mice by microinjection of fertilized eggs.
  • Figure 7 shows a transgenic mouse that expresses c-ski and a normal litter mate.
  • the c-ski transgene appears to segregate normally in crosses.
  • the photograph shows a heterozygous mouse that displays the muscular phenotype (foreground) and a DNA-negative litter mate. Double blind DNA analyses confirmed that the muscular phenotype segregates with the transgene.
  • Figures 8A-8B show Northern transfer analysis of transgene expression.
  • Panel A shows the analysis of RNA isolated from various tissues of a mouse of the line TG 8566.
  • the upper part of the panel shows an autoradiogram after hybridization with chicken c-ski.
  • the expected position of migration of the c-ski message appropriately transcribed from the transgene is 2.5 kb.
  • the position corresponding to 2.5 kb is marked (ski).
  • the lower panel shows an autoradiogram from the same filter following hybridization to a chicken 3-actin cDNA.
  • the 0-actin cDNA will hybridize not only with 0-actin mRNA but also with other actin messages.
  • the expected position of migration of both 0-actin and ⁇ -actin mRNAs are indicated on the right of the panel.
  • Panel B shows the autoradiograms shown in panel B are similar to those shown in panel A except that the RNAs derive from three other transgenic lines. The lines used to prepare the RNAs are indicated at the top of the figure. The filters shown in panel B were done at the same time; those in panel A were done on a different day.
  • Figure 9 shows RNase protection of RNA from the transgene.
  • the top of the figure shows an autoradiogram of the gel.
  • the first lane contains the antisense RNA probe, without RNase digestion.
  • the next two lanes show the results of digestion following hybridization of the probe either without added RNA or with tRNA.
  • the next eight lanes show the results of hybridization to RNA isolated either from the hearts (heart) or the skeletal muscle (SK muscle) of the four transgenic lines.
  • the next lanes show the results of hybridizing the probe to RNA from skeletal muscle of a mouse that does not carry the transgene (control SK muscle) .
  • the last lane contains molecular weight markers.
  • FIG. 10 shows chicken c-ski protein expression in transgenic mice. Extracts were made from the liver
  • Figures 11A-11D show cross sections made precisely through the middle of the plantaris muscle: ( Figure 11A) from control mouse and ( Figure 11B) from a mouse of line TG 8566. Both illustrations are at the same magnifi ⁇ cation, the size marker is 200 ⁇ .
  • Figure 11C Higher power illustration from the plantaris of control and ( Figure 11D) from the plantaris of an affected mouse. Size markers in ( Figure IIC) and ( Figure IID) are 100 ⁇ .
  • Figures 12A-12B show distribution of fiber diameters in selected muscles from normal and transgenic Mice. Panel A shows the diaphragm appears normal in transgenic mice that express c-ski.
  • a diaphragm from a mouse that has the muscular phenotype (TG 8566) and a diaphragm from a normal control mouse were sectioned and the number of individual muscle fibers of a given cross- sectional area tallied.
  • Panel B shows the anterior tibial muscle is grossly enlarged in mice from the line TG 8566.
  • Transverse sections were prepared from both a transgenic mouse and a control mouse. The number of fibers of each given cross-sectional area were tallied.
  • This muscle is composed of two distinct types of fibers, some of which are smaller, others larger, than the fibers found in the controls (see also Figures 11A-11D) .
  • Figure 13 shows transgene expression in specific muscles from TG 8566.
  • RNA was fractionated by electrophoresis and transferred to nitrocellulose membranes. The transfers were first probed with chicken c- ski, then the filters were stripped and reprobed with a chicken ⁇ -actin cDNA (Cleveland et al., 1980, Cell 20, 95- 105) .
  • the RNA was isolated from the diaphragm, the soleus, or from bulk skeletal muscle (sk muscle) .
  • Figures 14A-14D shows immunofluorescence staining of sections made through the middle of the Rhomboideus capitis muscle of an affected mouse (transgenic line 8566) . ( Figure 14A) staining with monoclonal antibody NOQ7 5 4D, specific for slow MHC. Slow fibers are not hypertrophied.
  • FIG. 14B staining with monoclonal antibody SC 711 specific for Ila MHC. Ila fibers are not hypertrophied.
  • Figure 14C is with monoclonal antibody 2G3 which reacts with all fast MHC isoforms. All hyper- trophied fibers stain with this antibody.
  • Figure 14D is with m/a BF-F3 specific for IIB MHC. Many, but not all, hypertrophied fibers stain. Magnification x 230.
  • Figures 15A-15B shows transgenic pigs that contain c-ski and control pigs.
  • Figure 15A shows the photo- graph shows a transgenic pig (3-0102) (left) that displays the muscular phenotype of the shoulders and a control pig (right) .
  • Figure 15B shows the photograph shows trans ⁇ genic pigs that display the muscular phenotype of the rear quarters (3-0503 (left) and 3-0202 (second from left) ) and a control pig (3-0203, third from left).
  • the present invention relates to a DNA segment encoding all, or a unique portion, of a chicken c-ski protein.
  • the DNA segment may encode one of several chicken c-ski proteins, for example, FB29, FB28 and FB27.
  • a "unique portion" as used herein is defined as consisting of at least five (or six) amino acids or correspondingly, at least 15 (or 18) nucleotides.
  • the invention also relates to DNA constructs containing such DNA segments and to cells transformed therewith.
  • the present invention relates to DNA segments that encode the amino acid sequence of exon 6 given in Figure 1 or the amino acid sequence of exon 7 given in Figure 1.
  • the present invention also relates to DNA segments that, in addition to exon 7, further comprise at least four exons selected from the group consisting of: exon 1, exon 2, exon 3, exon 4, exon 5 or exon 6, given in Figure 1. Examples of such DNA segments include FB29, FB28, and FB27.
  • DNA segments to which the invention relates also include those encoding substantially the same proteins as those encoded in the exons of Figure 1 which includes, for example, allelic forms of the Figure 1 amino acid sequences.
  • the invention also relates to DNA fragments complementary to such sequences. A unique portion of the DNA segment or the complementary fragment thereof of the present invention can be used as probes for detecting the presence of its complementary strand in a DNA or RNA sample.
  • the present invention further relates to DNA constructs and to host cells transformed therewith.
  • the DNA constructs of the present inven ⁇ tion comprise a DNA segment encoding a c-ski protein of the present invention and a vector, for example, pMEX neo.
  • the DNA constructs comprise a DNA segment encoding a truncated c-ski protein having the function of c-ski (such as, for example, ⁇ FB29) and a vector (for example, pMEX neo) .
  • the DNA construct is suitable for transforming host cells.
  • the host cells can be procaryotic or, preferably, eucaryotic (such as, mammalian) .
  • the present invention further relates to animals, such as, for example, domestic livestock, having increased muscle size and/or reduced fat.
  • animals such as, for example, domestic livestock, having increased muscle size and/or reduced fat.
  • Domestic livestock refers to animals bred for their meat, such as, for example, pigs, chickens, turkeys, ducks, sheep, cows and fish, particu ⁇ larly, trout and catfish) .
  • mice having increased muscle size by introducing a DNA construct comprising ⁇ FB29 and the pMEX neo vector into fertilized eggs. Resulting founder mice and their offspring have the DNA construct in all their cells, somatic and germ.
  • a ski protein such as, for example a c-ski protein
  • fertilized eggs of animals such as, by microinjection
  • animals with increased muscle size of the present invention can also be produced using DNA encoding a ski protein from various species (chicken being just one such example) .
  • animals of the present invention can be produced using a DNA construct encoding proteins related to ski, such as, for example, the sno gene.
  • the DNA segment ⁇ FB29 generated by a frameshift mutation results in a truncated protein.
  • the transgenic animals of the present invention can also be generated by DNA constructs containing DNA segments encoding a full length ski protein, a portion of a ski protein, such as, one or two exons or a biological active deletion derivative, such as, for example, v-ski, which represents a truncated c-ski fused to a viral protein.
  • v-ski which represents a truncated c-ski fused to a viral protein.
  • the selective expression of the protein in muscle tissue may result from DNA constructs created in vectors other than pMEX neo.
  • the present invention also relates to a method of stimulating muscle growth and preventing muscle degenera- tion in an animal, such as for example, a human.
  • a possible treatment for injuries resulting in loss of muscle tissue and neurological injuries resulting in generation of the muscle would be to stimulate muscle growth. In the case of loss of muscle this would involve stimulating regrowth of the tissue. Whereas in the case of neurological injuries, the muscle growth would need to be rendered independent of the missing nerve stimulus.
  • muscle growth could be stimulated by delivering a DNA construct encoding a ski protein to the muscle tissue under conditions such that the protein encoded in the construct is expressed.
  • the construct can be targeted and delivered to the muscle using standard methods known to those skilled in the art.
  • the present invention further relates to a method of treating a muscle degenerative disease such as, for example, muscular dystrophy and amyotrophic lateral sclerosis (also known as Lou Gehrig disease) .
  • Treatment would comprise delivering a DNA construct of the present invention to the effected muscle under conditions such that the protein encoded in the construct is expressed and treatment effected. Examples Screening of cDNA Library
  • Two chicken cDNA libraries were screened with a v- ski probe using standard protocols (Maniatis et al., 1982, Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .
  • One library was made from poly A mRNA isolated from the body wall of 10-day embryos and the other from mRNA isolated from AEV- transformed chicken erythroblasts.
  • Four distinct c-ski cDNAs were isolated; three were from the body wall library (these cloned were designated FB27, FB28 and FB29) and one cDNA clone from the erythroblast library (designated CEL) .
  • These cDNAs included sequences extending both 5' and 3' of the portion of ski present in the virus.
  • the cDNAs demonstrate that v-ski derives from a single cellular gene and suggest that multiple c-ski mRNAs, encoding distinct ski proteins, are produced from the c-ski locus by alter ⁇ nate splicing (Leff et al., 1986, Ann. Rev. Biochem.. 55, 1091-1117) , adding to a growing list of oncogenes known to produce multiple mRNAs in this fashion (Ben-Neriah et al., 1986, Cell. 44, 577-586; Levy et al., 1987, Mol. Cell. Biol. , 7, 4142-4145; Martinez et al. , 1987, Science 237, 411-415; McGrath et al., 1983, Nature. 304, 501-506). Structure of the cDNA clones.
  • exon 6 Differential splicing of exon 6, however, affects the coding potential of exon 7. If exon 5 is spliced to exon 7 (seen in FB27 cDNA) , a trans- lation termination codon is generated at the splice junction and exon 7 becomes a noncoding exon. However, if exon 5 is spliced to exon 6 and exon 6 to exon 7, as in FB28/29, then the open reading frame continues in exon 7 for 417 nucleotides encoding an additional 129 amino acids.
  • cDNAs that derive from the body wall (FB) library have long 3' untranslated regions that contain a 95-base pair (bp) AT- rich region from nucleotide 2803 to 2898. Within this region there are two copies of a sequence ATTTA that has been implicated in mRNA destabilization in a variety of transiently induced mRNAs including c-myc, interferon, c- jun, and c-fos (Meijlink et al., 1985, Proc. Natl. Acad. Sci. USA. 82, 4987-4991; Ryder et al., 1985, Proc. Natl. Acad. Sci. USA.
  • c-ski cDNAs contain two potential poly(A) signals (AATAAA) located at positions 3348 and 4167. Although all three clones isolated from the FB library end at the same position, none has a poly(A) tail; therefore, it is likely that the 3' ends of the c-ski mRNAs are not contained in these clones.
  • the 4.2-kb cDNAs that were isolated and characterized are smaller than the 5.7 -8.0 and 10.0-kb mRNAs detected by Northern transfer analysis (Li et al., 1986, J. Virol.. 57, 1065-1072). This dis ⁇ crepancy has not been explained, however, it is suggested that the clones isolated so far, which lack poly(A) tails, also lack sequences from the 3' ends of the mRNAs. The 5' End of c-ski mRNA(s)
  • FB28 primer extension analysis was carried out. Copying the 5' segment present in FB28 should give an extension product of about 280 bases. However, a primer extension product of 220 bases was seen. SI analyses data have shown that this segment is expressed in RNA. It is possible that the observed primer extension product is the result of premature termination; however, it is also possible that there are multiple 5' ends of the c-ski mRNAs.
  • RNA samples were isolated from 8, 10, 12, 15 and 17-day-old chicken embryos using standard protocols (Chirgwin et al., 1979, Biochemistry f 18, 5294-5299). Approximately, 20 to 30 ⁇ g of total RNA was used for nuclear SI analysis using standard procedures (Maniatis et al., 1982, Molecular Cloning , A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .
  • a uniformly labeled single-stranded probe spanning the region between the Kpnl and Hindlll sites of FB29/27 or FB28 was hybridized with total cellular RNA.
  • hybridization to mRNA and subsequent SI digestion of a probe derived from FB29 produced protected fragments of 645 bp indicat ⁇ ing hybridization to mRNA of the FB27/29 type and the 262/272 bp fragments expected if the probe hybridized with mRNA of FB28 type.
  • FB28 (probe B) protected a fragment of 534 bp (see Figure 4B) .
  • sequences absent from FB27 are bounded by apparent splice junctions (as judged by an examination of both the cDNA sequences and the available genomic DNA sequence) .
  • cDNAs are occasionally obtained that contain one or more introns, presumably because a partially processed mRNA was reverse transcribed, isolating cDNA artefactually missing an exon is less likely on theoretical grounds, and seems to occur rarely, if at all, in the manufacture of a cDNA library.
  • the interpretation that FB27 represents a real, if relatively rare, c-ski mRNA is currently favored. Comparison of c-ski with v-ski
  • 18 of 20 bp are identical between c-ski and the pl9 region of gag in the parental ALV.
  • This region of homology contains the 5' junction between viral sequences and v-ski. With such a long stretch of homology, it is impossible to assign the 5' recombinational joint precisely. No substantial homology can be seen precisely at the 3' ski/ALV joint. However, just downstream of the 3 ' junction, there is an ALV sequence that is closely homologous to a segment of c-ski found just 3 ' of the v-ski/AVL junction. It is possible to invoke this region of homology in the alignment of the nucleic acids involved in the recombinant event. Transduction mechanism
  • retroviral and cellular sequences can then be fused by DNA deletion events (Bishop, J.M. 1983, Ann. Rev. Biochem. , 52, 301-354; Czernilofsky et al., 1983, Nature. 301, 736- 738) or RNA read through (Herman et al., 1987, Science, 236, 845-848; Nilsen et al.
  • FB29 which contains sequences that derive from all seven coding exons of c-ski, and judged by DNA sequence, encodes a c-ski protein of 750 amino acids
  • ⁇ FB29 has a frameshift mutation at position 1475 in the fifth coding exon (one C in a run of five Cs was lost in the frameshift mutant) , and is pre- dieted to give rise to a protein of 448 amino acids of which the first 436 are identical to the first 436 amino acids of the FB29 form of c-ski (the last 12 amino acids are past the frameshift mutation and thus differ from those of the FB29 form of c-ski) .
  • ⁇ FB29 used in the generation of the transgene is shown schematically in Figure 6.
  • ski portion of the trans ⁇ gene is already described (Sutrave and Hughes, 1989, Mol. Cell. Biol.. 9, 4046-4051; Sutrave et al., 1990, Mol. Cell. Biol.. 10, 3137-3144). Briefly, a truncated chicken c-ski cDNA called ⁇ FB29 had been previously cloned into the adaptor plasmid Clal2Nco. The ⁇ FB29 segment was released from the adaptor plasmid by Clal digestion and the 5' overhangs filled in using the Klenow fragment of E. coli DNA polymerase I and all four dNTPs.
  • This blunt- ended fragment was ligated to the pMEX neo vector which have been digested with EcoRl restriction enzyme and blunt-ended with the Klenow fragment. Clones were selected that had inserts in the correct orientation and were digested with both Pvul and Nrul restriction endonu- cleases. These enzymes release a segment that contains the ⁇ FB29 cDNA flanked by an MSV LTR and the SV40 polyA signal (see Figure 6) . This fragment was gel purified and used to inject fertilized mouse eggs [Hogan et al., 1986, Manipulating the Mouse Embryo. A Laboratory Manual.
  • the ⁇ FB29 clone was placed in the pMEX expression plasmid in such an orientation that the truncated c-ski cDNA between an MSV LTR and an SV40 polyadenylation site (see Figure 6) .
  • the plasmid (named MSV-SKI has been deposited on June 29, 1989 at the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A. and has been given ATCC accession number 68044) was digested with Pvul and Nrul to release the expression cassette.
  • the expression cassette was purified by gel electrophoresis and introduced into fertilized mouse eggs by microinjection. Forty-four founder mice were obtained after two independent injections.
  • mice were identified by dot blot analysis of DNA isolated from tail clips. This analysis was confirmed by Southern transfer. Three of the 44 founder mice showed a distinct muscle phenotype (TG 8566, TG 8821, and TG 8562) . These three founders and a single mouse that contained unrearranged copy of the complete transgene but did not show any phenotype (TG 8542) were used to generate lines. Southern transfer analysis of DNA from TG 8566, TG 8821, TG 8562, and TG 8542 suggests that the site of integration of the transgene in each line is different and the copy number varies from approximately 5-35 copies per genome.
  • mice from the three lines had a similar distinct appearance resulting from abnormal muscle growth. Although the three lines of mice carry an oncogene, none of the lines appears to have an increased incidence of tumors. This result is not totally unexpected, since the v-ski virus is not tumorigenic in chickens unless the birds are injected with infected cells (E. Stavnezer, 1988, in The Oncogene Handbook. E. P. Reddy, A. Skalka, and T. Curran, eds. (Amsterdam: Elsevier Science Publishing Co.), pp. 393- 401) . The three strains of mice do not express high levels of the transgene except in skeletal muscle.
  • c-ski cDNAs FB27 and FB29 were cloned into the pMEX expression plasmid, as previously described for ⁇ FB29, in such a manner that the c-ski cDNA was between the MSV LTR and an SV40 polyadenylation site in the proper orientation.
  • the plasmids were designated pMNSK27 and pMNSK29.
  • the plasmids were each separately digested with Pvul and Nrul to release the expression cassette.
  • the two expression cassettes were each purified by gel electrophoresis and independently introduced into fertilized mouse eggs by microinjection [Hogan et al., 1986, Manipulating the Mouse Embryo , A Laboratory Manual.
  • the c-ski expression cassette ( Figure 6, contain ⁇ ing ⁇ FB29) was introduced into pigs.
  • the plasmid DNA (named MSV-SKI, ATCC accession number 68044) was digested with Pvul and Nrul and the expression cassette ( Figure 6) was purified as described for mice.
  • Microinjection of pig ova was similar to that described for mice except for the modification that pig ova were centrifuged as de- scribed by Wall (1985) Biol. of Reproduction r 32:645-651 prior to microinjection (Hammer et al. (1985) Nature 315:680-683).
  • Twenty-nine pigs containing ⁇ FB29 were obtained after injection (Table 1) .
  • the pigs were identified by dot blot analysis of DNA isolated from tail clips (Siracusa et al. (1987) Genetics. 117:85-92).
  • Three of the transgenic pigs had distinct muscle hypertrophy
  • RNA and RNA were isolated by standard procedures. For RNA isolation, tissues were frozen in liquid nitrogen immediately following dissection and homogenized in RNAzol (Cinna Biotex) and processed according to the manufacturer's recommendation. For Northern transfer analysis, approximately 20 ⁇ g of total RNA from different tissues was fractionated by electropho ⁇ resis on 1.5% agarose gels containing 2.2 M formaldehyde. The RNA was transferred to nitrocellulose membranes and probed either with a nick-translated chicken ski cDNA or a chicken j8-actin cDNA. The coding region of the 3-actin cDNA cross reacts with the messages for the other actions and can be used to validate the quantity and quality of RNA from most tissues.
  • RNA from spleen, lung, brain, kidney, liver, stomach, heart, and leg (skeletal) muscle of transgenic mice was isolated as described and the results are shown in Figure 8. All three lines with the phenotype (TG 8566, TG 8821 and TG 8562) expressed a 2.5-kb chicken c-ski specific transcript at high levels in skeletal muscle; however, some lines of mice showed low levels of chicken c-ski RNA in other tissues.
  • the TG 8562 line has RNA from the transgene in the heart, although at a lower level than in skeletal muscle. Histopathology of hearts from TG 8562 mice showed that there is no significant effect on this tissue.
  • Line TG 8542 which does not show any phenotype, had a much lower level of RNA from the transgene in muscle than did the lines that showed the phenotype.
  • the observa- tion that expression was restricted to muscle was unex ⁇ pected since the MSV LTR has been shown to express in a variety of tissues when linked to other genes. (Khillan et al., 1987, Genes Dev.. 1, 1327-1335).
  • the hybridizations were carried out overnight at 50°C in 80% formamide and lx buffer (5x hybridization buffer is 0.2 M Pipes, pH 6.4, 2 M sodium chloride, 5 mM EDTA) .
  • the samples were diluted in ribonuclease digestion buffer (10 mM Tris Cl, pH 7.5, 0.3 M sodium chloride, 5 mM EDTA) and treated with RNase Tl at a concentration of 1 u/ ⁇ l for 60 in at 30°C.
  • the RNase digestions were stopped by adding 10 ⁇ l of 20% SDS and 4 ⁇ l of Proteinase K (stock 10 mg/ml) and incubating at 37°C for 15 min.
  • the digested samples were extracted with phenol chloroform (1:1 mixture) and ethanol precipitated with carrier tRNA.
  • the pellet was rinsed once with 70% ethanol, dried and dissolved in formamide containing bromophenol blue and xylene cyanol dyes.
  • the samples were denatured at 100°C and separated on 6% polyacrylamide gels containing 7.5 M urea. Uniformly labeled antisense RNA was generated by
  • T7 RNA polymerase from a fragment that spans the MSV LTR and c-ski (see Figure 9) .
  • RNA from the three positive transgenic lines of mice a protected fragment of approximately 980 bases was seen, which is the expected size if the transcript is initiated at the authentic initiation site within the MSV LTR ( Figure 9) .
  • This analysis also gives a more quantitative estimate of the level of transgene RNA in the heart and skeletal muscle of both the phenotypically positive and the phenotypically negative lines of mice.
  • Figure 9 shows that the level of transgene RNA in the heart of TG 8821 is much lower (estimated at perhaps 1/10-1/20) than the level found in the skeletal muscle.
  • Indirect labeling proce ⁇ dures using, for example, labeled rabbit anti mouse detect not only the anti ski monoclonal but also the endogenous mouse heavy chain, which comigrates with the 50-kd form of c-ski made from the transgene.
  • extracts of muscle and control tissue (liver) from normal controls and from the transgenic mice were prepared. The endogenous mouse antibodies were removed from these extracts by precipitation with rabbit anti mouse antibody as described below.
  • tissue was homoge ⁇ nized in 1 ml of RIPA buffer, 20 mM Tris Cl pH 7.5, 150 mM NaCl, 0.5% SDS, 0.5% NP40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, and 35 ⁇ /ml of aproteinin.
  • the homo- genate was clarified by centrifugation at 10,000 rpm for 10 min.
  • Mouse IgG was removed from 100 ⁇ l of the super ⁇ natant by incubation with 10 ⁇ l of 1 mg/ml rabbit anti mouse IgG (in PBS) for 2 hr on ice.
  • the complex was removed by adding 100 ⁇ l of 40% protein A sepharose beads in RIPA buffer. The resulting supernatant was collected and 20 ⁇ l was fractionated on 10% SDS polyacrylamide gels. The proteins were transferred to nitrocellulose membranes overnight in buffer containing 0.125 M Tris Cl, 0.092 M Glycine and 20% Methanol, pH 8.3. The filters were blocked with 4% dry nonfat milk in TBS buffer (0.5 M Tris Cl, pH 7.4 and .2 M sodium chloride for 2 hr at room temperature and incubated with a mixture of three anti-ski monoclonal antibodies at a dilution of 1:3000 for 2 hr at room temperature and were then washed 3x with TBS.
  • TBS buffer 0.5 M Tris Cl, pH 7.4 and .2 M sodium chloride
  • FIGS 11A and 11B compare cross sections made precisely through the middle of the plantaris muscle from mature male controls and transgenic mice. Cross sectional area of the control is 2.7 ⁇ 2 and that of the TG 8566 mouse is 9.4 ⁇ 2 , more than twice the control value. This massive growth is generalized. Comparable increases in cross section were found in almost all axial and appendicular muscles throughout male and female mice of line TG 8566. Only three muscles were found that appear to be normal: the tongue, the diaphragm, and the soleus; these are the same size in transgenic as in control muscles (see Figure 12A and 12B) .
  • Northern transfer analysis shows that the level of chicken c-ski RNA is much lower in these two phenotypically normal muscles than in the effected muscles from the same line ( Figure 13) .
  • the most obvious additional gross morphologic abnormality is that transgenic animals were almost totally devoid of fat whereas control animals contained substan ⁇ tial amounts of subcutaneous and intraperitoneal fat. For this reason, there is little difference in weights between control and TG 8566 mice.
  • MHC slow myosin heavy chains
  • Figure 14 shows immunofluorescent staining of sections from the rhomboideus capitis muscle with these three antibodies. No evidence was found that slow fibers are enlarged ( Figure 14A) and the total number of slow fibers in the rhomboideus capitis from a TG 8566 mouse (120) approximates the number found in rhomboideus capitis from control mice (117) . Type Ila fibers are also not affected ( Figure 14B) . Many of the type Ila fibers lie between hypertrophied fibers and are frequently distorted in shape as if compressed by the expansion of their neighbors ( Figure 14B) .

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Abstract

La présente invention concerne des segments d'ADN codant la protéine c-ski du poulet, des constructions d'ADN comprenant les segments d'ADN ainsi que des cellules transformées avec ces segments. La présente invention concerne également des animaux trasngéniques ayant des muscles de dimension augmentée et/ou moins de graisse. De plus, la présente invention concerne des procédés de stimulation du développement des muscles et de prévention de la dégénération des muscles, ainsi que des procédés de traitement de l'obésité et des maladies de dégénérescence musculaire.
PCT/US1991/008881 1990-12-03 1991-12-03 Augmentation de la musculature chez les animaux WO1992009616A1 (fr)

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US5857961A (en) * 1995-06-07 1999-01-12 Clarus Medical Systems, Inc. Surgical instrument for use with a viewing system
US5869037A (en) * 1996-06-26 1999-02-09 Cornell Research Foundation, Inc. Adenoviral-mediated gene transfer to adipocytes
US9821114B2 (en) 2012-02-07 2017-11-21 Global Bio Therapeutics, Inc. Compartmentalized method of nucleic acid delivery and compositions and uses thereof

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US4736866A (en) * 1984-06-22 1988-04-12 President And Fellows Of Harvard College Transgenic non-human mammals

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JP2706697B2 (ja) * 1989-06-30 1998-01-28 アメリカ合衆国 動物における筋肉組織の増大

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US4736866A (en) * 1984-06-22 1988-04-12 President And Fellows Of Harvard College Transgenic non-human mammals
US4736866B1 (en) * 1984-06-22 1988-04-12 Transgenic non-human mammals

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Cell, Volume 59, issued 20 October 1989 (Cambridge, USA), COLMENARES et al., "The ski oncagene induces muscle differentiation in quail embryo cells", pages 293-303, see the entire document. *
Gene and Development, Volume 4, Number 9, issued September 1990, SUTRAVE et al., "Ski can cause selective growth of skeletal muscle in transgenic mice", pages 1462-1472. *
Journal of Virology, Volume 57, Number 3, issued March 1986 (Washington, USA), LI et al., "Unique sequence, ski, in Sloan-Kettering Avian Retroviruses with properties of a new cell-derived oncogene", pages 1056-1072, see the entire document. *
Molecular and Cellular Biology, Volume 10, Number 6, issued June 1990 (Washington, USA), SUTRAVE et al., "Characterization of chicken c-ski on oncogene products expressed by retrovirus vectors", pages 313-3144, see the entire document. *
Molecular and Cellular Biology, Volume 9, Number 9, issued September 1989 ( Washington, USA), SUTRAVE et al., "Isolation and characterization of three distinct cDNAs for chicken c-ski gene", pages 4046-4051, see the entire document. *
Molecular and Cellular Biology, Volume 9, Number 9, issued September 1989 (Washington, USA), STRAVNEZER et al., "The v-ski oncogene encodes a truncated set of c-ski coding exons with limited sequence and structural relatedness to v-myc", pages 4038-4045, see the entire document. *
Proceedings of the National Academy of Sciences, Volume 85, issued May 1988 (Washington, USA), ST.LOUIS et al., "An alternative approach to somatic cell gene therapy", pages 3150-3154, see the entire document. *
Science, Volume 240, issued 10 June 1988 (Washington, USA), WHITE et al., "The human as an experimental system in molecular genetics", pages 1483-1488, see the entire document. *
Science, Volume 242, issued 16 December 1988 (Washington, USA), ROSENBERG et al., "Grafting genetically modified cells to the damaged brain restorative effects of NGF eocpression", pages 1575-1578, see the entire document. *
Science, Volume 244, issued 16 June 1989 (Washington, USA), FRIEDMANN, "Progress toward human gene therapy", pages 1275-1281, see the entire document. *
See also references of EP0561965A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5857961A (en) * 1995-06-07 1999-01-12 Clarus Medical Systems, Inc. Surgical instrument for use with a viewing system
US5869037A (en) * 1996-06-26 1999-02-09 Cornell Research Foundation, Inc. Adenoviral-mediated gene transfer to adipocytes
US9821114B2 (en) 2012-02-07 2017-11-21 Global Bio Therapeutics, Inc. Compartmentalized method of nucleic acid delivery and compositions and uses thereof

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EP0561965A4 (en) 1997-03-19

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