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  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates 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.
• Viruses that contain the v-ski oncogene are not only capable of causing morphological transformation in vitro, but also can induce yogenic 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).
• the present invention relates to a DNA segment encoding a chicken c- ⁇ ki 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 to of the two DNA constructs 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 - k - 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, 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 l 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. The numbers next to the arrows correspond to the exon number.
• Figure 2 Complete coding sequence and the potential coding region of a cDNA of. the FB29 type.
• t indicates the site where FB28 and CEL diverge.
• the 25 bases found only in the CEL clones are not shown.
• I indicates the boundaries of v-ski .
• the 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 lines.
• 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 Diagrammatic illustration of the alternate mRNAs generated for c- ⁇ ki locus as deduced from the cDNA sequence analysis. The exons are not drawn to scale. The c- ⁇ ki mRNAs are shown in relation to v-ski . The dark areas are noncoding regions while the open boxes are the protein coding regions of the cDNA. The dotted boxes on both ends of v-ski are the gag regions of the gag-ski fusion in transforming ski viruses. The relative positions of the putative translational initiation codon and the translation termination codons are also shown. F gure 4. SI nuc ease protect on analys s of total RNA.
• Fiqure 4A shows the- 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
• Figure 4B showsProbe A (see Figure 1) 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.
• Figure 4C showsProbe B (see Figure 1) 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.
• Figure 4D showsprobe C (see Figure 1) contains a 497-bp Hindlll fragment of the FB27 type linked to M13 sequences.
• 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.
• FIG. 1 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.
• 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 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.
• 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- ⁇ ki .
• the expected position of migration of the c- ⁇ ki message appropriately transcribed from the transgene is 2.5 kb.
• the position corresponding to 2.5 kb is marked ( ⁇ ki) .
• the lower panel shows an autoradiogram from the same filter following hybridization to a chicken 9-actin cDNA.
• the ⁇ - 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 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 - fi ⁇ 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.
• 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.
• Below the autoradiogram is a diagram that shows a drawing of the MSV LTR c- ⁇ ki expression cassette in relation to the antisense RNA probe.
• the T7 transcript begins in the middle of the c- ⁇ ki coding region and goes entirely through the MSV LTR into adjacent sequences that derive from pBR322 (marked pBR) . If the transcripts deriving from the transgene initiate appropriately, then a fragment of 984 bases should be protected.
• FIG. 10 chicken c- ⁇ ki protein expression in transgenic mice. Extracts were made from the liver (Liv) or skeletal muscle (Sk.M) of control mice (control) or " mice carrying and expressing the c- ⁇ ki transgene. The positions of migration of radioactively labeled molecular weight standards are shown to the left of the figure. _ Q _
• FIG. 11 Cross sections made precisely through the middle of the plantaris muscle: (a) from control mouse and (b) from a mouse of line TG 8566. Both illustrations are at the same magnification, the size marker is 200 ⁇ . (c)
• FIG. 12 Distribution of fiber diameters in selected muscles from normal and transgenic Mice
• Panel A The diaphragm appears normal in transgenic mice that express c- ⁇ ki .
• 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 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 Figure 11) .
• RNA was isolated from the diaphragm, the soleus, or from bulk skeletal muscle (sk muscle) .
• FIG. 14 Immunofluorescence staining of sections made through the middle of the Rhomboideus capitis muscle of an affected mouse, (a) staining with monoclonal antibody NOQ7 5 4D, specific for slow MHC. Slow fibers are not hyper- trophied. (b) staining with monoclonal antibody SC 711 specific for Ila MHC. Ila fibers are not hypertrophied. (c) is with monoclonal antibody
• 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 l which includes, for example, allelic forms of the Figure l 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 invention 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.
• animals such as, for example, domestic livestock, having increased muscle size.
• Domestic livestock refers to animals bred for their meat, such as, for example, pigs, chickens, turkeys, ducks, sheep, cows and fish, particularly, 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.
• DNA constructs encoding 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 protiens related to ski , such as, for example, ⁇ no 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 ⁇ ki protein, a portion of a ski protein, such as, one or two exons or a biological active deletion derivative, such as, for example, v- ⁇ ki , which represents a truncated c-ski fused to a viral protein.
• v- ⁇ ki 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 degeneration 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 degeneration of the muscle would be to stimulate muscle growth. In the case of lose 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.
• 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 alternate 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-Neviah et al., 1986, Cell 44 , 577-
• the first ATG with a substantial downstream open reading frame is located at nucleotide position 168. Upstream of this ATG no reading frames are open, suggesting that these sequences represent the 5' untranslated region. Based on the cDNA sequence analysis and comparisons with the positions of the splice donor and acceptor sites known from the genomic sequence
• Differential splicing of exon 2 deletes 37 amino acids without affecting the coding potential of the open reading frame downstream. 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 translation 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.
• the CEL clone is missing 3' sequences.
• 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-rayc, interferon, c-jun , and c-fos (Meijlink et al., 1985, Proc. Natl.
• the 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 discrepancy 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.
• 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 for 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 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.
• FIG 4B hybridization to mRNA and subsequent SI digestion of a probe derived from FB29 produced protected fragments of 645 bp indicating 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 4C) .
• SUBSTITUTESHEET co a segment (exon 6) that is absent in FB27. Examination of the genomic DNA at a position corresponding to the position where the cDNAs diverge reveals a consensus splice donor
• FIG. 3 A schematic illustration of the differential splicing of three c-ski mRNAs as deduced from the nucleic acid sequence analysis of the cDNA clones is shown in Figure 3.
• SI analysis of total RNA derived from chicken embryos has confirmed the existence of two classes of mRNAs, those that do, and those that do not, contain exon 2.
• the existence of mRNA that contains exon 6 was confirmed. Utilizing this technique, the existence of mRNA lacking exon 6 which would correspond to FB27 cDNA, could not be demonstrated; however, several lines of argument suggest that the FB27 cDNA is not a simple cloning artefact.
• 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- ⁇ ki mRNA is currently favored.
• 18 of 20 bp are identical between c-ski and the pl9 region of gag in the parental ALV.
• This region of ho ology contains the 5 1 junction between viral sequences and v-ski .
• 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 recombination event.
• 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 fra eshift 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 predicted 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 a the transgene is shown schematically in Figure 6.
• ski portion of the transgene is already described (Sutrave and Hughes, 1989, Mol. Cell. Biol. 9 , 4046-4051; Sutrave et al., 1990, Mol. Cell. Biol. 10 , 3137- 3144) .
• 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 endonucleases. 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. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory) ] .
• 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 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. The 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) .
• 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 trans- gene except in skeletal muscle.
• the expression cassette was also introduced into fertilized pig eggs using standard procedures known to those in the art. Founder pigs show the expected muscle specific selection of expression of the gene and accordingly, it is expected that the pigs will have the same muscle phenotype as that seen in the founder mice.
• 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 fractioned by electrophoresis 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 ⁇ - 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 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 observation that expression was restricted to muscle was unexpected 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) .
• RNase protection assays were carried out as described by Melton (Melton et al., 1984, Nucl. Acids Res. 12 , 7035-7036) .
• a 1.8-kb Pv ⁇ l to Bgll fragment was subcloned in Bluescript KS vector and used to generate radioactively labeled RNA from the T7 promoter.
• Approximately 10 ⁇ g of total RNA was hybridized with 5 x 10 5 cpm of probe.
• 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 CI, pH 7.5, 0.3 M sodium chloride, 5 mM EDTA) and treated with RNase TI at a concentration of l u/ ⁇ l for 60 min 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.
• RNA 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 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 pheno- typically 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.
• tissue was homogenized in 1 ml of RIPA buffer, 20 mM Tris CI pH 7.5, 150 mM NaCl, 0.5% SDS, 0.5% NP40, 0.5% - 2 Q -
• the proteins were transferred to nitrocellulose membranes overnight in buffer containing 0.125 M Tris CI, 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 CI, 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 3 with TBS. Secondary incubations with rabbit anti mouse IgG were done for 2 hr at room temperature (1:2000 dilution from a l mg/ml stock).
• the filter was washed as described above and finally incubated with 5 ⁇ Ci of ,J5 I protein A (Amersham, sp. act. 30 mCi/mg) for 2 hr at room temperature.
• the filter was washed 3x with TBS and exposed to XAR Kodak film at -70°C for 6 days.
• Histology For histology, selected muscles from the line TG 8566 were isolated so that they remained attached at their origin and insertion and they were then fixed in 2% formaldehyde, 2% gluteraldehyde. Fixed muscle were transected precisely through the middle of the muscle belly and embedded in JB4 plastic (Polysciences, PA) . For im unocytochemistry, tissues were snap frozen in isopentane cooled in liquid nitrogen. The procedure for immunochemical staining was as outlined in Narusawa et al. [(1987), J. Cell. Biol. 104 , 447-459].
• transgenic animals were almost totally devoid of fat whereas control animals contained substantial amounts of subcutaneous and intraperitoneal fat. For this reason, there is little difference in weights between control and TG 8566 mice.
• skeletal abnormalities the tibia of transgenic animals is normal in size but was bowed cranially, apparently as an adaptation to accommodate the more than two ⁇ fold increase in size of the anterior tibia and extensor digitorum muscles.

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