JPH04502559A - Increased muscle tissue in animals - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Increased muscle tissue in animals Background of the invention field of invention The present invention relates to the c-ski gene. In particular, the invention provides chicken c-ski proteins. a DNA segment encoding a protein; a DNA construct containing the DNA segment; and regarding cells transformed thereby. The present invention further provides muscle size Concerning animals with increased growth.

Background information Virus containing the v-ski oncogene causes morphological transformation in vitro. Not only can it be rubbed, but it is also possible to induce myogenic differentiation (5t avnezer et al.

1981, J. Virol, 39.920-934; Li et al., 198 6. J, Virol.

57, 1065-1072; 5tavnezer et al., 1986. J, V irol, 57. 1073-1083; Colmenares and 5t avnezer, 1989. Ce1259゜293-303), c- Viruses harboring and expressing skicDNA also induce focal and myogenic differentiation. (5utrave et al.

1990, Mo1. Ce11. Rial, 1θ, 3137-3144 ), this means that the two known effects of ski, namely transformation and differentiation, are Given the seemingly contradictory properties, it is possible that the ski oncogene is bifunctional. This suggests the possibility of Genome for c-ski using v-ski probe A clone has been isolated and partially sequenced (5tavneze r et al., 1989. Mo1. Ce11. Biol, 9.4038-40 45), two forms of c-ski related by one splicing, and comparison of various y-ski and c-ski deletion mutants revealed that the trait The parts of ski required for conversion and differentiation were shown to be quite similar. child These results demonstrate that c-ski and V-ski cause transformation and induce differentiation. ski's abilities are related to a single characteristic of ski, rather than two separate functions. This suggests that this may be the case.

Not much is known about the biochemical function of the ski protein. studied All biologically active forms of C-5kt and v-ski are primarily localized within the nucleus. (Harkas et al., 1986. Virology 151. 13 1-138; 5utrave et al., 1990, Mo1. Ce11. Bio l, 10, 3137-3144), c-ski protein is present in chicken When overexpressed within cells, the different forms of c-ski localize within the nucleus. However, while these differences may be important, they remain irrelevant. It is clear. Overexpressed c-sk when chromatin condenses for cell division i proteins are condensed with condensed chromatin (5utrave et al., 1990 ．． Mol, Ce11. Biol.

10, 3137-3144), biochemical studies have shown that c-ski It is also possible that both forms can bind to DNA in the presence of other proteins. As shown (Nagase et al., 1990〜Nucl, Ac1ds Res, 18, 337-343).

Regarding its role in growth and development (if any) or mechanism of action It is possible to infer the normal functioning of c-ski to gain direct insight into the Capable data does not enable data.

Summary of the invention It is an object of the present invention to provide an isolated and characterized C-5kic DNA. Is Rukoto.

Another object of the present invention is to provide genes that increase muscle size in animals. That is.

Another object of the invention is to have increased muscle size and reduced adipose tissue. The aim is to provide livestock that

Another object of the invention is to treat patients suffering from severe muscle injuries or muscle degenerative diseases. It is to provide medical treatment.

Various other objects and advantages of the invention will be apparent from the drawings and the following description of the invention. It will become clear from this.

In one embodiment, the invention provides D It relates to an NA segment or an NA fragment complementary to said segment.

In another embodiment, the invention encodes a chicken c-ski protein. Relates to DNA constructs including DNA segments and vectors. Other embodiments In this regard, the present invention provides a truncated chicken C-5ki protein with the function of c-skl. A DNA construct containing a DNA segment encoding a protein and a vector. related. The present invention also provides for stable production of either of the two DNA constructs described above. A host cell that has been transformed to allow expression of the protein encoded by the product. Regarding cells.

In yet another embodiment, the invention relates to an animal with increased muscle size; 5121 protein introduced into the animal or its ancestors. A DNA construct containing a DNA segment encoding a quality and a vector. Ru. A DNA segment can encode the entire protein or a truncated variant. It can become a standard.

In other embodiments, the invention relates to an animal with increased muscle size; All of an animal's cells develop the functions of ski, which were introduced in the animal or its ancestors. The DNA segment and vector encoding the truncated ski protein are Contains a DNA construct containing.

In another embodiment, the invention provides that the proteins of the DNA constructs of the invention are expressed The DNA construct of the present invention is delivered to muscle under conditions that induce muscle growth. How to stimulate muscle growth or prevent muscle degeneration by reaching related.

In other embodiments, the invention provides that the proteins of the DNA constructs of the invention are The DNA constructs of the present invention can be used under conditions such that treatment is achieved. The present invention relates to a method of treating muscle degenerative diseases, which involves transmitting to the muscles involved.

Brief description of the drawing Figure 1: c-ski c DNN clone structure.

The length of the cDNA is drawn to scale and restriction sites are indicated.

y-ski is shown for comparison. Dotted four in y-ski The corner is the gag region of the gag-ski fusion during sensitive transformation of the virus SKV. shows. A, B, C and D are Ichikicho blocks for S1 nuclease protection analysis. indicates the area used to generate the curve. The number next to the arrow is the exon number. Match.

Figure 2: Complete coding sequence and partial coding region of FB29 type cDNA.

This indicates that the 5' end sequence of FB29 is at the 51 end of FB28 and CEL clones. It is assumed that they are similar.

↑ indicates the area where FB28 and CEL are active.

The 25 bases found only in the CEL clone are not shown. The number is v-skl indicates the boundaries of Exon boundaries are numbered and alternately spliced. Exons that have been removed are boxed. One base of c-ski and v-ski and amino acid changes are also boxed with dashed lines.

The translation stop codon is surrounded by a bold line. Possible polyadenylation signals are It is boldly underlined. Contains an ATTTA sequence that can be included in mRNA stability AT-rich regions are underlined with dashed lines.

Figure 3: For the C-5ki locus as predicted from cDNA sequence analysis. Explanatory diagram of generated alternating mRNAs.

Exons are not drawn at a constant rate. c-ski m RN A is v-ski shown in relation to. The black area is the non-coding region, while the white square is the cD This is the protein coding region of NA. With dots at both ends of y-ski The square is the gag region of the gag-ski fusion when transforming the ski virus. Ru. The relative positions of the putative translation start and stop codons are also indicated. There is. Figure 4 = 81 nuclease protection analysis of total RNA.

Figure 4A shows the uniformly labeled Ichiki probes used for hybridization. are shown diagrammatically below each photo, with thick lines representing cDNA sequences and thin lines representing cDNA sequences. Figure 2 shows a drawing representing the M13 arrangement. Overall length of probe and protection fragment Expected lengths are also shown. RNA hybridization is indicated above each column. It is. The numbers on the column (8, 10, 12, 15 or 17) indicate where the RNA was prepared. Indicates the age of the embryo. Figure 4B shows FB27/29 type Kpn 1-HindT! i hula Probe A (see FIG. 1) containing a component is shown. This probe is indicated by an arrow. One fragment of 645 base pairs (bp) and 262 and 272 b Two shorter fragments of l) were produced.

Figure 4C shows a protein containing the FB28 type Kpn I-H7ndm:7 fragment. 3 shows tube B (see Figure 1). This probe has a length of 534 bp as indicated by the arrow. One fragment was produced. No smaller fragments were detected.

Figure 4D shows a 497 bp Hindm fragment of type FB27 linked to the M13 sequence. Probe C (see FIG. 1) containing . The probe is a 497 bp fragment. yielded one fragment and two smaller fragments of 243/254. 479 Only bp fragments are indicated by arrows. r;14EjtFB28/29 110) A probe of length 111 abp containing the H4ndm 5'j Figure 1 shows part D (see Figure 1). Probe D is a 799bp flag indicated by an arrow ment occurred.

Figure 5: Alignment between c-ski and the p19 region of gag from avian leukemia virus. Column homology. The C-skl sequence is located at positions 218 to 242, and the p of the gag fl region. The 19 sequence is from position 63g to position 658. Homologous regions are boxed.

Figure 6: c-ski expression cassette.

Pvu I to Nru shown in the figure [segments are double digested plasmids] followed by isolation by gel electrophoresis.

Linear DNA is generated into transgenic mice by microinjection of fertilized eggs. was used to create a

Figure 7: Transgenic mice expressing c-ski and their normal littermates Child.

It is thought that the c-ski transgene will normally segregate through mating. The photo is muscle Heterozygous mice exhibiting a well-developed phenotype (forward) and DNA-negative littermates were show. Double-blind DNA analysis shows that the muscular phenotype coexists with the transgene. It was confirmed that the two were separated.

Figure 8 = Northern transcriptional analysis of transgene expression Panel A of TG8566 mice. Figure 2 shows an analysis of RNA isolated from various tissues of the United States. The top of the panel is a chicken. The autoradiogram after hybridization with c-ski is shown. to The predicted movement position of the c-ski message correctly transcribed from the Lance gene is 2. ．． It is 5kb.

A mark (Ski) is attached to a position corresponding to 2.5 kb.

The lower panel shows the results following hybridization to chicken β-actin cDNA. An autoradiogram from the same filter is shown below. β-actin cDNA is β -Hybridizes not only actin mRNA but also other actin messages tion will be made. Movement of both β-7 cutin and α-actin mRNA The predicted position of is shown on the right side of the panel.

Panel B: The autoradiogram shown in panel B shows that the RNA was Similar to that shown in the panel except that it is derived from a transgenic line It is.

The system used to prepare the RNA is shown on the far right of the figure. Panel BJ :The filters shown were performed at the same time, those shown in panel A were performed on different days. I was disappointed.

Figure 9. RNase protection of RNA from Nidorans genes.

The autoradiogram of the gel is shown at the top of the figure. The first column is after RNase digestion. Contains no antisense RNA probe. Next two columns do not add RNA or digestion after hybridization of the probe with addition of tRNA. Show the results. The next 8 columns are 4 types of transgenic hearts (Heart) or for RNA isolated from either skeletal muscle (Sk, Muscle). The results of hybridization are shown. The next column contains no transgenes. The results of hybridization of probes to RNA from skeletal muscle of mice (Control Sk, 1iuscle). Last column is molecular weight marker Contains. The bottom of the autoradiogram contains information about the antisense RNA probe. There is an explanatory diagram of the MSVLT Rc-ski expression cassette. T7 transcription is pBR1 begins in the middle of the c-ski coding region and via the MsV LTR 22 (denoted pBR). Transfers derived from transgenes If the transcription starts correctly, then the 984 base fragment will be protected. Probably.

Figure 10 Chicken c-ski protein expression in Nidoranszienik mice .

The extract was prepared from control mice (Control) or c-ski carrying the lance gene. ms from mouse liver (Liv) or skeletal muscle (Sk, M) that expresses Ta. The migration position of the radiolabeled molecular weight standard is shown on the left side of the figure.

Figure 11: (a) Control mouse and (b) 0 legs from T2O566 strain mouse Transverse sections prepared from the exact center of the fundus muscle. Both drawings are at the same magnification and are The size marker is 200μ. (C) Foot sole of control mouse and (d) affected Higher magnification drawing from the bottom of the mouse foot. Size in (C) and (d) The marker is 100μ.

Figure 12: Tissues in selected muscles from normal and transgenic mice. Distribution of fiber diameter.

Panel A: Diaphragm in transgenic mice expressing c-ski appears normal. Mice with a muscularly developed phenotype (TG8566) diaphragms from mice and from normal control mice were cut and obtained The number of individual myofibers in the transverse section area was recorded.

Panel B: Tibialis anterior muscle is greatly enlarged in mice from the T08566 strain. There is. Longitudinal sections were prepared from both transgenic and control mice. It was.

The number of fibers in each obtained transverse section area was recorded. This muscle is of two different types. type of fibers, some of which are more small, others larger (see also Figure 11).

Figure 13: Transgene expression in specific muscles from TG8566 20 μg of total RNA was fractionated by electrophoresis and transferred to a nitrocellulose membrane. Ru. Transcripts were first probed in chicken c-skz, then filtered into strips, The chicken β-actin cDNA was then searched again (C1eveland et al., 1980. Ce1l 20, 95-105), RNA is diaphragm, Isolated from plantar or large skeletal muscle (Sk Muscle).

Figure 14 = Prepared from the center of loam void isocavitis muscle of affected mice. Immunofluorescent staining of the sections.

(a) Staining with monoclonal antibody N0Q7 5 4D specific for slow MHC . Slow fibers are not hypertrophied.

(b) Staining with monoclonal antibody 5C711 specific for I[aMHC. I[a Fibers are not enlarged. (C) Monochrome that reacts with all fast MHC isoforms Staining with local antibody 2G3. All enlarged fibers stain with this antibody. (d) ffbMHc-specific m/aBP-F3 staining. Most, but not all, Large fibers are dyed.

Magnification: 230x.

Detailed description of the invention The present invention encodes all or a unique portion of the chicken c-ski protein. Concerning DNA segments. The DNA segment is derived from various chicken c-ski may encode one of the proteins, such as FB29, FB28 and FB27 . As used herein, a "unique portion" means at least 5 (or 6) amino acids, or at least 15 (or 18) equivalent nucleic acids. Defined as consisting of punch lines. The present invention also provides for such NA upper segments. and cells transformed with the same.

The present invention relates to the amino acid sequence of exon 6 shown in FIG. It relates to a DNA segment encoding the amino acid sequence of protein 7. The present invention also provides In addition to exon 7, exon 1, exon 2, exon 3, and at least 4 selected from the group consisting of exon 4, exon 5, or exon 6 A DNA segment further comprising two exons. That kind of NA top seg Examples include FB29, FB28 and FB27.

The DNA segments to which the present invention relates also include, for example, alleles of the amino acid sequence of FIG. Substantially similar proteins encoded in the exons of Figure 1 encompassing Contains things that encode quality. The present invention also provides an NA sequence complementary to the above sequence. Regarding ragment. Unique portions of the DNA segments of the invention or their complements The target segment is a probe for detecting the presence of complementary strands in a DNA or RNA sample. Can be used as a robe.

The invention further relates to the DNA construct or a host cell transformed therewith.

In one embodiment, the DNA construct of the invention comprises a c-ski protein of the invention. consisting of an encoding DNA segment and a vector, e.g. pMEXneo . In another embodiment, the DNA construct has a truncated c-ski function. -sk! DNA segments encoding proteins (e.g. ΔFB29) and and a vector (eg, pMEX+5eo). The DNA construct shapes the host cell. Suitable for transformation. The host cell is prokaryotic or preferably It may be a nuclear cell (eg, a mammalian cell).

The invention further relates to animals, such as livestock, with increased muscle size. increased The development of such breeds of muscular livestock is a major objective of normal breeding programs. continue. (As used herein, livestock is an animal raised for its meat, e.g. For example, pigs, chickens, turkeys, ducks, sheep, cows and fish, especially trout and Regarding catfish).

It is reasonable to introduce various genes into the germ line of mice and various types of livestock. This is a relatively normal operation for contractors. The present inventor has identified ΔFB29 and pMEX*eo vector. By introducing a DNA construct containing a protein into a fertilized egg, muscle size increases. created a new mouse. The founder mice generated and their descendants will have all of their The DNA construct is present in somatic cells and reproductive cells.

DNA constructs encoding ski proteins (e.g. c-ski proteins) introduced into fertilized eggs of animals (e.g., by microinjection). Resulting in a breed of animals with reduced muscle development and adipose tissue. As understood by those skilled in the art As would be expected, the animals with increased muscle size of the present invention also have various chicken that can be created using DNA encoding the ski protein from the species (Li is just such an example). Furthermore, the animals of the invention have proteins associated with ski. can be created using a DNA construct encoding a protein, such as the sno gene. .

The DNA segment ΔFB29 created by frameshift mutation is truncated. Produces deproteinization. However, as one of ordinary skill in the art would appreciate, Ming transgenic animals also contain the full-length ski protein, ski protein a DNA segment that encodes a part of a quality, e.g. one or two exons or a biologically active deletion derivative, e.g. a tip fused to a viral protein. A DNA construct containing v-ski expressing a truncated c-ski can be produced.

Furthermore, selective expression of proteins in muscle tissue was demonstrated using vectors other than pMEXneo. It is also understood that the DNA construct may be derived from a DNA construct created in a computer.

The present invention also stimulates muscle growth and prevents muscle degeneration in animals such as humans. Regarding how to prevent it. Injury resulting in loss of muscle tissue and muscle degeneration A possible treatment for neurological disorders would be to stimulate muscle growth. muscle loss In the case of loss, this treatment will include stimulation of tissue regeneration. On the other hand, neurological disorders In some cases, muscle growth may need to be independent of the evanescent nerve stimulant. Main departure According to Akira, muscle growth is encoded in a DNA construct that encodes the ski protein. The DNA construct is delivered to muscle tissue under conditions such that the encoded protein is expressed. It can be stimulated by reaching. The above constructs were constructed using standard methods known to those skilled in the art. Can be targeted and delivered to muscles.

The invention further relates to muscle degenerative diseases such as muscular dystrophy and amyotrophic lateral stiffness. Concerning how to treat Louis Gulick syndrome (also known as Louis Gulick disease). Osamu Treatment is achieved by expressing the protein encoded by the DNA construct of the present invention. The DNA construct of the invention is delivered to the affected muscle under conditions such that Consists of things.

Example Screening of cDNA libraries Two chicken cDNA libraries were Standard protocol (Maniatis et al., 1982. Molecular Cloning, A Laboratory Manual, Cold Spray Ring Harbor Institute.

v-ski probe (Cold Spring Harbor, NY) was screened. One library was isolated from the body wall of a 10-day-old embryo. The other is AEV-transformed chicken erythroblasts created from polyA'mRNA. It was created from mRNA isolated from.

Four different c-ski cDNA species were isolated, and eight were found in the body wall library. (These clones are designated as FB27゜FB28 and FB29. ), and one cDNA clone was from an erythroblast library (CE (marked as L). These cDNAs contain both of the ski parts present in the virus. It contained sequences extending 51 and 3' of. The cDNA is shown to be derived from a single cellular gene and encode a distinct ski protein. The multiple c-ski mRN A that converts into C-5ki by alternate splicing Leff et al. suggest that it is possible to produce from the genetic locus! 1986. Ann, Rev, Biochet 55, 1091-1117”), this is A growing list of oncogenes known to produce heavy RNA in a manner similar to (Ben-Neviah et al., 1986. Ce1l 44, 577-586; Levy et al., 1987+Mo1. Ce11. Bi ol, 7.4142-4145; Martinez et al., 1987.　5ci ence 237, 411-415; McGrath et al., 1983°Na ture 304.501-506).

Structure of cDNA clone DNA sequencing (Sanger et al., 1977, Proc. Natl., A. cad.

c- characterized by Sci. USA 74, 5463-5467) All structures of ski cDNA clones and their relationships with v-ski are shown in the figure. 1.

Only FB28 and CEL have the 5' sequence of y-ski; FB27 and FB Both of 29 are truncated at the 5' end. The missing sequence at the 5' end of FB29 is FB 28 and CEL, the hybrid nucleotide sequence and The predicted amino acid sequences for the and FB29 type cDNAs are shown in FIG. Really The first ATG with a natural downstream reading frame is located at nucleotide position 168. this There is no open reading frame upstream of ATG, and these sequences cover the 5' untranslated region. Suggest to represent.

Known splicing donors and splicing donors from cDNA sequence analysis and genome sequence Comparison of the positions of one part of the receptor (5tavnezer et al., 1989. Mo1. Ce11. Biol, 9.4038-4045), exon boundaries (See Figure 3). As seen in this drawing, the C-ski array is 7 distributed over two exons. FB29 contains all seven exons. FB28 and FB27 lack exon 2 and exon 6, respectively. There is. This unique splicing affects the protein-coding ability of the three cDNAs. echo.

Unusual splicing of exon 2 affects the coding ability of the open reading frame stream Deletes 37 amino acids without causing any damage. However, the unique sequence of exon 6 Pricing affects exon 7's coding ability. Exon 5 is an exon 7 (as seen in the FB27 cDNA), the translation A translation stop codon is generated at the splicing junction and exon 7 is a non-coding become sex exons. However, as in FB28/29, exon 5 and if exon 6 is spliced to exon 7, then this The open reading frame encodes an additional 129 amino acids in exon 7. 17 nucleotides follow.

The missing 51 end of FB29 and FB27 mRNA is present in FB28 and CEL. Assuming that the cDNA contains the same sequence as the existing one, then three different types of cDNA Translation of mRNA corresponding to the type will lead to the production of three types of proteins. . The first contains 750 amino acids (from FB29), the second contains 718 amino acids (from FB28) and the third protein contains 510 amino acids (from FB28) of amino acids (from FB27).

The CEL clone has lost the 3' sequence. It comes from the body wall (FB) library. The cDNA consists of 95 base pairs (bp) from nucleotides 2803 to 2898. ) has a long 31-untranslated region including an AT-rich region. Within this region, c- temporarily induced myc, interferon, c-jun and c-fos, etc. 2 copies of the mRNA deaminated sequence ATTTA in various mRNAs (Meij Hnk et al., 1985. Proc, Natl, Aca d, Set, USA82, 4987-4991; Ryder et al., 198 5. Proc, Natl, Acad.

Sci, USA 85, 1487-1491; Shaw et al., 1986. Ce1l 46.

659-667). Addition or deletion of these sequences may affect the transforming ability of c-fos. (Meijlink et al., 1985. Pro c, Natl, Acad, Sci, USA 112.4987-499 1).

c-ski c DN A has two strong positions located at 3348th and 4167th Contains a poly(8) signal (AATAAA). All three clones are FB Lyrics. Although it was isolated at the same location from A. braryi, it does not have a poly(A) tail.

Therefore, the 3' end of c-skimRNA is included in these clones. It seems that there is no such thing. 4.2 kb cDNA isolated and characterized A is 5, 7-8.0 kb and 10°0 kb detected by Northern transcription analysis. mRNA (Li et al., 1986. J, Virol, 57.1065-1 072) is smaller than. This discrepancy is not explained, but the lack of a poly(A) tail The clones isolated so far also lack sequences from the 3' end of the mRNA. It is suggested that

c-ski m 5' end of RN A Sequence comparisons at the 5' end indicate that both FB28 and CEL are linked to the putative translation initiator AT. This shows colinearity up to the 89 bp position upstream of the G codon. FB28 is an additional 7 6 bp, but CEL has 2 5 bp different from that found in FB28 exists. This region where the two sequences differ is compared with the sequence from the genomic clone. (5tavnezer et al., 1989. Mo1. Ce11. Biol, 9.4038-4045), and the upstream sequence in FB28 is the genomic sequence It is collinear with .

Examination of the genomic DNA sequences at the points where CEL and FB28 differ does not reveal consensus sequences for splicing sites. clone's S1 nuclease protection analysis was performed to confirm authenticity. This result is This shows that the sequence present in FB28 is expressed as mRNA in normal embryos. did. A similar 81 analysis was carried out in the 25 bp mRNA at the extreme 5' end of the CEL clone. gave no evidence for the existence of This allows the initial The 25 bases are the result of a cloning artifact and are found in the FB28 clone. It is suggested that the derived sequence is expressed in c-ski mRNA.

How much of the 5' untranslated region of c-skimRNA is contained in FB28? Primer extension analysis was performed to determine whether Exists in FB2B Replication of the 5' segment should yield an extension product of approximately 280 bases. . However, a 220 base primer extension product was observed. 81 analysis data showed that this segment is expressed in RNA. Observed pla Although the imer extension products may be the result of premature termination, c-ski mR It is also possible to have multiple 5' ends for NA.

Mechanism of internal exons Sequence ratio in the core of FB27, FB28 and FB29 and CEL cDNA The comparison is 1ll of 1079-1191 where FB28 would lose 37 amino acids. bp (exon 2).

Genomic sequences in the corresponding regions (5tavnezer et al., 1989.M.

1, Ce11. R7a1.9.4038-4045) has separate cDNAs at the border of the deletion, suggesting that it is derived from an mRNA spliced into reveals the existence of consensus splicing donor and acceptor sequences for Shown below.

81 analysis to confirm the presence of FB28 and FB28/29 type mRNA. was conducted for all RNs.

Total RNA was extracted using standard protocols (Chirgwin et al., 1979. Bioch emistry 1B, 5294-5299) using 8.10, 12. It was isolated from 15 and 17 day old chicken embryos. Total RNA approximately 20 to 30μ g is the standard protocol (Maniatis et al., 19B2. Cloning, A Laboratory Manual.

Cold Spring Harbor Laboratory 2 Cold Spring, New York ・Haber) was used for nuclear S1 analysis.

The region between the Kpn I and Hindm sites of FB29/27 or FB28 is A uniformly labeled Ichiki probe (probes A and B in Figure 1) was applied to the whole cell. Hybridized with RNA. As shown in rM4B, mRNA Hybridization to 645 bp showing hybridization to FB27/29 type mRNA. and the probe hybridize with FB2a type mRNA. The expected 262/272bp fragment was obtained when .

FB28 (probe B) protected a 534 bp fragment (see Figure 40) . Hybridization of FB28 probe to FB27/29 type mRNA Sl is outside the loop of the RNA, although smaller fragments from does not always cut effectively opposite DNA probes to the region of these The test consisted of mRNA corresponding to both FB27/29 types containing the second exon and The mRNA corresponding to the FB28 type lacking the second exon is expressed in normal cells. Show that.

Mechanism of 3′ coding exons Sequence comparison of three fibroblast clones shows that FB28 and FB29 are similar to FB27. It was shown to contain the missing segment (exon 6). cDNA is different The test of genomic DNA at a position corresponding to the position of the consensus splicin Revealing Gudner (5tavnezer et al., 1989.Mo1. Ce11. Biol, 9.4038-4045), the two cDNAs are thought to derive from splicing to include or exclude exon 6 It will be done. Downstream of this mutual exon, the 3' portions of all three cDNAs are identical. be.

To examine whether both types of cDNA represent normal cellular mRNA In total, 81 analyzes were performed. The uniformly labeled Ikki style probe is at the 3' end of FB28. 799b pH1ndT from the end! i fragment (see Plow 102 Figure 1) and 49 from the 3' end of FB27 subcloned into M13mp18. 7b pH1ndTIi”: Produced from l fragment (probe C).- Heavy chain probes were prepared and total R prepared from whole chicken embryos of different ages. Hybridization was performed with 20 μg of NA.

Hybridization of RNA to probe D (FB28) as shown in Figure 4E. tion and subsequent S1 treatment, if FB28/29 type mRNA is present, A 799 bp fragment was obtained, as would be expected in this case.

Generated by the FB28 probe that hybridizes with FB27 type mRNA. Smaller fragments that would have formed were not detected. FB27 blow 81 digestion of probe C) resulted in a 497 bp fragment and a 243 bp fragment. Two smaller fragments of 254 bp were obtained (see Figure 4D). child These smaller fragments indicate that the FB27 probe is similar to the mR of the FB28/29 type. This is thought to result from 81 cleavage of the probe at a site different from NA. these The data confirm the presence of FB28/29 type mRNA, but FB27 mRNA If A exists, it is considered to exist at a lower level than FB 28/29. It will be done.

unique splicing Three types of c-skim as predicted from nucleic acid sequence analysis of cDNA clones An illustration of the unique splicing of RNA is shown in FIG. Chicken embryo? By S1 analysis of total RNA derived from The existence of two classes of mRNA was confirmed. Using a similar protocol, The presence of mRNA containing exon 6 was confirmed. Using this technology, FB27 Exon 6, which would correspond to cDNA, is missing; the presence of mRNA cannot be demonstrated. However, a series of arguments have been made regarding whether the FB27 cDNA is simply a lonning artifact. It suggests that something is wrong. Sequences not found in FB27 are linked by well-defined splicing junctions. combined (testing both cDNA and available genomic DNA sequences) to be judged more). Perhaps partially processed mRNA is reversed cDNAs containing one or more introns are sometimes obtained from However, the isolation of cDNA with artificially deleted exons is based on theory. Probably (very few, if any) in the creation of cDNA libraries. It seems to be a rare occurrence. For these reasons, FB27 If relatively rare, the understanding that it represents an actual C-5kimRNA is currently supported.

Comparison between c-ski and v-ski Retroviral transduction of c-onc involves tip excision in c-onCi; often result in deletions or point mutations (Bishop, J. M., 19 83. Ann Rev, Bjochem, 52, 301-3”4) e Comparison of the c-ski and v-ski sequences revealed that the v-ski gene was truncated at both the and 3′ ends, and only part of the c-ski coding region (see Figures 1 and 3). Seask! The array is 244 starting at position (the putative initiator ATG is at position 168), and 154 C that ends in 1st place (see Figure 3).

The biological significance of the 51 and 3' tip truncation is unknown. v-ski is “C ” and c-skr grows rTJ at position 1284, relative to c-ski. Tev-ski has only one base change. This base change occurs in c-ski. Change from Trp to Arg in Cski. Deletion analysis of v-ski , the above amino acid changes do not play an important role in the transforming ability of v-s’ki. It means that. The biological activity of cDNA is determined by replication-competent retroviral vectors. (Hughes et al., 1987. J, Vjrol, 61.3004- 3012) was evaluated by expressing code polarization from cDNA. Ru.

As shown in FIG. 5, 18 bp out of 20 bp are C-sk2 and l! to ALV It is identical to the p19 region of gag. This region of homology with the viral sequence Contains a 5l joint between the If there is such a long range homology For example, it is not possible to accurately identify the 5' recombination junction. Substantial homology is The 3'5kil ALV junction cannot be precisely identified. However, 31 joints Immediately downstream, c-ski was found at 3' of the v-ski/A L V junction. There are human LV sequences that are highly homologous to the segment.

It is possible to cause this region of homology in the juxtaposition of nucleic acids involved in recombination. It is.

Transduction mechanism The exact mechanism by which retroviruses require cellular oncogenes remains unclear. Ru. In the first step, the viral DNA is placed next to or within the cellular oncogene. (Bishop, J. M., 198 3, Ann Rev, Biochem, 52, 301-354) o The retrovirus and cell sequences were then determined using the DNA deletion method (B15hop, J. M.

1983, Ann Rev. Biochem, 52, 301-354; C Zernjlofsky et al., 1983. Nature 301.736-7 38) or RNA read-through (Herman et al., 1987. ce 236, 845-848; Ni1sen et al., 1985. Ce1 41, 1719-1726), but the homology is Not known to be related to any of the methods. In the case of ski, c- Comparison of the SKI sequence and the p19 coding region shows the number of steps involved in the generation of the SKI virus. This means that the process involved homologous recombination. However, homologous sets The replacement may be secondary. the original process is non-homologous and involves direct repetition If a viral genome is generated, as observed with Louis malignant tumor virus, Perhaps through copy selection during reverse transcription, sequences between the repeats are assembled during virus culture. It would be expected that it could be eliminated by changing the However, oncogene model in which c-onc is required by homologous recombination between c-onc and a replication-competent virus. It is possible to make a proposal. A short length of homology is present at both ends of the viral m tumor gene (B Anner et al., 1985. Mo1. Ce11. Biol, 5.140 0-1407; Van Beveren et al., 1983. Ce1l 32, 1241-1255) or the left or 5' recombination junction (Besmer etc., +986°Nature (London) 320. 415-421; Roebroek et al., 1987. J.

Virol, 61.2009-2016). This data is , 51 does not allow determination of whether homologous recombination is primary or secondary. stomach.

Creation of transgenes and transgenic mice. FB29 contains sequences derived from coding exons and is determined by DNA sequence. isolated and encodes the 750 amino acid c-ski protein. The largest of the cDNAs was used to create the derivative ΔFB29. ΔFB29 is the There is a frameshift mutation at position 1475 within the coding exon of 5-Continued 1 out of 5 Cs disappeared in the frameshift mutant), and The first 436 amino acids are the first 436 amino acids of c-ski FB29 body. It is predicted to yield a protein of 448 amino acids that is identical to the amino acid ( The last 12 amino acids undergo a frameshift mutation, so c-ski (Different from those of 29 FBs). Used in transgene preparation ΔFB29 is schematically shown in FIG.

The construction of the ski portion of the transgene has been previously described (5utrave and and Hughes, 1989. Mo1. Ce11. Biol, 9゜4 046-4051: 5utrave et al., 1990. Mo1. Ce1l, Biol, 10.

3137-3144). In short, a tip-truncated chicken C called ΔFB29 -5ki CD NA is pre-loaded into adapter plasmid C1a 12Nc　o. Loaned. The ΔFB29 segment is converted into an adapter plug by C1a1 digestion. The 51 protrusions were cut out from the Sumid f:, coLi DNA polymer. Fill in using the Flenow fragment of enzyme I and all four dNTPs. child The plant-terminal fragment of was digested with EcoRI restriction enzyme and It was ligated to the pMEXneo vector which had undergone plant termination with the fragment. Positive Select a clone that has inserted in the correct orientation and insert both pvu1 and NγuI restriction endonuclease. These enzymes are MSV LTR and S Segment containing the FB2ecDNA person flanked by V40 polyA signals (see Figure 6). This segment was gel-purified and inserted into fertilized mouse eggs. used for injection (Hogan et al., 1986. Manipulatin g the mouseembryo, A Laboratory Manu al. (Cold Spring Harbor Laboratory, Cold Spring, NY) Pulling Harbor)].

ΔFB29io-:z+tMSV between LTR and SV40 polyadenylation site pMEX expression plus orientation such that there is a tip truncated c-ski cDNA It is placed in the middle (see Figure 6). This plasmid was used as pvu l and Nru The expression cassette was released by digestion with [. The expression cassette was determined by gel electrophoresis. It was purified and introduced into mouse fertilized eggs by microinjection.

Forty-four founder mice were obtained after two separate injections. The mouse was isolated from the tail amputation. Identification was made by dot plot analysis of isolated DNA. This analysis was performed using Southern transcription. Confirmed by.

Three of the 44 founder mice showed a clear muscle phenotype (TG8566, TG8821 and TG8562). These three founder mice and the complete trans one animal that contains an unrearranged copy of the gene but does not exhibit the phenotype. Mice (TG8542) were used to generate the system. TG8566, TG Southern transcriptional analysis of DNA from 8821, TG8562 and TG8542 The integration site of the transgene in each prefecture is different, and the copy number is different per genome. It is suggested that it ranges from about 5-35 copies.

DNA positive from three lines (TG8566, TG8821 and TG8562) The mice had a similar distinct appearance resulting in abnormal muscle growth. three types of ma Both lines have an increased incidence of tumors, although mice carry oncogenes. It doesn't seem like it does. If the chicken is not injected with infected cells, v-ski The above results are entirely expected since the virus is not tumorigenic in the bird. No CB, 5tavnezer, 1988. The Oncogene Handbook, B, P, Reddy, A, Akalka and T , edited by Curran.

(Amsterdam: Elsevier Science Publishers) pp. 393-401]. 3 Two strains of mice do not express high levels of the transgene except in skeletal muscle. this This means that c-ski directly affects skeletal muscle cells and is a modified locomotor. This is consistent with the finding that it is not a secondary outcome of menstrual function. Most skeletal muscles are covered Included. Mice with this phenotype are characterized by the appearance of enlarged forelimbs and jaw muscles. can be easily identified (Figure 7). Since the phenotype is obtained in eight separate founders, The most plausible explanation is that the phenotype is caused by the chicken c-ski transgene. It means that it will be done. Therefore, all four mouse systems, developed expressions of muscle Three lines with this type and one line with no observable phenotype were found to be transgenic. tested for the expression of

Expression cassettes were also introduced into fertilized pig eggs using standard methods known in the art.

Founder pigs show a predicted muscle-specific selection for gene expression, thus that the pigs would have a muscle phenotype similar to that seen in the founder mice; is predicted.

DNA/RNA analysis Total cellular DNA and RNA were isolated by standard methods. for RNA isolation , the tissue was cut into small pieces and homogenized in RNA sol (Cina Biotex). were immediately frozen in liquid nitrogen and processed according to the manufacturer's recommendations. . For northern transcriptional analysis, 20 μg of total RNA from different tissues was collected at 2.2 Fractionated by electrophoresis on a 1.596 agarose gel containing M formaldehyde. Ta. Transfer RNA to nitrocellulose membrane and nick-translate chicken 5 using either kicDNA or chicken β-actin cDNA as a probe. I tested it. The coding region of β-actin cDNA is a messenger for other effects. for the quantitation and qualification of RNA from most tissues. can be used.

Total RNA from spleen, lungs, brain, kidneys, liver, stomach, heart, and leg (skeletal) muscles and the results are shown in FIG. All three species with different phenotypes All systems (TG8566, TG8821 and TG8562) are highly expressed in skeletal muscle. Although some systems expressed the chicken c-ski specific transcript at a level of 2.5 kb, mice show low levels of chicken c-ski RNA in other tissues. did. The T08562 line is transgenic in the heart, although at a lower level than in skeletal muscle. It has RNA from offspring. Histopathology of the heart from TO8562 mice shows that the group It was shown that there was no significant effect on the texture. TG85 does not show any phenotype The 42 line showed a lower yield from the transgene in muscle than the line that showed the phenotype. It had high levels of RNA. MSV LTR was linked to other genes (Khillan). et al., 1987. Genes Dev, l, 1327-1335), The observation that expression was restricted to muscle was unexpected.

ski) Transcription is initiated at the appropriate site in the tissue expressing the lance gene. An RNase protection assay was described by MeltOn to determine whether (Melton et al., 1984. Nucl, Ac1d s Res, 12, 7035-7036), Pvu I to BgL A 1.8 kb segment of I was subcloned in the Bluescript KS vector. and used to generate radiolabeled RNA from the T7 promoter. . 10 μg of total RNA was hybridized with 5 x 10’ cpm of probe. I set it. Hybridization was performed in 80% formamide and 1x buffer (5x buffer). Hybridization buffer was 0.2MP Pipes, pH 6,4, 2M NaCl. The reaction was carried out overnight at 50° C. in 200° C., 5 mM EDTA). hybridization After digestion, the sample was dissolved in ribonuclease digestion buffer (10mM) Lys CI, pH 7, 5,0,8M sodium chloride, 5mM EDTA). ZeT1 and Iu/μ! It was treated at 30°C for 60 minutes at a concentration of

20% SDS 10μ! and proteinase K (preservation solution 10 mg/mA) 4 μm! RNase digestion was stopped by adding and incubating at 37°C for 15 minutes. . The digested sample was extracted with phenol-chloroform (1:1 mixture) and the carrier tRN A was precipitated with ethanol. Rinse the pellet once with 70% ethanol and dry. and a hotel containing bromophenol blue and xylene cyatool dyes. Dissolved in Lumamide. The samples were denatured at 100°C and 7.5M urea-containing. Separated on a 6% polyacrylamide gel.

Uniformly labeled antisense RNA spreads across MSV L, TR and c-ski. (See Figure 9). 3 Using RNA from a positive transgenic line of the species, a protective flap of approximately 980 bases A fragment is seen, indicating that transcription is opening at the actual start site within the MSV LTR. This is the expected size when the system is started (see Figure 9). This analysis also transgenes in cardiac and skeletal muscle of both positive and phenotype-negative mouse strains. Provides a more quantitative assessment of gene RNA levels. Figure 9 shows the inside of the heart of TG8821. levels of transgene RNA are lower than those found in skeletal muscle. (approximately 1/10 to 1/20). Furthermore, this analysis transgene R with low but detectable levels in skeletal muscle, Indicates that N A exists. These data indicate that the muscle-developed phenotype is similar to that of chickens. c-ski) not only associated with lance gene expression, but also producing muscle phenotype. A minimum threshold level of c-ski RNA must be achieved in order to It suggests that there is no. In fact, these data are useful for looking at the effects of transgenes. The minimum threshold level for chickens is high, probably due to the endogenous c- ski expression (5utrave and Hughes, 1989.Mo1.C e11. Biol, 9.4046-4051). It suggests that.

protein analysis The underlying assumption is that C-5ki RNA expression directly produces a muscular phenotype. This does not necessarily mean that the expression type is due to the presence of c-ski protein. That is to say. Therefore, the c-ski protein is observed in Sei. Rabbit anti-antibody that specifically recognizes the 50kd form of c-skt Although serum was prepared (Sutrave et al., 1990. Mo1. Ce 11. Biol, 10, 3137-3144), these antisera were Does not work well in Stan transcription assays. Recognizes c-ski and Mouse monoclonal antibodies have been developed that work well in transcriptional assays. However, the use of these reagents presents technical problems. These monoclonal antibodies were not available in sufficient quantities to allow direct labeling. For example, labeled rabbit anti-mouse The indirect standard method used is not only anti-ski monoclonal, but also endogenous murine stalwarts. was also detected, together with the 50kd body of c-ski produced from the transgene. Move to. To solve this problem, control and transgenic Extracts of muscle and control tissue (liver) from mice were prepared. endogenous mouse anti Bodies were removed from these extracts by precipitation with rabbit anti-mouse antibody as described below. It was.

Detection of ski protein in tissues from transgenic and control mice To prepare, add 1-5 mg of tissue to RIPA buffer (1 mf, 20 mM) 5,150mM NaCl, 0.5%SDS, 0.5%NP40.0.5%De Sodium oxycholate, 1mM EDTA, 1mM PMSF and 35μ/ mi! Homogenized in abroteinin. Homogenate to 11000Orp The cells were clarified by centrifugation for 10 minutes. 1mg/mi rabbit anti-mouse lgG Mouse IgG was removed from the supernatant by incubation for 2 hours on ice with 10μi’ (in PBS). was removed from 100 μl. The complex was prepared using 40% protein A in RIPA buffer. It was removed by adding 100 μl of Sepharose beads. The supernatant produced Collected, and 20μ! was fractionated on a 10% SDS polyacrylamide gel. It was done. The protein was purified with 0.125M Tris C1, 0,092M glycine and Transfer to nitrocellulose overnight in buffer containing 20% methanol, pH 8.3. did. TBS buffer (0,5M) Lis C1, pH 7,4 and 0.2M NaCl Block the filter for 2 hours at room temperature with 4% dry skim milk in and a mixture of three anti-ski monoclonal antibodies at a dilution of 3000 for 2 hours. It was kept warm and then washed three times with TBS. Second round with rabbit anti-mouse IgG Incubation was carried out at room temperature for 2 hours (1 mg/m1 from the stock solution 1 = 200 0 dilution). The filters were washed as above and finally washed with 5I protein A. (Amalsham, specific activity 30 mCi/mg) and incubated at room temperature for 2 hours with 5 μCi .

Filters were washed three times with TBS and placed on MAR Kodak film at -70°C. Exposure was made for 6 days.

Only the affected tissue (skeletal muscle) from the transgenic animal has a 50 kd c -ski protein (Figure 10). These data also indicate that three positive suggesting that there may be differences in the levels of c-ski protein in the muscles of the system. However, the operational complexity of this experiment makes quantitative understanding an uncertain issue.

histology For histology, selected muscles from the TG8566 line were analyzed at the point of origin and engraftment. Separated to remain attached, then 2% formaldehyde, 2% glutaric Fixed in aldehyde. Cut the fixed muscle precisely through the center of the muscle ampullae. and into JB4 Plastics (Polysciences, Pennsylvania). Embedded.

For immunocytochemistry, tissues were flash frozen in isopentane cooled in liquid nitrogen. did. The procedure for immunochemical staining roughly followed Narusawa et al. ((198 7), J, Ce11. Bi.

1, 104.447-459).

The results show that there is little transgene expression and that its action is restricted to muscle. At least in the TG8566 system, the expression and action of showed that it is limited to specific fiber types. However, it was affected Fibers are not all of the same type.

Myocardium was normal and there were no visceral smooth muscle abnormalities in these animals. . Figures 11a and 11b are from adult male control mice and transgenic mice. Compare transverse sections accurately made from the center of the plantaris muscle.

The cross section area of the control was 2.7 μ2, and that of the TC;8566 mouse was the control value. It is 9°4μ2, which is more than twice the angle of 9°4μ2. This increased growth is generalized. beside Comparable increases in cross-sectional sections were observed through male and female mice of the TG8566 strain. It was found in almost all axial and appendiceal muscles. Only 3 muscles: tongue, diaphragm and The sole of the foot was found to be normal. These are used in transgenic mice. and are the same size as those of control mice (see Figure 12). RNA is TG856 isolated from the diaphragm and plantaris muscles of 6 mice. Northern transcriptional analysis revealed that chicken Levels of avian c-ski RNA were significantly lower in these two groups than in muscles that received syngeneic action. is shown to be lower in two phenotypically normal muscles (Figure 13).

The most obvious other gross morphological abnormalities are those found in transgenic animals. Control animals had a substantial amount of subcutaneous fat and intraperitoneal tissue, whereas control animals had almost no total fat. Contains fat. For this purpose, control mice and T08566 mice were given a body weight There is almost no difference. The tibiae of Nidoransgenic animals, which also have skeletal abnormalities, Normal in size, but clearly more than twice the size of the anterior tibia and extensor digitorum. It is curved like the skull as an adaptation to accommodate the growth above.

Muscles in control mice consist of fibers with a range of cross-section areas. big The range of growth is greatly increased in TG8566 mice (Figs. 11 and 1). 2).

Not all fibers are affected. hypertrophy is limited to a selected number of fibers (Fig. 11d). Hypertrophy of these fibers increases muscle mass in the TG8566 system. Clearly explains the increase. The number of fibers is said to increase significantly in individual muscles. There was no evidence. For example, the number of fibers in the control sole is 912 ± 111 (n = 3), and for TG8566 it is 991±87 (n=3). Similarly, TG8 No difference was observed in the total number of fibers in the extensor digitorum longus muscle between 566 and control mice. won.

To investigate which types of fibers are affected in the TG856B system, lo-myosin heavyweight (MHC) (NOQ7 5 4D, Narusawa et al., 19 87. J, Ce11. Biol, 104.447-459), all Fast MHC (2G3゜Narusawa et al., 1987. J, Ce11 ．． Biol, 104. 447-459) It type fast MHC (S C 711, 5chiaffino et al., 1989°J, Muscle Res, and Ce1l Motil, 10, 197-205) and nb type filter. Fast MHC (BP-F 3, 5chiaffino, etc.).

1989, J. Muscle Res, and Ce1l Motil, 10 , 197-205) were used. It was. Figure 14 shows loam void is cavitides muscle with these three types of antibodies. Immunofluorescent staining of the sections is shown.

There was no evidence of increased slow fibers (Fig. 14a), and the TG8566 matrix The total number of slow fibers (120) in the rhomboid iscavitis muscle from the Number of slow fibers found in the loam-voidois cavitis muscle from the mouse Approximate to (117). I [Many type A fibers are located between hypertrophic fibers, and The shape is often distorted, as if being compressed by the spread of the Figure 14b).

Small fast type 1A fibers and all hypertrophic fibers were treated with monoclonal antibody 2G3. (rEI14c), all indicating that the hypertrophic fibers are fast. B Most, but not all, large fibers are also In addition, I [bMHC-specific monoclonal antibody BP-P3 (stained with monoclonal antibody BP-P3) Figure 14d). In the plantar foot, there are many differences in reactivity, and 50% of hypertrophic fibers stained with BF-P3. These results in TG85613 mice The hypertrophic transformation of the fibers is shown to involve at least two types of fast fibers. these One of them is the mb type. By exclusion, we are the other ■.

Fibers (5chiaffino et al., 1989. J, Muscle Res, and CellMotil, 1θ, 197-205; Termin et al. , 1989. Histochemistry 92, 453-457; Gorza, 1990. J, Histochem, Cytochem, 38.257-265).

Staining of hypertrophic fibers after acid pre-incubation with actomyosin ATPase histochemical reaction Differences in DPNH staining and PAS staining for mitochondrial enzyme activity In this strain of transgenic mice, all of the fast fiber types are 1 or more. This supports the conclusion that it is affected.

These results also support the interpretation that both mb and πX fibers are enlarged. hold

Occasional necrotic and regenerative fibers were present in but not all muscles of T08566 mice. Found a few. In the hind leg, these are most extensively located in the tibialis anterior muscle. They appear to be loamoid cavities, and the superficial muscles of the neck are indeterminate. It was not found.

*Zero Zero Zero Ne Ne All publications mentioned above are incorporated herein by reference in their entirety.

Although the present invention has been described in some detail for clarity and understanding, After reading the disclosure of the specification, those skilled in the art will realize that various changes in form and detail can be made to the invention. It will be understood that what may be done without departing from the true scope and scope of the claims. cormorant.

death.

ATG TACAAG AAA GACAAT GGCAA GACCCA GCCW CCT GTA CTG CATMetTyrLysLysAspA snGlyLys^5pProAlaGluProValLeuH4sCTG CCCCCV ATCCAG CCCCCCCCCGTG ATG CCT G GT CCCTrCTTCATG CCC395Leu Pro Pro I @ Gln Pro Pro Pro Val Met Pro Gly Pr o Ph@Phe Met oro 76 ATCTCCTGCTTCGTG GTG GGT GGG (aAA AAG CGCCTT TGCTrG CCCCAG ATC14X4 11e S@r Cys Phe Val Vat Gly Gly Glu Lys Arg Leu Cys Leu Pro Gln@lie 109 CTG AACTCG GTG (7CAGG IIJc TTCTCCCTG CAG CAG ATCAAT TCG GTGLeu Asn Ser V al　L@u　Arg^sp　Phe　Ser　Leu　Gin　Gln　II s　Asn　Ser　ValTGCGAT　CAG　CTA　CACATr　T ACTGCTCCAGA TGCACCGCT (aAc CAG CTG G AG 5X3 Cys Asp Glu Leu H4s lie Tyr Cys Ser^ rg Cys Thr^1sho^sp Gln Leu Gl Fu@! 42 FjG, 2 GACTCTGCA AACTGI:t AGG TCCTACATCCTCC TT AGCCAG 1. AT TACACT GGG 9W9 Asp Ser^la Asn Trp Arg Ser Tyr X1e L eu Leu Ser Gln ALP TF Thr G attack @274 N) me FIG. 4A FIG.5 FIG. 6 FIG.7 TG 8566 FIG, 8A FIG. 8B FIG.9 Control TG 8821 FIG. 10 TG 8566 monthir/j FIG. 13 FIG, 14A FIG, 14B Submission of translation of written amendment (Article 184-8 of the Patent Law)

Claims (32)

[Claims] 1. A DNA segment encoding the chicken c-ski protein or the D DNA fragment complementary to the NA segment. 2. DNA segment containing exon 6 described in Figure 1. 3. DNA segment containing exon 7 described in Figure 1. 4. Exon 1, exon 2, exon 3, exon 4, and exon described in Figure 1 at least four exons selected from the group consisting of exon 5 and exon 6; 4. The DNA segment of claim 3, further comprising: 5. i) a DNA segment according to claim 1, and ii) a vector. A DNA construct containing. 6. 6. The construct according to claim 5, wherein the vector is pMEXneo. 7. i) encodes a truncated c-ski protein with the function of c-ski; and ii) a vector. A DNA construct containing. 8. 8. The DNA construct according to claim 7, wherein the DNA segment is ΔFB29. 9. The DNA construct according to claim 7, wherein the vector is pMEXneo. 10. 6. Enables expression of said protein encoded in the construct of claim 5. A host cell stably transformed with said construct in a manner that allows said construct to function. 11. 8. Enables expression of said protein encoded in the construct of claim 7. A host cell stably transformed with said construct in a manner that allows said construct to function. 12. The host cell according to claim 10 or 11, which is a mammalian cell. 13. all of the cells have the ski protein introduced into the animal or the progeny of said animal. a DNA construct consisting of a DNA segment encoding a vector and a vector; Animals with increased muscle size. 14. 14. The animal according to claim 13, wherein the ski protein is a c-ski protein. 15. 14. The animal according to claim 13, wherein the protein is chicken c-ski protein. . 16. 14. The animal according to claim 13, wherein the vector is pMEXneo. 17. 14. The animal according to claim 13, which is a domestic animal. 18. 18. The animal according to claim 17, which is a petfish. 19. 14. The animal according to claim 13, which is a mammal. 20. 20. The mammal according to claim 19, which is a mouse. 21. All of the cells have the function of ski introduced into the animal or the progeny of the animal. DNA segments and vectors encoding the truncated ski protein An animal with increased muscle size containing a DNA construct consisting of. 22. 22. The animal according to claim 21, wherein the protein is chicken c-ski protein. . 23. 22. The animal of claim 21, wherein the DNA segment is ΔFB29. 24. 22. The DNA segment encodes a v-ski protein. animals. 25. 22. The animal according to claim 21, wherein the vector is pMEXneo. 26. 22. The animal according to claim 21, which is a domestic animal. 27. 27. The animal according to claim 26, which is a petfish. 28. 22. The animal according to claim 21, which is a mammal. 29. 29. The mammal according to claim 28, which is a mouse. 30. A DNA segment encoding the ski protein and a vector. under conditions such that the protein of the DNA construct is expressed and muscle growth is induced. stimulating muscle growth, comprising transmitting said DNA construct to the muscle; or how to prevent muscle degeneration. 31. A DNA segment encoding the ski protein and a vector. under conditions such that the protein of the DNA construct is expressed and therapy is achieved; A muscle degenerative disease consisting of transmitting said DNA construct to the affected muscle. How to treat. 32. A claim in which the muscle degenerative disease is muscular dystrophy or amyotrophic lateral sclerosis 31. The method described in 31.