JP2003274961A - Vector for mammary gland expression - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a vector for mammary gland expression for transduction of a gene so as to exhibit expression having no position effect and expression dependent on the number of copies in the production of a transgenic animal. <P>SOLUTION: The vector for mammary gland expression is specified by the restriction enzyme cleavage map (B is BamHI; C is ClaI; E is EcoRV; C is SmaI; X is Xhol). <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a mammary gland expression vector for introducing a gene so as to express mammary gland specifically in the production of transgenic animals.

[0002]

2. Description of the Related Art With the establishment of gene manipulation technology, a drug (biopharmaceutical product) has been developed that produces rare human proteins, which have not been obtained in the past, by using them in recombinant microorganisms or recombinant animal cells, and has excellent efficacy. Sex is recognized in the medical field. However, in the production of biopharmaceuticals, it is difficult to produce the target protein encoded by the transgene with high efficiency, and the production cost is high. Therefore, research and development has been conducted on a technology (transgenic milk) for efficiently producing useful proteins in milk that can be used for biopharmaceuticals, etc., by utilizing the excellent protein synthesizing ability of mammals. For example, in Bio / Technology 9, 830-834 (1991), production of human α1-antitrypsin in sheep milk, WO Publication 9
4/05796 discloses human protein C in pig milk.
, Bio / Technology 12, 699-702 (1994) reported the production of human and tPA in goat milk. Since these animals can efficiently produce useful substances in milk (high-concentration production), it is expected to provide a very inexpensive method for producing useful substances.

However, there are still problems to be overcome in producing livestock producing these transgenic milks. That is, the efficiency of gene transfer in livestock is as low as about 1% in pigs and sheep and about 0.1% in cattle. Furthermore, even if the gene is introduced, only a part of the individuals recognize the expression of the introduced gene. This is called the position effect, which is a phenomenon in which the expression level varies greatly depending on the transgenic animal line, and expression that is not proportional to the copy number of the introduced gene occurs. It is thought that this is because the introduced gene is introduced into the heterochromatin region of the chromosome, or the expression of the gene is suppressed by being heterochromatinized by introducing multiple copies of genes in a lined form. However, it is believed that the original gene has sites in upstream and downstream regions that prevent position effects. In addition to such low production efficiency, the breeding cost of the livestock that does not express the introduced gene makes the production cost of the desired livestock enormous.

As described above, the enormous cost of producing livestock for producing transgenic milk makes it impossible to take advantage of the advantage of transgenic milk, which is a low production cost for producing useful substances. . In order to solve such problems, a yeast artificial chromosome (YAC) vector (Molec. R) containing a wide control region of the human α-lactalbumin gene, which can be expected to be highly expressed in all introduced transgenic animals.
eprod. Dev. 1997 47, 158-163) has been developed and is being improved. However, the YAC vector has drawbacks that it is difficult to purify high molecular weight DNA, is difficult to handle, and only one copy of the gene is introduced. Also, Y
It is also known that the DNA inserted into the AC vector is unstable. Therefore, it was considered necessary to improve the technique in order to highly express a useful gene in the production of transgenic milk.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a mammary gland expression vector for introducing a gene so as to exhibit position-independent expression and copy number-dependent expression in the production of transgenic animals. With the goal.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have conducted diligent research on the position effect of gene expression in transgenic animals into which a gene has been introduced so as to specifically express the mammary gland, and as a result, suppress the position effect. We succeeded in identifying a region that has a function, that is, a region that is considered necessary for expression without position effect. Further, they have found that the gene can be expressed without receiving the position effect by introducing the gene by using a vector containing a region required for expression without the position effect, and have completed the present invention. That is, the present invention is a mammary gland expression vector defined by the following restriction enzyme cleavage map.

[0007]

[Chemical 2] [In the formula, B is BamHI, C is ClaI, E is EcoRV, S is SmaI, and X is X.
Indicates hoI. Examples of the mammary gland expression vector include (a) 1 to 5340 bp and 84 of the nucleotide sequences shown in SEQ ID NO: 1.
Expression that hybridizes under stringent conditions to a DNA consisting of a nucleotide sequence containing 35 to 32110 bp or (b) a sequence complementary to all or part of the nucleotide sequence shown in SEQ ID NO: 1 and has no position effect And a base sequence containing

The mammary gland expression vector further comprises:
A gene encoding the target protein may be included. Here, as the target protein, α1 antitrypsin, acid α-glucosidase, antithrombin, bile salt-stimulating lipase, bovine tracheal antibacterial peptide, erythropoietin, factor VIII, factor IX, fibrinogen,
Granulocyte macrophage colony stimulating factor, growth hormone, insulin-like growth factor, interferon γ, interleukin-2, lactoferrin, lysozyme, nerve growth factor, parathyroid hormone, protein C, procollagen, salmon calcitonin, serum albumin, superoxide dismutase , Surfactant protein
B, tissue plasminogen activator, urokinase and the like. In addition, the mammary gland expression vector contains a mammary gland-specific promoter, a secretion signal and / or polyA.
Additional signals may be included. Here, as the mammary gland-specific promoter, α-lactalbumin, αS1-casein, αS2-casein, β-casein, κ-casein,
Wey acidic protein, β-lactoglobulin, lactoferrin promoter and the like can be mentioned. Examples of secretory signals include IL-2, growth hormone, and serum albumin secretory signals.
A additional signals include SV40, rabbit β-globin, mP
1. Thymidine kinase, bovine growth hormone poly A addition signal and the like. The vector may be a bacterial artificial chromosome (BAC) or cosmid. The present invention also provides an animal cell into which the above vector has been introduced.

Further, the present invention is a transgenic animal obtained by growing the above-mentioned animal cells, which secretes the target protein in milk. The present invention is also the milk produced by the transgenic animal. Further, the present invention is a method for producing the target protein, which comprises collecting the target protein from the milk.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The mammary gland expression vector of the present invention is characterized by containing a region necessary for expression without position effect. When a gene encoding a target protein is introduced into animal cells using this mammary gland expression vector and the resulting transgenic animal is bred and bred, the gene encoding the target protein in the transgenic animal has a position effect. The protein of interest is highly expressed in a copy number-dependent manner without being suppressed by and the protein of interest is secreted in a large amount in milk.

1. Region Required for Expression without Position Effect The present inventors first identified a region required for expression without position effect in a transgenic animal that expresses a target protein in a mammary gland-specific manner. “Position effect” refers to a phenomenon in which a phenotype is changed by a change in the position of a gene on the chromosome. For example, in the case of a transgenic animal, the transcription level of the foreign gene is located at which position on the gene. It is known that the phenomenon depends on whether or not is inserted. Further, the “region required for expression without position effect” means a region where high expression is expected when a gene having the region is introduced into a chromosome regardless of the introduced position.

As used herein, "-○○ kb" and "+ ○○
“○ kb” is a numerical value in ○○, and unless otherwise specified, ○ αkb upstream or 5 ′ side (−) and α ○ kb downstream or 3 ′ side (+) from the α-lactalbumin gene, respectively. ) Indicates the base. Therefore, -5 kb represents a base located 5 kb upstream (5 'side) of the α-lactalbumin gene, and +5 kb represents a base located 5 kb downstream (3' side) of the gene.

The inventor of the present invention has a human α-lactalbumin gene and an approximately 200 kb sequence having an upstream region and a downstream region of 100 kb.
It was considered that the kb yeast artificial chromosome (YAC) clone yLABLA has all the regions required for expression without position effect. This is because the transgenic rat produced using this vector shows high expression in all strains (Molec. Reprod. Dev. 1997 47, 158-16).
3). A high proportion (40-60%) of YAC clones have been reported to be chimeric, unstable and prone to deletion of internal regions. On the other hand, bacterial artificial chromosome (B
AC) is believed to avoid the above problems.
Therefore, the BAC library was screened by the PCR method to obtain a 130 kb clone (bLA) containing the human α-lactalbumin gene, and an attempt was made to identify a common sequence by comparing both DNAs. The restriction enzyme map of bLA was constructed using SmaI, ClaI, BamHI and XhoI (FIG. 1). bLA contained a 3 kb α-lactalbumin gene, a 6 kb 5 ′ region and a 121 kb 3 ′ region (FIG. 1).

Then, Southern blotting using a DNA fragment of bLA (BAC) as a probe and yLABL
PCR marker for A (YAC), bLA and yLABL
A common sequence with A was determined (Figure 2). As a result, -6kb
It was found that about +26 kb and about 32 kb are common. It was considered that this approximately 32 kb contained all the region necessary for expression without position effect. Therefore, the DNA base sequence of all about 32 kb was determined. The base sequence is shown in SEQ ID NO: 1. Therefore, the region required for expression without position effect can be represented by the following restriction enzyme cleavage map.

[0015]

[Chemical 3] [In the formula, B is BamHI, C is ClaI, E is EcoRV, S is SmaI, and X is X.
Indicates hoI. ]

In consideration of the presence of the human α-lactalbumin gene, promoter and poly A-added signal sequence, the region required for expression without position effect is 1 to 5340 bp of the nucleotide sequence shown in SEQ ID NO: 1 and 8435 ~ 32
It may be a nucleotide sequence containing 110 bp. However, as long as the base sequence shown in SEQ ID NO: 1 imparts expression without position effect,
Mutations such as deletion, substitution and addition may occur in a plurality of bases in the base sequence, preferably one or several bases.
To impart expression without position effect refers to a function of highly expressing the target gene to be introduced in transgenic animal production without being affected by the position effect. This function can be confirmed by constructing a vector containing a region considered necessary for expression without position effect, producing a transgenic animal into which the vector is introduced, and examining the expression of the target gene. If the gene of interest is highly expressed as compared to the vector having only the human α-lactalbumin gene promoter and the poly A addition signal sequence, the region is considered to confer position-independent expression.

For example, a base sequence which hybridizes with a DNA consisting of a sequence complementary to all or a part of the base sequence shown in SEQ ID NO: 1 under stringent conditions and imparts expression without position effect is also included. It is included in the region required for expression without position effect. The stringent condition means a condition in which a specific hybrid is formed and a non-specific hybrid is not formed. For example, it refers to conditions under which DNA having high homology (homology is 60% or more, preferably 80% or more) is hybridized. More specifically, the sodium concentration is 150-900 mM, preferably 600-900
mM, and the temperature is 60 to 68 ° C., preferably 65 ° C.

Once the nucleotide sequence of the region required for expression without position effect has been determined, this region can be obtained thereafter by chemical synthesis. In addition, by site-directed mutagenesis or the like, there is a mutation in the nucleotide sequence of the region specified by SEQ ID NO: 1 which is required for expression without position effect and imparts expression without position effect. It can also be synthesized. In addition, to introduce mutations into the base sequence, Kunk
A known method such as the el method and the Gapped duplex method or a method similar thereto can be adopted. For example, a mutagenesis kit using site-directed mutagenesis (eg Mutan-
Mutation is introduced using K (manufactured by TAKARA) or Mutan-G (manufactured by TAKARA). Utilizing the region of about 32 kb required for expression without position effect, the introduced gene can be expressed in transgenic animals without position effect and in a copy number-dependent manner.

2. Construction of Mammary Gland Expression Vector In the mammary gland expression vector of the present invention, a gene encoding a target protein can be introduced into an animal cell such that the gene is expressed specifically in the mammary gland. Therefore, the mammary gland expression vector contains a region required for expression without position effect and a gene encoding a target protein.
Here, the “target protein” may be a heterologous protein of the animal to be introduced, or a homologous protein. Heterologous protein refers to a protein that is not naturally expressed in the animal to which it is introduced, including, but not limited to, α1 antitrypsin, acid α-glucosidase,
Antithrombin, bile salt-stimulated lipase, bovine tracheal antimicrobial peptide, erythropoietin, factor VIII, factor IX, fibrinogen, granulocyte-macrophage colony-stimulating factor, growth hormone, insulin-like growth factor, interferon-γ, interleukin-2, It may be a useful protein such as lactoferrin, lysozyme, nerve growth factor, parathyroid hormone, protein C, procollagen, salmon calcitonin, serum albumin, superoxide dismutase, surfactant protein B, tissue plasminogen activator, and urokinase.
A homologous protein is a protein that is naturally expressed in the animal to which it is introduced, but may be a protein that is not normally expressed in the mammary gland, or is expressed in the mammary gland during the lactation period or is expressed only in a small amount.
Homologous proteins include, but are not limited to, bovine lactoferrin, bovine growth hormone, bovine kappa casein, and the like.

Furthermore, the vector of the present invention has a mammary gland-specific promoter, a secretion signal and / or a polyA addition signal operably linked so that the target protein is secreted into milk. You may build by. The term "inserted in a operatively linked manner" means that in a transgenic animal into which a gene encoding a target protein is introduced, the gene is expressed under the control of a region and promoter required for expression without position effect. So that its expression product (protein of interest) is secreted into milk, a region necessary for expression without position effect, promoter and / or secretion signal and / or poly A addition signal, gene encoding the protein of interest and others It means that the components of (4) are ligated and integrated into a vector.

[0021] The mammary gland-specific promoter refers to a promoter which has been shown in the present invention to be specifically expressed in the mammary gland in cultured cells or transgenic animals. For example, without limitation, α-lactalbumin, αS1-casein, αS2-casein, β-casein,
κ-Casein, wey acidic prot
ein), β-lactoglobulin, lactoferrin gene promoter and the like. In addition, examples of sequences that act to secrete the expressed protein into milk include a secretion signal and a poly A addition signal. The secretory signal refers to a protein region required for the protein synthesized in the cell to be transported to the outside of the cell, and examples thereof include, but are not limited to, IL-2, growth hormone, and serum albumin secretion signals. To be In addition, the poly A addition signal means that the poly A chain is added to the expressed mRNA,
A sequence having a function of enhancing protein synthesizing ability by enhancing stability of A, for example, but not limited to, SV40, rabbit β-globin, mP1, thymidine kinase, poly A addition signal of bovine growth hormone gene, etc. Is mentioned.

Furthermore, the mammary gland expression vector of the present invention contains a matrix attachment region (Matrix Attachment Regio).
n; MAR), scaffold attachment area (Scaffold Attachment Regio
n; SAR), insulator, etc. may be included.
The MAR and SAR are structural regions that contribute to expression without position effect in association with functional regions such as the locus regulatory region, and refer to AT-rich regions of the eukaryotic genome that serve as nuclear matrix binding sites. . The insulator refers to a sequence having an action of avoiding the position effect of the transgene. Regarding the MAR and SAR and the insulator, those skilled in the art can determine the relevant area and use it.

In order to insert the above-mentioned constituents into a suitable vector, first, the purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and ligated to the vector. The method of doing is adopted. Vectors that can be used as the mammary gland expression vector of the present invention include cosmid DNA, bacterial artificial chromosome (BAC) DNA, retrotransposon DNA,
Yeast artificial chromosome (YAC) DNA, P1 phage-derived artificial chromosome (PAC) DNA, mammalian artificial chromosome DNA
Etc., but bacterial artificial chromosome DNA is particularly preferable.

As cosmid DNA, SuperCos1, pWE
B, pAC3, PWEX15, pcosAX, etc. BAC vectors include pBeloBAC11, pECBAC1, pCLD04501, pB
Examples include iBAClac1, BiBAC2, V41 and the like. Examples of the P1 phage include pCYPAC2 and pPAC4, and examples of the retrotransposon include Ty factor. Examples of the YAC vector include pYAC2, pYAC3, pYAC4, pYACNeo and the like.

3. Production of transgenic animal The transgenic animal of the present invention can be produced by introducing the mammary gland expression vector constructed above into animal cells and growing the animal cells. The transgenic animal thus produced secretes the target protein into milk. The animal into which the mammary gland expression vector is introduced is not particularly limited as long as it is a mammal, and examples thereof include cattle, sheep, goats, pigs, rabbits, mice and rats. Particularly preferred are mammals that produce a large amount of milk and have a long lactation period, such as cows, sheep, goats, pigs, and the like.

In order to introduce the mammary gland expression vector of the present invention into animal cells, it is preferable to inject it into the fertilized egg of the target animal. As a result, the gene encoding the target protein and its regulatory region are integrated into the cells of the fertilized egg, and the gene encoding the target protein and its regulatory region are copied to cells throughout the body as the fertilized egg divides. .

As a method for introducing a gene into an animal cell,
In addition to the microinjection method into a fertilized egg, a method of introducing into an ES cell, a method of introducing a cell nucleus introduced into a cultured cell into a fertilized egg by nuclear transfer, and the like can be mentioned. The vector DNA for mammary gland expression of the present invention is introduced into ES cells or other cultured cells by electroporation, lipofection or the like, and positively selected with neomycin, puromycin or the like to obtain the desired introduced cells. ES cells are
Inject into embryo placental vesicles or 8-cell stage embryos using a capillary tube or the like.
Nuclear transfer is performed by injecting cells into which DNA has been introduced into a fertilized egg from which the nucleus has been removed, and performing cell fusion by electrical stimulation.

Thereafter, embryo placental vesicles or 8-cell stage embryos are directly transplanted into the oviduct of the foster mother, or those that have been cultured for one day to develop into blastocysts are transplanted into the uterus of the foster mother. The foster mother is bred and given birth to obtain a transgenic animal that is a child in which the desired trait has been expressed. To confirm the expression of the desired trait in the animal, a part of the body of the animal (eg tail tip)
Cleavage is performed, DNA in somatic cells is extracted, and the presence of the introduced gene is confirmed by PCR or Southern blotting. If the individual confirmed to have the introduced gene is the first generation, the introduced gene is transmitted to 50% of the offspring (F1). That is, by mating this transgenic animal with a normal animal, a heterozygous animal (F1) is obtained,
Homozygous animals (F2) by mating heterozygotes
Can be obtained.

4. Production of Target Protein The transgenic animal produced as described above specifically expresses the introduced gene encoding the target protein in the mammary gland and secretes the target protein into milk. Therefore, the protein production method of the present invention is characterized by collecting the target protein from the milk produced by the transgenic animal.

To collect the target protein from milk,
A general biochemical method used for protein isolation and isolation, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., can be used alone or in combination. Alternatively, if the target protein is a protein that enhances the nutritional value of milk, the milk can be recovered and used as it is as milk with high nutritional value.

Since the mammary gland expression vector of the present invention contains a region required for expression without position effect in transgenic animals, the introduced gene can be expressed reliably, and a multicopy gene is introduced. Is something that can be done. Therefore, the gene introduced using the mammary gland expression vector of the present invention is highly expressed in the transgenic animal, and the expression product is secreted in large amounts in the milk.

[0032]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, the technical scope of the present invention is not limited to these examples. [Example 1] Screening and analysis of α-lactalbumin BAC DNA BAC library (Genome systems Inc., St. Loui
s, MI) was screened by PCR (Fujiwar
a et al., 1997). BAC DNA was isolated by the conventional alkaline method and treated with InCert agarose (FMC BioProduct
s, Rockland, ME) to avoid mechanical cutting. The DNA embedded in the agarose block was digested with a restriction enzyme and subjected to pulse field gel electrophoresis (PFGE) (CH
EF-DRII, BIO-RAD, Hercules, CA) were used to construct a restriction enzyme map. The DNA fragments were separated by PFGE using low-melting point agarose, the DNA band in agarose was melted at 65 ° C, and β agarase (New England
Biolabs. Hitchin, England) Digestion and ethanol precipitation were performed to recover DNA. The DNA fragment is a plasmid
Ligated in pBluescript SK- (Stratagene, La Jolla, CA). The subcloned fragments were mapped and several fragments were cloned into the ABI 373 Auto Sequencer (Per
Kin Elemer, CT, USA). The restriction enzyme map was constructed using SmaI, ClaI, BamHI and XhoI (FIG. 1). bLA contained a 3 kb α-lactalbumin gene, a 6 kb 5 ′ region and a 121 kb 3 ′ region (FIG. 1).

Example 2 Identification of Region Required for Expression without Position Effect BAC and YAC clones were compared as follows. Digesting YAC and BAC DNA with restriction enzymes,
After electrophoresis, the nylon film Hybond N ⁺ (Amersham Pharmacia Biotech, Lit
tle Chalfont Buckinghamshire, England). The probe DNA (20 ng) was used for the Rediprime labeling system (Amersham Pharmacia Biotech, Little Cha).
lfont Buckinghamshire, England) [ ³² P] -dCTP
Labeled with. To adsorb repetitive sequence DNA, the labeled DNA is boiled in 5 × SSC 0.1% SDS for 5 minutes and kept at 65 ° C.
Incubate for 20 minutes at 50 μg COT1-DNA (Roc
he Dianostics GmbH, Mannheim, Germany). The solution was then hybridized to the membrane at 65 ° C. Hybridize the membrane with 2 x SSC
It was washed with 0.1% SDS at 65 ° C. The hybridized band was recorded by Fuji Bio-Image Analyzer BAS 2000 (Fuji Pho
to Film Co., Tokyo, Japan).

PCR primers were designed from the bLA sequence. The primer pairs used are shown below: A1: AGGAAAGGAAAAACTACCAT (SEQ ID NO: 2) -GGCTTATTTTGGATTGCTAT (SEQ ID NO: 3), C2: CTCAAAGTGCTGGGATTACA (SEQ ID NO: 4) -CCGAGTCTGGTGGATCACTT (SEQ ID NO: 5), A3: GGAAGCTTCAGTCATGAGCTACCA (SEQ ID NO: 6). AAGGATCCTGGAAGCATCACTCAG (SEQ ID NO: 7), C4: GGCTCGAGCTCCGCTCCTTGGTA (SEQ ID NO: 8) -TCACAAAGCCACTGTTCCAG (SEQ ID NO: 9), A4: TGGAGTTTTGCCCTTGTTGC (SEQ ID NO: 10) -CTGGATAAAGGAGGCAGTGT (SEQ ID NO: 11GA), GT6: GAAC: ACA: A6: GAAC: SEQ ID NO: 13), A7: TATTCTCGTGCCTCAGTCCA (SEQ ID NO: 14) -AAAACAGTGGGCATCATAGA (SEQ ID NO: 15), A8: AACTAACCAGCCTTTCTCAC (SEQ ID NO: 16) -AAGACCAGTTGAATGACCTT (SEQ ID NO: 17), A9: ACTGGGACTTCTTTGACCAG (SEQ ID NO: 18) -SEQ ID NO: 18). 19), A10: ACAAGTGAACAAGGGTCTCT (SEQ ID NO: 20) -TCTGCCCCACCGCTCCATCT (SEQ ID NO: 21), A11: AGAGATTTTGTCACCACCAG (SEQ ID NO: 22) -CCTGTATCCCTTTATTTTGA (SEQ ID NO: 23), A12: GGCTGGCTCAACATACACAA (SEQ ID NO: 24) -ATCCCTGTCTTATGCCAGTT (SEQ ID NO: 25), A14: GTTGGTGGGACTGTAAACTA (SEQ ID NO: 26) -TGTCCCCCACCCTGCG NO: CCTGCCCCACCCTG. .

PCR was performed with 3 pmol of each primer, 0.25 U
Taq polymerase (Gene Taq, Nippon Gene, Toky
o), including 200 nM of each NTPs and 1x auxiliary buffer 2
DNA Thermal Cycler TP-3000 (T
akara, shuzou, Kyoto) at 94 ℃, 55 ℃ and 72 ℃
35 cycles of 30 seconds were performed at each temperature.

YLABLA and bLA DNA are shown in FIG.
The fragment of the bLA insert shown in (4) was analyzed by Southern blotting to determine how much region was a common sequence. The results are shown in Table 1.
Shown in.

[0037]

[Table 1]

YLABLA of the size predicted by probes X7, X9, C10 and C2 was detected, but S2, S10
And R5 showed no band in yLABLA (FIG. 1 and Table 1). Probes X5 and R4 are yLABL
A positive band was shown in A, but a different size band. From the above results, yLABLA and bL
A was shown to have a common region, namely X5-R4, suggesting that the common region contains all the elements necessary for position-independent expression.

To prove the above hypothesis, the X5-R4 region was sequenced and further analyzed. I.e. 32,110 b
The nucleotide sequence of p was sequenced. This sequence data is registered as accession number AB049976 in DDBJ, EMBL and Genbank nucleotide sequence databases. There are many repetitive sequences belonging to the AluI family in this database, and Line1 is an unidentified element (Fig. 2). These repeats account for 58% of the region. A sequence 7 kb downstream of the α-lactalbumin gene has a sequence highly homologous to the olfactory receptor gene (OR37).

[Example 3] Generation of α-lactalbumin BAC transgenic rats and analysis of milk thereof A region (about 32 kb) required for expression without position effect, α-lactalbumin gene, α-lactalbumin promoter and signal sequence were prepared. The contained BAC DNA was digested with NotI, the insert DNA was separated by PFGE and purified by the procedure described previously (Fujiwara et al., 1997). DNA (1 ng / μl) was used for fertilization of rat (Wistar strain,
(Charles River, Japan) was microinjected into the pronucleus and the embryo was transferred to the oviduct of pseudopregnant Wistar rat.
DNA was extracted from the tail of newborn rats and the presence of transgene was detected by PCR. The copy number of transgene was determined by Southern blotting. Six strains of transgenic rats were produced. The experimental results are shown in Table 2.

[0041]

[Table 2]

Transgenic female rats were mated with wild-type Wistar rats at 10 weeks of age. Rat milk was collected only once on the 9th to 11th day after delivery. Transgenic rats were weaned from their pups 4 hours before milking and hand-milked after injection of oxytocin (5 U / rat, Sigma, St. Louis, MO). Proteins in rat milk were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were either stained with Coomassie Brilliant Blue for Western blotting or transferred to PVDF membranes (Immobilon, Millipore, Bedford, MA). Membranes were incubated with anti-human alpha lactalbumin rabbit serum (Zymed Laboratories, CA),
Antiserum coupled ECL detection system (Amersham Pharmacia Bi
otech, Little Chalfont Buckinghamshire, England)
Was visualized using. Band intensities were quantified with a Model GS-670 imaging densitometer (BIO-RAD, Hercules, CA) using purified human alpha lactalbumin as a reference.

All 6 lines produced human α-lactalbumin at 0.6 mg / ml or more per copy of transgene. The transgenic line 1875-3 had 10 copies of the transgene and produced 41.0 mg / ml α-lactalbumin. In conclusion, bLA, a BAC construct containing the region required for expression without position effect (approximately 32 kb).
Can have all the sequences required for position-independent expression and copy number-dependent expression.

[0044]

INDUSTRIAL APPLICABILITY With the mammary gland expression vector of the present invention, a gene encoding a target protein can be introduced into animal cells such that the gene is expressed specifically in the mammary gland and is not affected by the position effect. Since the transgenic animal thus obtained secretes the target protein in a large amount in milk, the present invention is useful for producing a protein which is usually difficult to produce.

[0045]

[Sequence list]                                SEQUENCE LISTING         <110> National Agricultual Research Organization      Japan Science and Technology Corporation                   Yoshihiro Fujiwara <120> Mammal grand specific vector <130> P01-0636 <140> <141> <160> 27 <170> PatentIn Ver. 2.0 <210> 1 <211> 32110 <212> DNA <213> Homo sapiens <400> 1 aagctttttg atgtgctgct ggattcggtt tgccagtatt ttactgagga tttttgcatc 60 aatgttcatc aaggatattg gtctaaaatt gtcttttttg gttgtgtctct tgccaggctt 120 tggtatcagg atgatgctgg cctcataaaa tgagttaggg aggattccct ctttttctat 180 tgattggagt agtttcagaa ggaatggtac cagttcctcc ttgtacctct ggtagaattt 240 ggctgtgaat ccatctggtc ctggactctt tttggttggt aagctattga ttattgccac 300 aatttcacag actggcaaat tggataaaga gtcaagaccc gtcagtgtgc tgtattcagg 360 aaacccatct catgtgcaga gacacacata ggctcaaaat aaaaggatgg aggaagatct 420 accaagcaaa tggaaaacaa aaaaaggcag gggttgcaat cctagtctct gataaaacag 480 actttaaacc aacaaagatc aaaagagaca aagaaggcca ttacataatg gtaaagggat 540 caattcaaca agaggagcta actatcctaa gtatatatgc acccaataca ggagcaccca 600 agttcataaa gcaagtcctg agtgacctac aaagagactt agactcccac acaataataa 660 tgggagactt taacacccca ctgtcaacat tagacagatc aacgagacag aaagttaaca 720 aggataccca ggaattgaac tcagctctgc accaagcaga cctaatagac atctacagaa 780 ctctccaccc caaatcaaca caatatacat tttttttcag caccacacca cacctattcc 840 aaaattgacc acatagttgg aagtaaagct ctccttcagc aaatgtaaaa gatcagaaat 900 tataacaaac tgtctctcag accacggtgc aatcaaacta caactcagga taaagaaact 960 cactcaaaac cgctcaacta catggaaact gaacaacctg ctcctgaatg actactgggt 1020 acataacgca atgaaggcag acataaagat gttctttgaa accaacgaga acaaagacgc 1080 aacataccag aatctctggg acacattcaa agcagtgtgt agagggaaat ttatagcact 1140 aaatgcccac aagagaaagc aggaaagatc caaaattgac accctaacat cacaattaaa 1200 agaactagaa aagcaagagc aaacacattc aaaagctagc agaaagcaag aaataactaa 1260 aatcagagca gaactgaagg aaatagagac ataaaaaacc cttcaaaaat taatgaatcc 1320 aggagctggt ttttttgaaa ggatcaacaa aattgataga ccgctagcaa ggctaataaa 1380 gaagaaaaga gagaagaatc aaatagatgc aataaaaaat gataaagggg atatcaccac 1440 cgatcccata gagatgcaaa ctaccatcag agaatactat aaacatctct acgcaaataa 1500 actagaaaat ctagaagaaa tggataaatt ccttgacata tacaccctcc taagactaaa 1560 ccaggaagaa gttgactctc tgaatagacc aataacaggc tcttttttgt tttttaaatt 1620 ttggtgggta catcatagct gtgtatattt atggggtaca taaaatgttt tgatacaggc 1680 atgcaatgtg aaataaatac tttatgggga atgcggtggt agattgttaa tatgagttgc 1740 caggatgatg tttggcaagg aagaaatgag gaggaagaaa gggaagccat tcctaaaagg 1800 aaaggaaaaa ctaccatgtt cacaaaaaat aggatgtaag attctatcaa aggtgttgat 1860 gtaaaattat gtaaatatgt ttatttaaaa ataaacattt tataaattaa aaatgaaaaa 1920 tcaattaaaa tttgcataga aattttttta gcttcttggt aattacatgt gtatcggttt 1980 gttttagcta atattcagtt aaaaaggtaa aatttatttt agtatctttt aaaatcattt 2040 ttgtgttata atttatattt ccatgcttgc attttttggt tgatactatc cccaattcac 2100 acaaatgaat caatggttca tttaagtata aaagcagtga tataaatagt aatgcaaata 2160 tagcaatcca aaataagccc atataaattg caagcaggcc tttggtgtgg gatatagaat 2220 gtgaatctat aatgctgagt aactttgtaa ggacttttgg acaagcagct gaaaaagaaa 2280 aatgccaata aaaaatcact ccctttctaa atcttaatta ctttaattaa ctctttaatt 2340 tggttaaaca ttttcatgaa atttgggttt caagatctag catcattgtc tacctagtga 2400 taattttcct gaattatgag agaaagtaga acaagatgag gatataagtg tattttaaaa 2460 tagagacagg gtcttgctct attgcccagg ctagagtgag tggcacaatc aaagcccact 2520 gcattcttga actcctgggc tcaagcaatc ctcgtacctc agtagctagg actataggca 2580 cgtgccacta tgcctggcta atttttattt ttttttgtag tgacagagtc ttgctatgtt 2640 gcccaggctg gtctccaact cctggcctca agtgatcctc ctgcctcagc ctcccaaaat 2700 gttgggagta taggcatgag ccactgcagg cacaaggtaa ggatattaac tgcaagatgt 2760 aatggccatt atgactgtgg ctctcagggt gttccctcta aatggcaggc ctaggctctg 2820 tctagaaact ccagctcacc tacagactac agtttcagat ggaaaacgtg ccttgaaaca 2880 catgctttca atttctttat tttcagaaat aaagatattt taattttatt tttattatta 2940 ttattatttt ttgagatgga gtcttgctct gtttcccagg ctggagtgca gtggcacaat 3000 cttggctcac tgcagccttc acctctgggt tcaagcgatc ctcctgcctc agctgcccga 3060 gtagctggga ttatagactc ctgccagcat gctcagctaa tttttgtatt tttagtagag 3120 acggaatttt gccatgttgg tcaggctggt ctcgaactcc tgacctcaaa taatctgcct 3180 accttgggct tctcaaagtg ctgggattac aggcatgacc caccatgccc ggcctgaact 3240 ttttatttta taaataaaga aatttacttt tagaaataaa atttttattt tgttcatctt 3300 caaaaaggtg atttctggtt ttagaaacct ggatatttcc ccacagcatc tgagagaatg 3360 aacataattt tctagtctat ttctaacaaa atctaggtaa gtgtattgta aatgcctctt 3420 caccatcttg attcagctct cgacctccat gcagagcacc ctgagtaaac ctctctggaa 3480 agggagattt tggaggaggt ttcttcctgg acaggaattg ttgagcagga gctttcttcc 3540 acgagctgtg cttaatgtct ttccacatac ttcctctttc agtgctgcga tcattgtgta 3600 ttgttctcct ttggacaatc tccaagaggc tgcatctttc tctggatgtt tgcagttgtt 3660 cccattagac actttctatc ttctttttca gatgaccccc acgtatccta ttttaagaac 3720 atttataggg aaataatggt tccttttgcc ggagacatgt ttattttctt ttctgcactt 3780 agttgtgatt cctgacctgt atgcttattt ttattgctta tagggaaggg ccaaggtata 3840 atcaaatgat aggcaagcag gcagctgcct taggtcttga cttggctgaa agtgtagaaa 3900 acccctgtga ttcttgagac cctggcccac ccttttactc tatcacaggt acttagtcaa 3960 tagcctaggg caggaggcat tttacacaag actccactat tggaaggact agtcctcagg 4020 actagctttt cttatctttc cctctcacac atggttcaag gtcactctca gccatattct 4080 caacaaagct tagagtgata gaattcccat tcctgtcgtg tacccttgca gtgcctctgg 4140 gtggaatgcg gagaaatgga gtggctccac ttctgttgtg tttctgaaca tgtatctctt 4200 gctatcagaa ctttctgctc atcccttctg gcacaccaag atcctccaca ttcccttcac 4260 tcatgccact tcatatactg gttatccatg gtacagaaga caggatttaa ctgagaggac 4320 ttttccctga ctctgaatac atgtaggaga taacgatatg gaagaccttc agtatgtaag 4380 tcttaaatag attggttggg ataaatgttc cctgaaacat aagaaacagc gcagcggctc 4440 ctgtctgtaa tcctagcact ttgggaggct gaggcccagg caggcaaatt gcctgagctc 4500 agaagtttga gaccagcctg gccaacatgc agaaactccg tctctactaa aaatacataa 4560 attaaccggg catggtaaca cgtgcctgta gtcccagcta ctcgggaggc tgaggcagga 4620 gaatcacttg agcctgggag gcagaggttg cagtgagcca agatcgcgcc actgcattcc 4680 agcctgggca acagagtgag acttggtcaa aaaaaaaaaa aaaaaaaaaa aaaggaagaa 4740 gaagaagaaa tcaggtttag agatgaggac aaagaagacg aatggtggca tgaaggagct 4800 aagagctact tgtcaccatg acatgaagct tcatgccagc aaattaaagg agctattcag 4860 aactagtatc ctcaactcta cttgctcagg ggcactgacc ttatagagat tccagacata 4920 agcttgttca gccttaaagt ccaatctttc cactggcttg ggtccttccc actttctgtg 4980 gccaactctg aggttgtcta caagttattg gtcttagatt tatgtaatgt ctcaatgcca 5040 gtgtagtatt tggttattta cggtaggagt ggttaggggt ggggaatctg ataatagctc 5100 gtaggatagc tagattcttt tttttttttt ttttttttaa agatagggtc tcactttgtc 5160 tcccaggatg gatggagtgc agtggagtga acatggctca ctgcagcctc gacctcctgt 5220 gctcaagtgt tcctcctgcc tcagcccctc aagtagctgg gactacaggc acatgtcacc 5280 atgcccagct aatttttttt gtagagatgg gattttacca tgttgcccag gctggtctcg 5340 agctcctggg ctcaagtgat ccaccagact cggcctccca aaatgccggg attacaggtg 5400 tgagccactg tgcctggcct agatgctttc atacaggctt ttcaattatg cattttcctt 5460 aagtaggaag tcttaagatc caagttatat cggattgttg tagtctacgt tcccatattc 5520 tattcctatt tctgagcctt cagtcatgag ctaccatatt aaagaactaa ttctgggcct 5580 tgttacatgg ctggattggt tggacaagtg ccagctctga tcctgggact gtggcatgtg 5640 atgacataca ccccctctcc acattctgca tgtctctagg ggggaagggg gaagctcggt 5700 atagaacttt attgtatttt ctgattgcct cacttcttat attgccccca tgcccttctt 5760 tgttcctcaa gtaaccagag acagtgcttc ccagaaccaa ccctacaaga aacaaagggc 5820 taaacaaagc caaatgggaa gcaggatcat ggtttgaact ctttctggcc agagaacaat 5880 acctgctatg gactagatac tgggagaggg aaaggaaaag tagggtgaat tatggaagga 5940 agctggcagg ctcagcgttt ctgtcttggc atgaccagtc tctcttcatt ctcttcctag 6000 atgtagggct tggtaccaga gcccctgagg ctttctgcat gaatataaat aaatgaaact 6060 gagtgatgct tccatttcag gttcttgggg gtagccaaaa tgaggttctt tgtccctctg 6120 ttcctggtgg gcatcctgtt ccctgccatc ctggccaagc aattcacaaa atgtgagctg 6180 tcccagctgc tgaaagacat agatggttat ggaggcatcg ctttgcctga atgtgagttc 6240 cctgcctctg tgtttcatcc attcctcata cgcttctctc ctccatcccc tctttcttcc 6300 acttcgcccc tccactttta cttaattatc taatcatcct cttttctgct catttgcata 6360 ctcttttatt tcatgtatgt atatatgtat gtatttattt atttttgagg tggagtttcg 6420 ctcttgttgc ccagactgga gtgcaatggt gtaatctcgg ctcactgcaa cctccgcctc 6480 ctcggttcaa gtgattctcc tgcctcagcc tcccaagtag ctggaattac aggcacccac 6540 caccatgcct ggctaatttt gtattttttg tagagacagg gtttcaccat gttggccagg 6600 ctggtctcaa acttctgacc tcaggtgatc cgccctcctc agcctcccaa agtgttggga 6660 ttacaagcgt gagccatcat gcctggcccc atttattttc ctatcctttc tttctcttat 6720 tgtctgattt ttttttggaa ttctccatct catcaagaaa ctctgagctt tgccatcttt 6780 ggagattggc tggaaagcat ttttgtctga gaattacagt tcctccttta tgcagatcct 6840 gtacatctct gtggtatctc tttctcatct ttccctcagt gatctgtacc atgtttcaca 6900 ccagtggtta tgacacacaa gccatagttg aaaacaatga aagcacggaa tatggactct 6960 tccagatcag taataagctt tggtgcaaga gcagccaggt ccctcagtca aggaacatct 7020 gtgacatctc ctgtgacagt gagtagcccc tataaccctc tttctctgtt tttctgaggc 7080 ctgcccttgg gataatctcc tttttagtgc caagcagacc tcaggcttca ttgccttggc 7140 tgggctctat aaaaattgtg ggacttgaat tggcagtact gagtaagaag ctgtttggat 7200 ttttcatggt catcaaatcc ccagacagtt ccttgaggtt cagtggtaga caatcggagc 7260 tgtctgagag tcttggaatc tgattgtctg cattttcagg gtaagtcagt tgatgaagct 7320 gatgattcct ccagagatat cccagggaaa tgaaggaagt ccctacccag ggttagacat 7380 taccacattg gtcctttcat atagaaagac aacaggcaca agccttgagt ttagagaacc 7440 cactggatcc aggggttagg ggaactcagt gcctttctgg gtaatacttg tcagctgtct 7500 caatcctttc cctgtaactc ctgccagagt tcctggatga tgacattact gatgacataa 7560 tgtgtgccaa gaagatcctg gatattaaag gaattgacta ctggtgaatc cttattctat 7620 tttctatttc cccatcctcc ttctccttac cccattagcc cagcacccct ttcctcttac 7680 cctatctctt ggtcatttaa tctagaatac agtgtctgaa acaaagctta cctagagact 7740 caggtttctg ttattaagcc tctctcgctc cgctccttgg tagcaatttt cctaataagg 7800 ggttgcctaa tggagggctc agacccaggc ctcctttcac ttagacttgg acatctaatt 7860 ccacttgttt agttctatgc cctaaagcaa gctgttggta acattgcatc tcttttttaa 7920 ccctacaatt ttcttggata ttttttatgg actgtattcc acttgatggc ttgtgtcgct 7980 tgacatcagg ccaggaatgt ctttctgtaa ttctcgtcca cgctcttcca cttcagccct 8040 cctgggaatg aatgtaaaga ttcagtcagc taactcacct tgtccccctt ctccattatc 8100 aggttggccc ataaagccct ctgcactgag aagctggaac agtggctttg tgagaagttg 8160 tgagtgtctg ctgtccttgg cacccctgcc cactccacac tcctggaata cctcttccct 8220 aatgccacct cagtttgttt ctttctgttc ccccaaagct tatctgtctc tgagccttgg 8280 gccctgtagt gacatcaccg aattcttgaa gactattttc cagggatgcc tgagtggtgc 8340 actgagctct agacccttac tcagtgcctt cgatggcact ttcactacag cacagatttc 8400 acctctgtct tgaataaagg tcccactttg aagtcactgg ctgtaatttt tttccccctg 8460 gagggaaggg gaagaaatag gatgagtagg tggacactga agccataggt catagccacc 8520 ttccatctct actgaagaag aagtaggctg aatttacaat agaaaggtga aggttactgt 8580 ctgtaccaac tcaatgcaac aaacttttat tgatcaccta atctattcaa ggaactgtag 8640 acggatccaa agttggctca acactggccg ggtgcagtgc ttccacctgt aattccagca 8700 ctttggaagg ctgaggtggg cagattgctt gaggtcagga gtttgagacc agcttggcta 8760 acagcaaaac cctgtctcta ccaaaagtac aaaaattatc tgggtgtggt ggcaggcgcc 8820 tgtaatccca gctactcggg aggctgaggc agaagaattg tttgaacctg ggaggcagag 8880 gttgtagtga gctgagatcg cgtcattgcg ctctagcctg ggtgctccat ctcaaaaaac 8940 aaaaacaaaa acaaaaaaca aaaacacaaa gttggctgaa tagccttttc cctcaaggac 9000 taatgggaaa gacaagataa acattaataa atagttacaa tataaggaag agagttataa 9060 gcatcatgag agaggttaga aatgtgcgct gggcattcac aaaaggaaag ataacttatc 9120 tttggagggg atgttgggaa aaaggaatca agaaagggtt cctgaaaggg agagcattta 9180 ggctggtttt aaagtctgtg tgggattttg acaagtggag atgtggggtg ggaaaaggaa 9240 gagcatggac gggaacccaa gaatctgaaa tgggtgactt gttgaaggat ttctttatcc 9300 ttcatgagga gctgtgaagg gaacccttcc tgtctctgga aaccagagag agaacaggga 9360 ccttgacagt cagcacagtg gtgctgaaga atctgagaaa atgggacctt ttggtttgga 9420 ccctccactc cattatttct tcttgtgctg tgcttggttt ttaaggagaa tatttgcaga 9480 acttcattca tttaaataat tatgggatat gtgaaagaag aagaatcagg aaatttgggt 9540 cctaaaatct gtttctgcta cttattgagg gtctgaacct gggcaggtca tctccattct 9600 ctgtgcctca gtttcctcat cttagaataa ggattttggt ctagacagtt ttctgagatc 9660 acttcctgct ttaacaatcc agcattccaa actacagttt aatttgtcac actctgaatg 9720 agctgagaat gacattcgca ttgttgctca gctccacagc cctggccttc atcccacaga 9780 gaagacagga aagttcacct gtgtgggcaa gtacaccgta tatgagttca tgatcttcag 9840 gagccgttgc tgaaggaatt gacagtaaga gatttatctg tagagagaac ctcagcctga 9900 ggtcagaaga tcaagaacag atagaagcca gggaactggc aggaaagaca tctttttctt 9960 tttttcaatt attcatttgt tacagtgggt tatgatacaa atgtttatag atgcctactc 10020 tgtactagta ctacagagca gtttttctgt gtttatattc agttcaattg tagtgtgttg 10080 agttgtaaaa taatccatgt attaaatcaa ataaacaaac aaaatgccat gttctttggt 10140 acaagcaaca ctaccaaagg catttggggt ctgcatttgg aattctcagg caaactctct 10200 cttgttccta gtctgtactt attttcccca cactagctta tgtatatata ttttttgaga 10260 tggagttttg cccttgttgc ccaggctgga gtgcagtggc acgatcttgg ctcactgaga 10320 cctccacgtc ttgggttaaa gcgtttctcc tgcctcagcc tcctgactag ctgggattac 10380 aggcgcctgc caccatgccc ggctaatttt tgtattttta gtagagatgg gctttcacca 10440 tgttgctcag gctggtcttg aactcccacc tcggccttcc aatgtgctgg gattacaggt 10500 gtgagccaca gtgactggcc tgtacatttt ttaaatttca atgtctaata tggtgtccac 10560 tgaattaaga attcttttga gaaaatgaat caataaatct atacactgcc tcctttatcc 10620 agtgaggtat ggctggatca gcttcatgac atacatgcca gtagtcttct tcgccttctc 10680 ctcctcctcc ttttttacaa ataaaaattg tatatgttga aggtgtacaa cttgatgttt 10740 tgttatatgt atacacttaa atgatcacca cagtcaagct cattaacata tcatcgcctc 10800 acatgattac ttttcttttt ctttggacat agaagcaggc ttgatagatg cagttctctt 10860 ggcatttggg tagaagcagg acaggaaact gctgttctct tcctgagaac tttcttgaga 10920 gctgaatagg aggcagcatg gcagctgagc atctgttctg ctttctctac ctctgtctct 10980 ttcccttagg cctaaaatga agctctaagc caagcaaagg tctgaagtca tccagactaa 11040 ttgggaagcg ggtaggctcc agggagtggc tctcagagag cagaccattt actgagctct 11100 gtgtgagaaa gactcaaaat ggttcacagc aatacagagt tttgttctct cctcttatcc 11160 tgctttctcc tccctgctac ttttccctga cacctatctt gttgtgaaga caggaattgc 11220 attagataaa atcaaatctt ttttattttt ttttgagatg gaatcttgct ctgttgccca 11280 ggctggagtg ctgtggcacg atcttggttc actgcaacct ccgcttcctg ggttcaagcg 11340 attctcctgc ctcagccttc tgaatagctg ggattacagg catgcgctgc cacgcccatc 11400 taatttttgt atttttagta gagacggggt ttcaccatgt tggccaggtg ggtcttgaac 11460 tcctgccctc aggtgatccg ccctcctcag ccttccaaag tgctgagatt acaggcatga 11520 gacaccgtgt ctggcctctt tttttttttt tttgatggag tctctctctc tgttgcccag 11580 gctagagtgc agtggcgtaa tctcagctca ctgcaacctc tgcctctcaa gttcaaacag 11640 ttttcctgcc tcagcctccc aagtagctgg ggttacaggt gcccaccacc atgcccagct 11700 aatttttgta tttttagtag acctggggtt tcaccgtgtt ggccgggttg gtcttgaact 11760 cctgacctca ggtgatccgc ccacctcagc ctcccaaagt gctgggatta caggtgtgag 11820 ccaccatacc cggccccaaa tatttctttt catgtttctt ctcccacttc tccttctctc 11880 tcatccattc atttttttca tttattgaac actaattaag agcctattag gtgacaggag 11940 gtactagata ttgggtatac agaagctatt agaggttttt ctcttaagac ccttatactt 12000 tagaggggga tactcccctc ctcccatttc ctgggatcat gcaagagaaa agacttcagg 12060 tgatccacat tcattccttc cttcactgct tgatttgtca ccaagtggtt attgaggata 12120 tgctgtttgc taggtactac tttacttatt tatttgttta tttagagatg gggtctcaca 12180 atgttgccca gtctacagga cagtggctat tcacaggtgt gagcacagca cactacagcc 12240 tcaaactcct gagttcaaga gatcctcctg cctcagtctc tcgagtagct gggactacag 12300 ggatgtgcca ccacacatgg cttaggctct actttagctg ctacttgtta aggatgaaga 12360 taggaggaga cactcttatt ttatttgatt tctttttttt tttttttttt tgacagagtt 12420 ttgctctgtt gccaggctgg agtgctcact gcaacctcca cctcccaggt tcaagcaatt 12480 ctcctgcttc agcctcccga gtagctggga ctacaggcat gaaccaccac accaggctaa 12540 tttctgtatt tttagtagag acagggtttc accatattgg ccaggatggt cttgatctcc 12600 tgaccttgtg atctgcccgc ctcggcctcc caaagtgcta ggattacagg tgtgagccac 12660 catgcccggc ctatttgact tctttctaag ttgttatggg atgtagatct ttccagggac 12720 tttttggcat gagaacaaaa gaggagttgg gtcacagtgc caagatggga ctatcccaga 12780 atgcagaagg acatttgcta gtggtctgat aggtcgattt accttttgtg catagacata 12840 agtaaaaggg agagggagag gcaggaagaa gaggggtcaa aaaggcaaga aatgagagga 12900 aaaagtatta gctgtagaaa tggccaggat gccctggatg ccctgaggat tttcttgtat 12960 aatgactgga ccccctctga tgggtgctac ctgctctgtg agagtttcct cattgccttt 13020 ggctgggaac agggcattgg aggaagataa actaaggtct ctgttttgga aggagatgaa 13080 tgttgaggta gctgagtggt gatagctctt cctccctcct tttttcccca taacttttcc 13140 tcctgctgag gcagggacgc tgggctacaa gggaagcctg gacttcctgg ccagcatcct 13200 cctcagagcc acctttacct cctggctctt cagactgtag atgagggggt tcagcagggg 13260 ggttatcaca ctgtactgca cggagagcac ttgttctagg gctgagccag atgctggaat 13320 catatacctt gaagggaatt gtacagaaaa gcaacttatg acaatggtga caattctcac 13380 tgttggcagc aataattcat cccctccctg caaagactct gcaaaggagg tcttcagagc 13440 tgactttctc tggtggcagc atctagaaat ggctgatctg tgaaaagtca gtcatttctg 13500 ccttcaaaca cttccaaaca taagtgagct cacacagaaa gttataggag ctgccccgag 13560 gaacaagcag ttctgcagca tatcaaatgt tgcaggttag aacagaatta atattgggga 13620 cagataaaaa gtaaaacaaa aaaatagaat gaataataat atcatacatt tccatagcat 13680 ttcatactct tcaaagcatt cttccttaat agctgtgatg atcatatgag gaatttttct 13740 ctttatacac aacagtacag aaacgcaatg ttctcacctt gtgatgactg ttcatgtaaa 13800 ctgtacactt agtttaaaat atcgtcatta ttcccaacct tcatggccct gggaattacc 13860 ctcagaatcc tcgcctgtta tttgtataaa gttgagtgga acaaacattt gcccttttag 13920 ttgcattttt ctttggtttg gaaaagaagc caaaactggt aaaatgggag ataaaaatgt 13980 gttaagcaca taatatgatt ccaaaataat aagattaaaa tcttgggtac attttgctaa 14040 gtaccactcg gagtatcttc caaagaagca ctagtattat tatttatttg cagtggcacc 14100 atcattattg gaatagacta tcccaaggat gttccttgtg ggttccaatg tgtttggtta 14160 taaaaaaaaa tagtcatttt actgttattt tttgtcatct aacttattct ttctaacatt 14220 tctatgaagt aagtagtcac atccctcatg gcagatgagg aaactgagac aaagggagat 14280 gaagagattg gtgcacagct caggtaagct ctcctgggct atttatttat ttatttattt 14340 tacttatttt tttttctctc ctagccactg gactacaggt actgggctat tcttaccatc 14400 catttccatc ctggacttct ctctgaatca caggcaggtg agaaaaatat tgaacaattt 14460 gtgcaaatct accttcaatt tactagtaga aagcacaggc atcgattgtt ttgtctcctg 14520 gtgagggagg actaggcaga cagagctgtc gatgtctgag aagcatggta aggaagggca 14580 cggatctcgg tccaacttat ggctccatta ctagcttgtt ctgggactgc tgagaaatta 14640 cctgattact ccccaaactc aggttcttta cctaaaagtg ggttgataat atttctcttg 14700 tagtgtgatg gatactcaaa ttggcttggc gatgtaaagc agcttgtgta tagtaaatgg 14760 ctagtaaatt gctacaacat atgtaagctt tggcgacatc tcccagtagg aaataaagat 14820 ggcatatgtt tcaaagaaag gttttatttt ttgtctaaca ctgattaaaa atctaacaaa 14880 gcttctctga tttctttttg cgaactccca aatcttaggt taatgaagta gaaaaatctt 14940 tatcttggtc taaattcatt ttccagaatg gacaattgaa gcctcagaaa gaaaatagat 15000 tctgtttttt tttttttttt gacctagtct cactctgta cccaggctgg agtgcagtag 15060 tgcaatttcc gctcactgca acctctgcct ccgaggctga agcaattctc ctgcctcagc 15120 ctcccgagta gctaggacta caggtgcacg tgaccacacc tggctgattt ttatattttc 15180 agtagagatg gggtttcgcc atgttgacca ggctggtctc caactcctgg gctcaagtga 15240 tccacccacc tcagcctccc aaagtgctgg ggttacaggc atgagccact gtacccagct 15300 gaaaatagat tctttacaat tcatacagct agtccatggt agagccagca ctagagccta 15360 ggccttttga atgtcctgct attttgtaat gctctcttct gccaacggag accacttagg 15420 gtaaggaata tcccgcattt atattcaggg gtatagtatg acatacctga gaactcctga 15480 agtataaaag atggtgacca caaggaaatg ggaagagcag gtggaaaaga tcttgctccg 15540 acctgtagca gagttgatcc tcagggctgt catgatgata cagctgtagg aacccagcaa 15600 aaggacaagg gtgccaaggc ccaggactga catggtagtg agaatggaga cgatgctagc 15660 gcaggggtca gaacaggtca ataagagcac cggaggaagc tcgcaggcaa aactgtggat 15720 gacattaggg ccgcagaagt gctgctgagc caggaggagg gtgttaatta ggccagtccc 15780 cattcctatg gcccaggatg cccccaccag gccagcacac accctcctgt ttataatcac 15840 cacatacaac agtggatgac acatggcctg aaaccggtca taggccatga ccgagaggag 15900 gcaagcctca gtagctccag aaaatatgac cagggagatc tgagtgaaac actcaaggaa 15960 ggatattgtc tcccccttag aaagaaggtt ctctagcagt ttaggcacaa taattgagga 16020 atagaaagca tccaggaagg agagttgtct caggaagaag tacatagggg tgcagaggtg 16080 ggaatcagta ctgatcacca gcaccatcag caggttcccc agtagagtca agaggtaaat 16140 caccaggaac agaacaaaga gcagtgcctg aacctgaggg ttgttggaca gtcctaggag 16200 aataaacaca gtgactgtgg tcatgttgct gacttccatg gagcattcag gtttcccttt 16260 tgactgacaa gaactaaaaa gaatgctcca gtgggttatc agtggcaggg catgcctggg 16320 gatgaggcat tggcagtaaa gcttcaaggc agacccagga cagattctgt agcagaggaa 16380 gccttgccat gtcaggctgc gtcagtcctt tccagctcag ggcagtaacc tattggccgt 16440 ggtggagaag gtggggagga aaagttcaga gaatcagacc tagaaaggaa ggagaagaaa 16500 gcagcaatta ttcagtctgg agaaggaagg gaagggagtt gggactcctg agtgctgaga 16560 agtgctattc tctacttcca aaaggaaccg agctcaaacc acaggaaggc taactggcat 16620 agtgaataag caataaacat tttggctgac catctgggtg gcagaccatg atgagtgtct 16680 gagtgactga tagaagtatt atgagacctg gtcatggtgc aagttgtctc tcagccactg 16740 taagctcata agccacttaa gtttctaaaa aaaaaaaaag tctgagaaaa atagatggtt 16800 tctaaagttc attccagatt agattttttt tttttttttt tttttttgag acagagtctt 16860 actcttttgc ccaggctgga gtgcagtggc acgatctcga ctcactgaag cctccacctc 16920 ctaggttgaa gctattctcg tgcctcagtc cagcttagat ttttttaaat ttaattttta 16980 atttaatttt tttctaggga tggggtctca ttatattgcc caggctagtt tcaaactctt 17040 ggccttaagt gatcatccct ccttagcctc ccaaagtact gggattacag gtgagtcaat 17100 atacctacta ccagcttaga aattaaaaag atggggaaac agtcttcctg tggtgtttct 17160 ggtactaaat agaagcccct tctcccattc tgtgtcaatc caagtagaaa tgacacatga 17220 atgtattggt ttcacagaag ccaggcaggg ggaaaccctt gaatagaaat gaggactgtg 17280 aacatcggtc attccaaggc cttcccattt cagcatttct gtgaatctat gatgcccact 17340 gttttcactg caatttcagg tcttaacatg ggcatccaca aagactatgc tattctttgg 17400 aatatttgga aaaagatttg gaaaaagcca aatcaaactt ggatattttt atgcatcaga 17460 aggaagttaa agaccaaaaa taggggtgag aaataaaaga acacctggta aaatggacag 17520 ttagaggatt tagtctagat cagaggaagg taaagtggct agtccccctc cccttgatgg 17580 ttaactcaag aaggttatca atgagaatct gatctcgatt catgacttgt accctcgtgg 17640 gttcccagag tgggtagttt taggagaaga ttgatttgaa tgagaaggaa atttgccttc 17700 acctgggctc cctaggccaa cgagatgccc tgaatggagg atccctgtat ggcatccttt 17760 ccaagagtgg agcaaaagcc acagatagga gagggctctg tgggcttcta tcggctttgt 17820 ccttccttta gagaatgcag gggtgaaact tctgagcgag gaaatatccc catagccttt 17880 ctgtccaagg ggaagccttg aatctacctc ccaaataata atggccagag ggacttgatc 17940 tcagctctag gccttggaga gtgagggagg tccctggaga ggtccttaag caggaggcct 18000 gttctgattc tgaacgggtt ctccagggcc aactacttct cccttgtatc attctcattt 18060 cagtgggttg catcatcaag agggccctag ggtggccaat aatttcattt tttcaagtct 18120 actattatgt tttaaagtat ctttgaaaaa aaattttttt acggtatttt caaattacat 18180 actattccta ttgagcatgt atggcagctt aactacccga atacaaagtt taagaagcca 18240 aaactaatct agctttgttt taaaaaattg tgctattgtt gtaatggtta acaaagcgag 18300 gagaagcacc actcaaatca taatgttaca atctctttgt tcagttggat tatgggttgg 18360 tcaaacatta gacttcacac aggcatggct atgatgcagt gattcctcag aagaagacga 18420 aattctcaaa tcacctcaga tgtaatatta ggagcagaat ttaacatttt caacataata 18480 caccataaaa gtcaggacta gttcttatct gatccccacc ttgtaacttc agtaaccaaa 18540 gacatactgg tctataatga attatttcta attaactaac cagcctttct cacaaagagg 18600 agattctttt tgatggggag aaatgcaaat ttaagtctga actgagaaat caacttaaca 18660 gttagaactt aattgtattt ctgcatttaa aaaaagttta ggctttagca agtattactt 18720 aaccctaagg aaccatttta gcttccaaaa agccaatcca aatcccccag caaaagagaa 18780 ccccatctgc tcaaatatgt gagtacttca ccccaggagc aaactcctct gcatttggat 18840 tgattgctgt ggatcacagg gtcttttaga gaggagtcat cattgataat aaggtcattc 18900 aactggtctt ggacttggtc cacagttcgt gggaagtctt gaaccggaat aaactactca 18960 tgttcttccc cttcagcatt tcttggaaac agtgtggtag gaattattaa gaaattgttt 19020 taggcagata gagaggaaaa ggggtccttg gaaagttttc atctctttta aagcagctcc 19080 agaaatgttt cctgtcttag agccaggctg gcaacttttg atatgcaaat gtcaggtgtg 19140 caacctttga tatgcaaatg caagccatta gaaattgggt ccacccaaca tggcgattcc 19200 caccattgtc ctctttccct tgcctcctta cgtgcctggc aacaaggcca cccccacata 19260 tccccatgtt tgtagaacat catggtgccc tgcctttgta tattaaaagg ctagagtggg 19320 agggccagtt tttttcgcgg gctatgtgaa tgacgtgcct ggtcaaacca atcccctaag 19380 ccctatgcaa atcagacacc acctcctcca gcctcttcat acacctggct ggtttccacc 19440 acacttgggg tctcttctgt cggctttaga gacccccacc ctctgtctct gtacggggag 19500 cttcttcttt ctttcttctc cattctttct tgcctattaa actctctgct ccttaaaacc 19560 actccatgtg tgtccctgac gttttatcta atttagcatg gggaccaaga atcctggtgt 19620 tcctctgctt atcagagcca tatcattttg gtgcattggc tgggaatctg aggtaaagcc 19680 tttgttggag tggtgaatat gaaagcaagc ttaaaacctg ttctatcatt cagaggtgct 19740 cttagcctct atattagaat aaatcaaata aatcaatagg catctgtcag cctagtacat 19800 atgcttagcg ttggctgccg tactgaagac acagatatga agctttctgg gaagaacatg 19860 gagaatcccc cattacccac tggtattggt aatgttggcc atgtttgaac tagcttcttt 19920 ttacagggag actttgctat catgcgaggc tgggaaaagt tctgaggcaa ctgagaattc 19980 cagaccaggg cctggtgtga ttcaaatgcc tctggaccga atgcagcctc tgacagtcca 20040 ttccagtgtt ggtagagaat cctcaactgt cctgttgcaa aactcttctt ccttctctat 20100 ctgtggcctc ttactctttc tctgcgtgaa atgtgcaagg atttttacag tctaggaaca 20160 gcattttccc cttttcccct gaggagaggt tacttgctac aacttctctg gtgagcacgt 20220 gatatttcta agctacctag tggaaataaa aatcctcttc atgagacaca ttgctggttc 20280 tctgtggtac attgcagctt tggaactttt ctattttgcg cttttctact gctacttctg 20340 taagtgggaa agctctgttt ttaacagtta ggagtaagat gtcttccata gccaaatttt 20400 agtcttgata ttgtcccact ggcaggaaaa tggccattcg gtttctacgt tcctttaagg 20460 cacctattgt gtctcctatt aagactactt aattagtaag gggattttaa gtccggaagt 20520 taaccagaac cattcttcta cgggtaagtg ctttagtatg agccataata gcatataaag 20580 ttcaatctag catgcccccc accttaaagg gggcttgccc aattaagtgg tttttcttga 20640 aatccatttt gttttttttt ttttggaaga cacacaggcc acagaagtct aggaggtcaa 20700 agggaaataa aaggcagagg actaagactt cttggggaca gcatgactaa ggcccaaaag 20760 tatagttcct ctggtgtcgt ggtgcagagg gtcatgcctg cagtcatggg cggcacattt 20820 aaatgggtgc cagggattca ggagccaagg agagaaaatt attgggggac gcctcctact 20880 gctttcttct ccatcctgga tcacatacca aaaggaagga gacgcttttc ttctcacttc 20940 tctttctaga tgggcaacag atcatcttca acatgcactc ccctggagtg tattttggag 21000 cactgggact tctttgacct ttaaactttg aagaaaaagt ggcttatttt cttctgcaca 21060 agggcatggc atttttatta gacctttgca agtgttgcaa aatcaaccca gctcttttag 21120 caaccatatt gggcaggccc agggaaaata gttccccaaa attaaaaaag taactttcag 21180 ggaaataatc tgagtgtccc ccttattttg ggccccttca cattcccttc tcattgcagg 21240 accttagtca agtgaaggag tcttagacca attttctgat aacactgata ggtgtataga 21300 agctttccaa agtttaactc aggtatttga cttctcatgg aggaatgtta tgctgctcct 21360 aagccaaacc ctactgcagc taaaacaggc agctctgcaa acaggagaaa attttgggga 21420 tgagcaatat gtcttctata gtaggccaaa agggaaagag aaaatagaga aggcaaagaa 21480 ataggggaaa caccattccc aataggaaaa gaggcaatac ctcttgacaa ccctaactgg 21540 aactcctttc catggtgttt ttccttcttt catggtttaa aaaggcttct atctctttta 21600 taatgttctc ccaacctgga aaagttaatt ttccaaaact taaaaatgtt tggcttagag 21660 ttgagctggg ggaagggaac ccagaagact aatatgctgg caaaagggta aaaatttctt 21720 accagtcagg cttttgtctt ctctctcact gtgcaaaatg gcaaaaggga taataagaat 21780 cattgttgga gaggttccaa gatggccaaa taggaacagc tccagtctag agctaccagc 21840 atgagtgatg cagaagatgg gtgatttctg catttccaac tgaggtacca ggttcatctc 21900 actggggctt gttggacagt aggtgcagcc cacagagcat gagccaaagc agggtggggc 21960 attgcctaac ccgggaagtg caaggggttg gggaattccc tttcctagcc tagggaagcc 22020 atgacagatg gtacccggaa aatagggaca ctcccaccct aatactgtgc ttttccaacg 22080 gtcttagcaa acggcacacc aggagattat atcttgtgcc tggctcagag ggtcccatgc 22140 ccacggagcc tcgctcactg ctagcccagc agtctgagat caaactgcaa ggcggcagcg 22200 aggctgaggg aggggcatcg ctgctgaggc ttgagtaggt aaacaaagcg gccgggaagc 22260 tcaaactggg aggagcccac tacagctcaa ggaggcctgc ctgcctctgt agactccacc 22320 tctgggggca gggtgtagct gaatcaaagg cagcagaaac ttctgcagac ttaagtgtcc 22380 ctgactgaca gctttgaaga gagtagtggt tcttccagca tggagtttga gatctgagaa 22440 tggacagact gcctcctcaa gtggggccct gacccctgag tagcctaact gggaggcact 22500 tccaagtagg ggccaactga cactcatatg gccgggtgcc cctctgagac aaggcttcca 22560 gaggaaggat caggcagcaa catttgccat tctgcaatat ttgctgttct gcagcctcca 22620 ctggtgatac ccaggcaaac agggtctgga gaggacctcc agaaaactcc aacagacctg 22680 cagctgaggg tcctgagtat tagaaggaaa actaacaaac agaaaggaca tccacaccaa 22740 aaccccatcg gtacgtcacc aacatcaaag accaaaggta gataaaacca caaagatggg 22800 gagaaaccag agcagaaaag ctgaaaattc taaaaatcag agagcctctt cttctccaaa 22860 ggagtgcagc tcctcactag caatggaaca aagctggatg gagaatgact ttaatgagtt 22920 gagagaaaaa ggcttcagat gattggtaat aacaaacttc tctgagctaa aggaggatgt 22980 tcgaacccat cacaacgaag ctaaaaacct tgaaaaaaga ttagacgaat ggctaactag 23040 aataaacagt gtagagaaga ccttaaatga cctgatggag ttgaaaacca tggcacaaga 23100 actacgtgac gcatgcacaa gcttcagtag ccaatttgat caactggaag aaaaggtatc 23160 agtgattgaa gaacaaatga atgaaatgaa gcaagaagag gagtttagag aaaaaagagt 23220 aaaaagaaat gaaaaaacct ccaagaaata tgggactatg tgaaaagacc aaatctatgt 23280 ctgattggtg tacctgaaag tgatggggag aatggaacca agttggaaaa caccctgcag 23340 gatattatcc aggagaactt ccccaaccta gcaaggcagg ctaacattca aattcaggaa 23400 atacagataa cgccacaaag atactcctcg acaacagcaa ttccaagaca cataatttca 23460 gatgcaccaa agttgaaatg agggaaaaaa tgttaagggc agccagagag aaagtttggg 23520 ttaaccacaa agggaagcct atcagactaa cagcagatct cttggcagaa gctctacaag 23580 ccagaagaga gtgggggcca atattcaaca ttcttttttt tttttttttt tttttttagt 23640 atttattgat cattcttggg tgtttctcag agagggggat ttggcagggt cataggacaa 23700 tagtggaggg aaggtcagca gataaacaag tgaacaaggg tctctggttt tcctaggcag 23760 aggaccctgt ggccttccgc agtgtttgtg tccctgggta cttgagatta gggagtggtg 23820 atgactctta acgagcatgc tgccttcaag catctgttta acaaagcaca tcttgcaccg 23880 cccttaatgc atttaaccct gagtggacac agcacatgtt tcagagagca cagggttggg 23940 gggtaaggtt atagattaac agcatcccaa ggcagaagaa gttttcttag tacagaacaa 24000 aatggagtct cctacgtcta cttccctcta cagagacaca gcaacaatct gatttctcta 24060 tcttttcccc acatttcccc cttttctatt cgacaaaacc accatcgtca tcatggcccg 24120 ttctcaatga actgttgggt acacctccca gacggggtgg ctgccgggca gaggggctct 24180 tcacttctca gaaggggcgg ctgggcagag acgctcctca cctcccagat ggggtcatgg 24240 ccaggcagag gcactcctca catcccagat ggagcggtgg ggcagaggcg ctccccacat 24300 ctcagacgaa gggcagccag gcagagacgc tcctcacttc ctagacagga tggcagctgg 24360 gaagaggcgc tcctcacttc ccagactggg cagccaggga gtgggtctct cacatcccag 24420 atgatgggtg gccaggcaga gacgctcctc acttcccaga cagggtggtg gccgggcaga 24480 ggctgcaatc tcggcacttt gggaggccaa ggcaggcggc tgggaggtgg aggttgtagt 24540 gagctgagat cactccactg cactccagcc tgggcaacac tgagcactga gtgagtgaga 24600 ctccgtctgc aatcccagca ccttgggagg ccgaggctgg cagatcactc gtggttagga 24660 gctggagacc agcccagcca acacagtgaa accccgtctc caccaaaaaa atatgaaaac 24720 cagtcaggcg tggcggtgca tgcctgcaat tgcaggcatt gggtaggttg aggcaggaga 24780 atcaggcagg gaggttgcag tgagctgaga tggtggcagt acagtccagc ttcagctcgg 24840 catcagaggt agaccataga gagagaggga gagggagagg gagaccatgg ggagagggag 24900 agggagaggg agctcaacat tcttaaagaa aagaattttc aacccagaat ttcatatcca 24960 gccgaactaa gcttcataag tgaaggaaaa ataaaatcac ttacagacaa gcaaatgctg 25020 agagattttg tcaccaccag gcctgcctta caagagctcc tggaggaagc actaaacatg 25080 gaaaggaaca accggtacct ttccactgca aaacatgcca ctgcaaaaac atgccaaatt 25140 gtaaagacca tcgatgctag gaagaaattg cagcaactaa tgagaaaaat aaccagctaa 25200 catcataatg acaggatcaa attaacacat aacaatatta accttaaatg taaatgggct 25260 aaatgctcca attaagagac acagactggc aaattggata aagagtcaag acccatcagt 25320 gtgctgtatt caggagaccc atctcacatg caaagacaca tataggctca aaataaaggg 25380 atacaggaag atctaccaag caaatggaaa acaaaaaaaa agcagaggtt gcaatcctag 25440 tctctgataa aacagacttt aaaccaacaa agatcaaaag agacaaagaa ggccattaca 25500 taatggtaaa gggatcaatt caacaagaag agctaactat cctaaatata tatgcaccca 25560 atacaggagc acccaagttc ataaagcaag tccttagaga cctacaaaga gacttagact 25620 cccacacaat aataatggga gattttaaca ccccactgtc aacattagac agatcaacga 25680 gacagaaagt taacaagggt atccaggaat agaactcagc tgtgcaccaa gcagacctaa 25740 tagacatcta cagaactctc cactccacat caacagaata tacattcttc tcagcaccac 25800 atcacactta ttccaaaatt gaccacatag ttggaagtaa agcactcctc agcaaatgta 25860 aaagaacaga aattataaca aactgtctct cagaccacag tgcaatcaac tagaactcag 25920 gattaagaaa ctcactcaaa accactcaac tacatggaaa ctgaacaacc tgctcctgaa 25980 tgactactgg gtacataatg aaatgaaggc agaaataaag atgttctttg aaaccaatga 26040 gaacaaagac acaatatact agaatctctg ggacacatta aagcagtagg tagagggaaa 26100 tttatagcac taaatgccca caagagaaag caggaaagat ctaaaattga caccctaaca 26160 tcacaattaa aagaactgag aagtaagagc aaataaattc aaaagctagc agaaggcaag 26220 aaataactaa gaatagagca gaacggaagg agataaagac acaaaaaacc cttcaaaaaa 26280 tcaatgaatc caggagctgg ttttttgaaa acatcaacaa aattgataga cagctagcaa 26340 gactaataaa gaagaaaaga gagaagaatc aaatagacac aattaaaaat ggtaaagggg 26400 gatcaccacc gatcccacag aaatacaaag taccatcaga gaatactata aacacctcta 26460 tgcaaataaa ctagaaaatc tagaagaaat gaataaattc ctggacacac acaccctccc 26520 aagactaaac cagtaagaag ttgaatccca gaatggacca atatcaggct ctgaaattga 26580 ggcaataatt aagagcctac caaccaaaaa aagtccagga ccagacggat tcacagccaa 26640 attctaccag aggcacaaag agaagctggt accattcctt ctgaaactac tccaatcaat 26700 agaaaagaag ggaatcctcc ctaactcatt ttatgaggcc agcatcatcc tgataccaaa 26760 gcctggcaga ggcacaacaa ataaagagaa ttttaggcta acatccctga tgaacatcca 26820 tgcaaaaatc ctcaataaaa tactggcaaa ctgaatccag cagcacatca aaaagcttat 26880 ccaccacgat caagttggct tcatccctgg gatgcaaggc tggctcaaca tacacaaatc 26940 aataaacata atccatcata taaacaaaac caaagacaaa aaccacatga ttatctcaat 27000 agatgcagaa aaggcctcgg caaaattcaa caacccttca tgctaaaaac tctcaaaaat 27060 aggtatcgat gggatgtatc tcaaaataac aagagctatt tatgacaaac cctcagccaa 27120 tatcatactg aatgggcaaa aactggaagc attccctttg aaaactggca taagacaggg 27180 atgccctctc tcaccactcc tattcaacat agtgttggaa gttctggcca gggcaatcag 27240 gcaggagaaa taaataaaga gtatttgatt aggaaaagag gaagtcaaat tgtccctgtt 27300 tgcagatgac atgattgtat atttagaaaa ccccatcgtc tcagcccaaa atctccttaa 27360 gctgataagc acttcagcaa ggtctcagga tacaaaatca atgtacaaaa atcacaagca 27420 ttcttataca ccaataacag accaacagag agccaaatca tgagtgaact cccattcaca 27480 attgcttcaa agagaataaa atacctagga atccaactta caagggatgt gaaggacctc 27540 ttcaaggaga actacaaacc actgctcaac gaaataaaag aggacacaaa caaatggaag 27600 aacattccat gctcatggat aggaagaatc aatatcgtga aaatggccat actgcccaag 27660 gtaatttata gattcaatgc catccccatc aagctaccaa tgactttctt cacagaattg 27720 gaaaaaacta ctttaaagtt catatggaac caaaaaagag cctgcattga caagacaatc 27780 ctaagcaaaa agaacaaagc tggaggcatc acactacctg acttcaaact atactataaa 27840 gctacagtaa ccaaaacagc atggtactgg taccaaaaca gagatataga ccaatggaac 27900 agaacagagc ccgcacaaat aataccacac atctacaacc atctgatatt tgacaaacct 27960 gagaaaaaca agcaatgggg aaaggactcc ctgtttaata aatggtgctg gaaaaactgg 28020 ctagccatat gtagaaagct gaaactagat cccttcctta caccttatac aaaaaataat 28080 tcacgatgga ttaaagactt aaatgttaga cctaaaatca taaaaaccct agaagaaaac 28140 ctaggcaata ccattcaggc cataggcaca ggcaaggact tcatgactaa aacaccaaaa 28200 gcaatggcaa caaaagccaa aattgacaaa tgggatctaa ttcaactaaa gagcttctgc 28260 acagcagaag agactaccat cagagtgaac aggcaaccta cagaatggga gaaaattttc 28320 gcaacctact cgtctgacaa agggctaata tccagaatct acaatgaact caaacaaatt 28380 tacaagaaaa aaacaaacaa tcccatcaaa aagtgggtga aggacacgaa cagacacttc 28440 tcaaaagaag agatttatgt agccaaaaaa cacgtgaaaa aatgctcacc atcactggcc 28500 atcagagaaa tgcaaatcaa aaccacaatg agagaccatc tcacaccagt tagaatggca 28560 atcattaaaa agtcaggaaa caacaggtgc tggagaggat gtggagaaac aggaacactt 28620 ttacactgtt ggtgggactg taaactagtt caacccttgt ggaagtcagt gtggcgattc 28680 ctcagggatc tagaactaga aataccattt gacccagcca tcccattact gggtatatac 28740 ccaaaggact ataaatcatg ctgctataaa gacacatgca cacgtatgtt tattgcggca 28800 ctattcacaa tagcaaagac ttggaaccaa ctcaaatgtc catcaatgat agactggatt 28860 aagaaaatgt ggcacatata cactatggaa tactatgcag ccataaaaac tgatgagttc 28920 atgtcatttg tagggacatg gatgaaactg gaaataataa ttctgagcaa actattgcaa 28980 ggacagaaac tcaaacacct catgttctca ctcataggtg ggaattgaac aatgagaaca 29040 catggacaca gggtggggaa catcacacac cgggacctgt cttggggtgg ggggaggagg 29100 gagggatagc attaggagat atacctaatg taaatgacga gttaatgggt gtagcacacg 29160 aacatggcac atgtatacat atgtaacaaa cctgcacatt gtgcacatgt accctgaaac 29220 ttaaagtaca ataaaaaaag aaaaaaaaaa taaatccagt atacaacggc aagaaaaaaa 29280 aaaaagaatc attgtttata ttctttttaa atttataatt aatgacaaag aatttgtgag 29340 gttggttgta agctgtagac tatctggtgt gctttgcatg tctttctcgt tggttctgtc 29400 aaagaaagag tatcttagtt taggaagcag tcccaggacc ccataagcct gctgttcaag 29460 ccagcccagc aaaacgatca gtaacaaact gggtacagga ctccgtcttg tttcatgtcc 29520 ttgggagcat aacctgtaac cacgtggcaa tacttttgag gcagaaatca gagaaataag 29580 aaaataaaat taaaaagaaa aagaaataag ctttcctata tttcctatat tacccttccc 29640 agcctctggt agccatcctt ctactctatg ttcatgagtt caattgtttt gatttttaga 29700 tcctacaaat aaatgacaac atgcaatgtt tgtttttctg tgcctgggtt atttcacgtt 29760 acataatgat ctccagttcc atccatgctg ttacaaatga ctggatctca gtctttttaa 29820 tggctgaata gactccattg tgtatgtgtgg gtacaatagt cttgattacc ataactgtat 29880 agtaattctt agaattggat aatgtgaatc ttccaatttt gttgtttttt ctcagaattg 29940 tttgggctta ttctagtacc tttgccttta catattcacc agtttcctca gggtaaggtt 30000 ttgaatctgt tttcctggct acagtgggat tgcgccagta ccctcagagt aggggctttg 30060 atgaattacc tgcttctggt ttagcctttt gttctatacg ggagacaaga gaaccgggtc 30120 tgggaggatt taacagtggc ttctgttctc ctaccccagc tagaactatg gggagagatt 30180 ttcttgagag cctcatggag ctcctggagg aaaaggctgg aaaaagttgg gaacccctct 30240 atgattgtga ccctcaggaa cttcaaagtc tcatactacc atacactcag tcaccagcaa 30300 tttgtcaaaa tttccagttt aatcttccta cttctttaca taacacccag cagcttctgt 30360 cctaggtaag caaatgctca ggttcagtgt ctccttgcag gaacctgcct ctccagattg 30420 atttctgtat ggccatctgc cctgtgacct cagttctctt atggtccaag aaaagttact 30480 gattttcagt ttgttcagct tttttttttt tttttttttt ttttaaataa gagtgggagt 30540 gacatctttc cagccctctc cttttttttt tttttttttt tttttttttt tgagacagag 30600 tcttgctctg tcacctaggt tggagttcag tggcacgatc tcagctcact gcaacttcca 30660 tctcccaggc tcaagggatt ctcctgcctc agcctcctga gtagcttggg tcgcaggttc 30720 atgccaccac acctggctaa tttttgtatt tttagtaggg atggggtttt accatgttgg 30780 ccaggctggt ctccaactcc tgacctcagg tgatccgcct gcctctgcct cccaatttgc 30840 tgggattaca ggcgtgagcc accatgcctg gcctagccct ctgcgttttt gagtggaaac 30900 tggaagtctg agttttaaga taataaaatc agagctcctg gttccaggat gccttttatt 30960 gtgctgtatt tgaatctgta attcctcttc atatatattt ttttcaggcc aggcgcagta 31020 atcccagcac tttgggagac ctaggcaggt ggattgcctg aagtcaggag tttgagacca 31080 gcctggccaa cataggtaaa cctcatctct actaaaaata caaaaaatta gctgggcatg 31140 gtggtgggtg cctgtaatcc cagctactca ggaggctgag gcaggagaat cacttgaacc 31200 caggagatgg aggttgcagt gagccaagat catgccattg cactccagcc tgggcaacaa 31260 gagtgaaact tcatctcaaa aacattatat atatatatgt atttttttcc atggtagttg 31320 ttttggtcca tgtgtatctc cttcaacatt ttttaatggg gttgttctga ttattcctct 31380 tgatggttgt ctttcattgc ctgtttgtac tattgtttcg tttgctcatt gtggcaaaag 31440 actggctatt tcagttcttt aagtggtaac agtctcatga gagaagggaa tctccgcctg 31500 tagtgtagca ccagataagt tgccagtccc acttccaggc acccacctct gcactccagt 31560 tttatgcttt cccatcctca agacagggaa aacttcctct gttttagttc ttccttagat 31620 gcttgggagt cagatagaga ggtgcattta catggggcaa taccaacctt tcagctgtat 31680 atccatcacc cgagtgctaa tgttcctgga acatccatgt tcagcccccg gatagaaagg 31740 gtcacacacg ctgcagcagc tgctgctgct gctgcagacg ggacttccat gggacctggg 31800 gctttaccac tcccaccttc cgattgctgt agctgcagct tagctcagga aagccttgga 31860 cttgggatta gagagtatgg tgggacatgt tcatgttaga tatagataat attagatata 31920 ttaatggtca gtcatgctgc atcttcagtt gattttgcat atcattagct tattccatta 31980 aatactaatt atattattta atagaaaaat ctattatttt cacttcgaag ttttatgaga 32040 ctttattcaa gtccttgagt acttatgatt tattctccct ccagaaaaaa tttcctcaaa 32100 cccagatatc 32110 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 2 aggaaaggaa aaactaccat 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 ggcttatttt ggattgctat 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 ctcaaagtgc tgggattaca 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 ccgagtctgg tggatcactt 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 ggaagcttca gtcatgagct acca 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 aaggatcctg gaagcatcac tcag 24 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 8 ggctcgagct ccgctccttg gta 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 9 tcacaaagcc actgttccag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 10 tggagttttg cccttgttgc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 11 ctggataaag gaggcagtgt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 12 gaacaggtca ataagagcac 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 13 ccctcaggtt caggcactgc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 14 tattctcgtg cctcagtcca 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 15 aaaacagtgg gcatcataga 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 16 aactaaccag cctttctcac 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 17 aagaccagtt gaatgacctt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 18 actgggactt ctttgacctt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 19 agggttgtca agaggtattg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 20 acaagtgaac aagggtctct 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 21 tctgccccac cgctccatct 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 22 agagattttg tcaccaccag 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 23 cctgtatccc tttattttga 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 24 ggctggctca acatacacaa 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 25 atccctgtct tatgccagtt 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 26 gttggtggga ctgtaaacta 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 27 tgttccccac cctgtgtcca 20

[0046]

[Sequence list free text]

SEQ ID NO: 2: Synthetic DNA SEQ ID NO: 3 synthetic DNA SEQ ID NO: 4: Synthetic DNA SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic DNA SEQ ID NO: 7: Synthetic DNA SEQ ID NO: 8: Synthetic DNA SEQ ID NO: 9: Synthetic DNA SEQ ID NO: 10: Synthetic DNA SEQ ID NO: 11: synthetic DNA SEQ ID NO: 12: Synthetic DNA SEQ ID NO: 13: Synthetic DNA SEQ ID NO: 14: Synthetic DNA SEQ ID NO: 15: synthetic DNA SEQ ID NO: 16: Synthetic DNA SEQ ID NO: 17: synthetic DNA SEQ ID NO: 18: synthetic DNA SEQ ID NO: 19: synthetic DNA SEQ ID NO: 20: Synthetic DNA SEQ ID NO: 21: Synthetic DNA SEQ ID NO: 22: Synthetic DNA SEQ ID NO: 23: Synthetic DNA SEQ ID NO: 24: Synthetic DNA SEQ ID NO: 25: Synthetic DNA SEQ ID NO: 26: synthetic DNA SEQ ID NO: 27: Synthetic DNA

[Brief description of drawings]

FIG. 1 is a diagram showing a restriction enzyme map of a bacterial artificial chromosome bLA.

FIG. 2 is a diagram schematically showing a region required for expression without position effect.

Continued front page    (72) Inventor Kenji Sekikawa             2111-7 Kamenomuro, Tsukuba City, Ibaraki Prefecture (72) Inventor Yoshihiro Fujiwara             7-3-4 Sumiyoshi Yamate, Higashinada-ku, Kobe City, Hyogo Prefecture             Kobe University Dormitory A-303 F term (reference) 4B024 AA10 BA80 CA01 DA02 EA06                       FA02 FA18 GA11 HA20                 4B065 AA90X AA93Y AB01 AC15                       BA02 CA24 CA41 CA44

Claims (13)

[Claims] 1. A mammary gland expression vector defined by the following restriction enzyme cleavage map. [Chemical 1] [In the formula, B is BamHI, C is ClaI, E is EcoRV, S is SmaI, and X is X.
Indicates hoI. ] 2. A mammary gland expression vector comprising the base sequence shown in (a) or (b) below. (a) 1 to 5340 bp and 84 of the nucleotide sequences shown in SEQ ID NO: 1
A nucleotide sequence containing 35 to 32110 bp (b) An expression which hybridizes with a DNA consisting of a sequence complementary to all or part of the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions and has no position effect Base sequence to be given 3. The vector according to claim 1, which further comprises a gene encoding a target protein. 4. The target protein is α1 antitrypsin, acid α-glucosidase, antithrombin, bile salt-stimulating lipase, bovine tracheal antibacterial peptide, erythropoietin, factor VIII, factor IX, fibrinogen, granulocyte macrophage colony stimulating factor. , Growth hormone, insulin-like growth factor, interferon γ, interleukin-2, lactoferrin, lysozyme, nerve growth factor, parathyroid hormone, protein C, procollagen, salmon calcitonin, serum albumin, superoxide dismutase, surfactant protein B, tissue The vector according to claim 3, which is selected from the group consisting of plasminogen activator and urokinase. 5. The vector according to claim 1, which further comprises a mammary gland-specific promoter, a secretion signal and / or a poly A addition signal. 6. The mammary gland-specific promoter comprises α-lactalbumin, αS1-casein, αS2-casein, β-casein, κ-casein, and wey acidic protein.
vector), β-lactoglobulin or lactoferrin promoter. 7. The vector according to claim 5, wherein the secretion signal is a secretion signal of IL-2, growth hormone or serum albumin. 8. The signal associated with poly A is SV40, rabbit β-
The vector according to claim 5, which is a poly A addition signal of globin, mP1, thymidine kinase or bovine growth hormone. 9. The vector according to claim 1, which is a bacterial artificial chromosome (BAC) or a cosmid. 10. An animal cell into which the vector according to any one of claims 1 to 9 has been introduced. 11. A transgenic animal obtained by growing the animal cell according to claim 10, which secretes the target protein in milk. 12. Milk produced by the transgenic animal of claim 11. 13. A method for producing a target protein, which comprises collecting the target protein from the milk according to claim 12.