JP2006141404A - Transgenic mollusk and method for creating the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a transgenic mollusk which expresses a desired foreign gene, and also to provide a method for creating the same. <P>SOLUTION: This transgenic mollusk into which a desired foreign gene is transduced or which expresses the desired foreign gene. The method for creating the transgenic mollusk comprises micro-injecting a recombinant vector prepared by combining a vector with the desired foreign gene to be transduced or a nucleic acid containing the foreign gene in the male and female gonads of the mollusk, mating the male with the female to make descendants in the first generation, and then screening an individual expressing the desired gene from the descendants. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a transgenic mollusk and a method for producing the same.

  On the other hand, conventional pearl farming can only produce several kinds of naturally occurring pearls, and the color tone has been processed by dyeing after production. In these post-production processes, the color tone tends to fade, and it is almost impossible to process the color tone as desired because the nacre is strong.

  If a transgenic pearl oyster having virus resistance and the ability to produce colored pearls is obtained, it is advantageous for the cultured pearl industry. However, not to mention transgenic pearl oysters having such useful properties, transgenic molluscs that can express a desired foreign gene have not been produced so far in the entire mollusc. .

  Accordingly, an object of the present invention is to provide a transgenic mollusk capable of expressing a desired foreign gene and a method for producing the same.

  As a result of intensive research, the inventors of the present invention have microinjected a desired foreign gene into the male and female gonads of a mollusc and mating those males and females, or a promoter that functions in a mollusc. The present invention was completed by successfully producing a transgenic mollusk expressing a desired foreign gene by introducing a desired foreign gene linked downstream into a fertilized egg or embryo of the mollusk.

  That is, the present invention provides a transgenic mollusk into which a desired foreign gene (excluding a gene conferring virus resistance) is introduced and expresses the foreign gene. The present invention also provides a method for microinjecting a recombinant vector obtained by incorporating a desired foreign gene to be introduced or a nucleic acid containing the foreign gene into a vector into a male and / or female gonad of a mollusk. A method for producing a transgenic mollusk according to the present invention, which comprises mating females to form a first generation and selecting an individual expressing the desired gene. Furthermore, the present invention relates to a recombinant vector in which a nucleic acid operably linked to the desired gene is incorporated downstream of the original gene in a mollusk to be transformed or a promoter that exhibits modified promoter activity. The transgenic mollusc of the present invention, comprising introducing the fertilized egg or embryo of the mollusk, growing the fertilized egg or embryo into an individual, and selecting an individual expressing the desired gene. Provide production methods.

  According to the present invention, a transgenic mollusk capable of expressing a desired foreign gene is provided for the first time. According to the present invention, various industrially useful molluscs such as pearl shells that form colored pearls can be produced.

  The transgenic mollusk of the present invention may be any animal belonging to the mollusc, but preferred examples include shellfish such as mussel (Axopoda) and snail (Gastropoda), especially pearl oysters. Pearl shells such as white butterfly and black butterfly.

  The desired foreign gene to be introduced into the mollusk may be any gene that can give the trait to be imparted to the mollusc (except for genes that confer virus resistance). As a preferred example, when the mollusk is a pearl oyster, a gene involved in coloring such as a green fluorescent protein (GFP: green fluorescein protein) gene, an anthocyanin gene, a fluorescent luciferase gene, a β-galactosidase gene, a phosphatase gene, etc. Can be mentioned. As used herein, the “gene involved in coloring” is not only a gene encoding a dye (including a fluorescent dye) but also a reaction that produces a dye in the body, as is apparent from the above examples. In addition, a gene encoding a substance involved in a chromogenic reaction in the body, such as a gene encoding an enzyme to be used, is included.

  The transgenic mollusk of the present invention can be produced as follows. In the first method, a recombinant vector in which the desired foreign gene or a nucleic acid containing the foreign gene is incorporated into a vector is injected into a mollusk male and / or female gonad by microinjection, and these male and A female is crossed to produce a first generation individual, and an individual expressing the desired foreign gene is selected from the first generation individuals.

  Only the desired foreign gene may be incorporated into the recombinant vector to be microinjected, or a nucleic acid containing the foreign gene may be used. Examples of such nucleic acids include those in which a promoter that exhibits promoter activity in mollusc cells is linked upstream of the foreign gene, and fusion genes in which the foreign gene is fused with other genes. . Here, as a preferable example of the other gene fused with the desired foreign gene, when the foreign gene is a gene involved in coloring and the mollusk is a pearl shell, the nacreous protein gene, the prism layer Examples include skeletal protein genes and calcium carbonate crystallizing enzyme genes. Examples of the vector include animal cell vectors such as adenovirus vectors and retrovirus vectors. Since these vectors are commercially available, commercially available products can be used.

  A method for microinjecting the recombinant vector into the mollusc of a mollusk will be described. Basically, it can be performed by injecting a solution containing the recombinant vector directly into the gonad with an injection needle. The medium of the solution for microinjection may be a buffer solution such as TE buffer, and the concentration of the recombinant vector in the solution is preferably about 2 to 200 μg / ml, particularly preferably about 5 to 10 μg / ml. . The amount of the solution to be injected is preferably about 10 to 50 μl per site, and it is preferable to inject into about 2 to 4 sites for both the ovary and testis.

  After microinjection, the microinjected male and female are mated after standing at 10 to 25 ° C., preferably 15 to 20 ° C. for 24 to 72 hours, preferably 24 to 48 hours. The mating may be natural mating, but it is preferable to perform artificial insemination in order to ensure mating with good reproducibility. Artificial insemination can be performed basically by adding sperm from the microinjected testis to mature eggs in the microinjected female ovary. Microinjection is preferably performed on both male and female gonads to be mated, but a transgenic mollusk can be produced even if it is performed on either gonad. The method of artificial insemination of mollusks can be performed by the method described in Dev Biol 1994, 163 (1): 162-174 and the like.

  An individual can be bred from a fertilized egg by incubating the fertilized egg in seawater or artificial seawater in the normal growth temperature range of the mollusk.

  Next, a transformant is selected from the obtained individuals. This is to examine whether or not the desired foreign gene to be introduced is present in the mollusc cells by Southern blotting, and further to determine whether or not the foreign gene is expressed in the mollusc cells. This can be done by examining. The Southern blotting and Northern blotting methods themselves and the sample preparation methods themselves are well known in this field. For example, Nakayama / Nishikata, "Bio-Experiment Illustrated- (2) Basics of Gene Analysis", Shujunsha ( (1995).

  In order to establish the transformation line, the male and female of the first generation mollusk confirmed to be a transformant by the above method are mated in the same manner as described above to obtain a second generation individual, Among them, it is preferable to select a transformant in the same manner as described above. Furthermore, it is possible to establish a transformation line more reliably by making the third and subsequent generations in the same manner.

  In the second method for producing a transgenic mollusc, a recombinant vector in which a nucleic acid operably linked to the desired gene is incorporated downstream of a promoter that exhibits promoter activity in the mollusc to be transformed. The mollusc is introduced into an unfertilized egg, fertilized egg or embryo, and the unfertilized egg, fertilized egg or embryo is grown to an individual, and an individual expressing the desired gene is selected.

  The promoter that exhibits promoter activity in the mollusk to be transformed may be any promoter that exhibits promoter activity in the mollusk to be transformed, such as an actin gene promoter, heat Examples thereof include, but are not limited to, shock protein gene promoters. Further, “functionally linking” a desired foreign gene downstream of the promoter means that the foreign gene is linked in a reading frame so that the foreign gene is controlled by the promoter. In this case, the desired foreign gene can be linked to the downstream of another structural gene or fragment thereof functionally linked downstream of the promoter and expressed as a fusion protein. In addition, the method of functionally linking a structural gene downstream of a promoter is well known in this field. A recombinant vector can be prepared in the same manner as in the first method.

  Subsequently, the prepared recombinant vector is introduced into a mollusc unfertilized egg, fertilized egg or embryo, preferably an unfertilized egg. This can be done, for example, as follows. A vector solution having a concentration of about 100 to 200 mg / ml is prepared in a petri dish or well, and an unfertilized egg, a fertilized egg or an embryo is immersed therein. The soaked egg or embryo is injected into the egg or embryo so as to be sharply scratched for a moment using a micropipette for microinjection (not limited to this). At this time, it is important to make a hole to the extent that it does not cause a fatal wound as well as a rupture. An individual can be obtained from a fertilized egg or embryo into which a recombinant vector has been introduced in the same manner as in the first method, and a transformant can be selected and a transformation line can be established in the same manner as in the first method. .

  An unfertilized egg can be obtained as an individual in the same manner as in the first method after artificial insemination immediately after the above operation or in parallel.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Reference Example 1 Production of transgenic pearl oysters introduced with human interferon α gene Human or mouse interferon α gene (commercially available from BBL and RDS) is incorporated into an adenovirus vector (Takara Adenovirus expression vector kit) A replacement vector was produced. Specifically, this operation was performed as follows. Swa I site of the above-mentioned commercially available interferon α gene in the cosmid vector (pAxcwt (44,741 bp), Niwa, M. et al., (1991) Gene 108 , 193, included in the above-mentioned commercially available adenovirus expression vector kit) Inserted into. A cosmid vector having an integrated gene and the above-mentioned commercially available adenovirus-derived DNA-TPC treated with the above restriction enzyme (Miyake, S. et al., (1996), Proc. Natl. Acad. Sci. USA 93 1320) Were co-introduced into 293 cells (human embryonic kidney cells, manufactured by Dainippon Pharmaceutical Co., Ltd.). Specifically, coexistence was carried out as follows. 293 cells were cultured in DMEM medium supplemented with 10% FCS at 100% confluence under conditions of 5% CO ₂ and 37 ° C., and 10 μg of the cosmid vector DNA and 5 μg of restriction enzyme-treated DNA-TPC were mixed on a petri dish having a diameter of 6 cm. did. This transfection was performed by the calcium phosphate method. The cells after co-introduction were cultured at 37 ° C. and 5% CO ₂ for 24 hours, and then the fractions of the grown recombinant adenovirus were collected, and the amount of DNA (100 to 200 mg / piece) was injected into the ovary of the pearl oyster. It was fertilized by mixing in a test tube with sperm extracted from males (twice the amount of eggs). This was cultured in seawater at 25 ° C. for 24 days to obtain juvenile shellfish. Fluorescence (FITC) color of the interferon DNA probe was observed in 31 of the 200 juvenile shellfish. Further, the DNA of juvenile shellfish was purified and the sequence was confirmed with the same DNA probe. These shellfish were continuously cultured.

  In addition, it has been reported that the closed shell muscle changes to red due to virus infection, and 18 of 20 adult shellfish in which the presence of the interferon gene was confirmed did not show this product change.

  On the other hand, the recombinant adenovirus vector obtained as described above was transfected into Hela cells and propagated in Hela cells. Transfection and growth of Hela cells was performed by the method described in Nature 1995, 374 (6523): 660-662. Recombinant vector DNA was recovered from Hela cells by a conventional method, and this was recovered using 10 mM Tris-HCl (pH 7.5), 1 mM EDTA to 20 mM potassium phosphate, 3 mM potassium citrate, 2% PEG-6000 (pH 7). .5) was dissolved in DNA at a concentration of 50 to 100 μg / ml to prepare a microinjection solution. The solution was microinjected into female ovaries and male testes of pearl oysters. The amount of the solution injected was 100 μg in terms of DNA per site, and 3 sites were injected into the ovary or testis. After 24-48 hours, artificial insemination was performed using sperm from the testes and mature eggs from the ovaries. Specifically, artificial insemination was performed as follows. The testis from the male pearl oyster and the ovary from the female were excised, and the sperm and ovum were removed from the test tube and mixed at a ratio of 1: 2. The first generation pearl oyster individual was obtained from the fertilized egg by incubating the fertilized egg in seawater for 2-3 weeks at 25 ° C. Total DNA was collected from the obtained pearl oyster gonad by a conventional method, and Southern blotting was performed by a conventional method (“Bio-Experiment Illustrated”, supra) using the human interferon α gene as a probe. Furthermore, total mRNA was collected from the visceral mass and closed shell muscle cells of the pearl oyster by a conventional method, and the northern blot method was performed by a conventional method (“Bio-Experiment Illustrated”, supra) using the human interferon α gene as a probe. .

  Artificial insemination was performed in the same manner as described above using mature eggs and sperm collected from Southern blot positive and Northern blot positive female and male individuals, and second-generation individuals were obtained in the same manner as described above. By performing Southern blotting and Northern blotting in the same manner as described above, three lines of transgenic pearl oysters into which the human interferon α gene was introduced were selected.

  From the state of the cultured pearl oysters, the transgenic pearl oysters produced as described above and the conventional pearl oysters were cultivated for 180 days in the waters considered to be virus-contaminated and the waters not considered to be virus-contaminated. The rates were compared. The results are shown in Table 1 below.

  As is clear from Table 1, in the transgenic pearl oysters (lines 1 to 3) of the present invention, the lethality in the uncontaminated sea area is equivalent to that of the conventional species, but the lethality in the contaminated sea area is higher than that of the conventional species. It was much lower. From this, the effect of lowering the lethality by introducing human interferon α gene was clearly recognized.

Reference Example 2 Production of transgenic pearl oyster into which human interferon β gene was introduced Instead of human interferon α gene, human interferon β gene (commercially available from BBL, HIGASHI, Y. et al. (1983) J. Biol. Chem. 258 : 92) and the probe used in the Southern blot and Northern blot was a probe obtained by modifying a sequence fragment of the 5 'end 40 base to 50 base of the interferon β gene with FITC fluorescence. The same operation was performed to produce a transgenic pearl oyster (second generation) into which the human interferon β gene was introduced.

  The effect of human interferon β gene introduction was examined in the same manner as in Reference Example 1. The results are shown in Table 2 below.

  As in Reference Example 1, the effect of human interferon β gene introduction was clearly observed.

Example 1 Production of pearl oyster with green fluorescent protein (GFP) gene (1)
A full-length GFP gene (Science 1994, 263: 802-805; GenBank No. U53602, commercially available from Wako Pure Chemical Industries) was incorporated into an adenovirus vector in the same manner as in Reference Example 1 (cells used for propagation were 293 cells) . The obtained GFP-containing recombinant vector was microinjected into the oyster oyster ovary in the same manner as in Reference Example 1 with TE buffer to a concentration of 100 mg / ml. Thereafter, a transgenic pearl oyster (second generation) was produced in the same manner as in Reference Example 1 (however, the probe used in the Southern blot and the Northern blot was a GFP gene).

  Whether fluorescence emission was observed in each tissue of the obtained transgenic pearl oyster was examined with a fluorescence microscope. The results are shown in Table 3 below. In the table, “+” indicates that fluorescence was observed, and the greater the number of “+”, the stronger the fluorescence.

Example 2 Production of pearl oyster with green fluorescent protein (GFP) gene (2)
The pearl shell prism protein is an important protein constituting pearls. In this example, an attempt is made to fuse a green fluorescent protein protein gene to a prism protein gene to cause the pearl to emit autofluorescence.

  The pearl oyster prism protein gene (Nature 1997, 387 (6633): 563-564; GenBank No. D860 / 3) was cloned, including the 5 kb upstream of the codon at the start of reading, and the GFP gene was fused to this promoter, and the adenovirus vector Introduced. The fusion gene of prism protein gene (including promoter) and GFP gene was prepared by the method described in M. Chalfie et al., Science 1994, 263: 802-805. That is, a linker (poly T) of 9 nt, 10 nt, or 11 nt is linked to the 10 nt from the start codon of the prism protein gene including the 5 kb upstream of the reading start codon, and 9, 10 and 10 nt are respectively attached to the 5 ′ ends of the commercially available GFP genes. Alternatively, a fusion gene was obtained by hybridizing DNA to which an 11 nt poly A linker was bound. The obtained fusion gene was inserted into the same adenovirus vector as used in Reference Example 1 using restriction enzymes NHeI and EcoRI. Subsequently, in the same manner as in Reference Example 1, a transgenic pearl oyster into which a fusion gene of a prism protein gene and a GFP gene was introduced was obtained. When the mantle tissue of the obtained transgenic pearl oyster was observed with a fluorescence microscope, fluorescence was observed.

  The transgenic pearl oyster obtained in this example has a GFP gene linked downstream of the promoter and structural gene of the prism protein, and since this is expressed and fluorescence is observed, this pearl oyster can be made to make a pearl. For example, since GFP is fused to the prism protein constituting the pearl, it is considered that a pearl that emits fluorescence is formed.

Example 3 Production of pearl oyster with green fluorescent protein (GFP) gene (3)
Mantle protein is an important protein that constitutes a pearl like prism protein. Using the pearl oyster mantle protein gene (Nature 1997, 387 (6633): 563-564; GenBank No. 86074) instead of the prism protein gene of Example 2, the same operation as in Example 2 was performed, and the mantle protein gene A transgenic pearl oyster in which a fusion gene of GFP and GFP was introduced was produced. The fusion of the pearl oyster mantle protein gene and the GFP gene and the integration of the fusion gene and the adenovirus vector were performed as follows. As in Example 2, 9 or 10 or 11 nt of poly-T was ligated to 15b from the start codon of the DNA fragment containing about 5 kb upstream of the start codon of the mantle protein, and the same GFP gene as used in Example 2 was linked to 9 A fusion gene was prepared by hybridizing 10, 11 nt of poly A. Using the obtained fusion gene, the same operation as in Example 2 was performed to obtain a transgenic pearl oyster.

  When the mantle tissue of the obtained transgenic pearl oyster was observed with a fluorescence microscope, fluorescence was observed.

  The transgenic pearl oyster obtained in this example has a GFP gene linked downstream of a mantle protein promoter and a structural gene. Since this gene is expressed and fluoresced, a pearl is formed on this pearl oyster. In this case, since GFP is fused to the mantle protein constituting the pearl, it is considered that a pearl that emits fluorescence is formed.

Example 4 Introduction of GFP gene or LacZ gene by promoter trap method Basically, the method of IA Hope, Development 113, 339-408 (1991) was used. First, a GFP gene (10 to 50 mg DNA / piece) was injected into the testis and ovary by the method described in Example 1, and then fertilized in a test tube. This was cultured in seawater at 25 ° C., and juveniles were obtained in 3 weeks. About 5 to 15% of larvae were colored with GFP. These were cultured for 12 months in a culture tank to obtain adult shellfish. Adult shellfish were dissected, and particularly well-developed organs were isolated and DNA was extracted. The promoter gene sequence that expresses the GFP gene in the extracted DNA was analyzed. Promoter genes for protein synthases and enzymes in the shell muscle were trapped.

  Swa I restriction site (site) of GFP or LacZ gene inserted downstream of the sequence containing the newly isolated promoter region and β-actin promoter excised from the expression vector pAxCAwt (Takara adenovirus expression vector kit) with restriction enzymes The resulting gene was introduced between the megalovirus enhancer sequence and the rabbit β-globin poly A signal. Using the obtained recombinant vector, the vector solution having a concentration of about 100 to 200 mg / ml was prepared in a petri dish, and unfertilized eggs were immersed therein. The vector solution was injected into the egg so that the soaked unfertilized egg was scratched sharply for a moment using a micropipette for microinjection. Thereafter, a transgenic pearl oyster was obtained in the same manner as in Reference Example 1. The expression level of each gene was detected spectroscopically.

As a result of observation of the obtained transgenic pearl oyster by autofluorescence emission (in the case of the GFP gene) and staining with the chromogenic substrate XG (in the case of the LacZ gene), fluorescence emission or staining was observed in various tissues of the pearl oyster.

Claims (8)

  A transgenic mollusk into which a desired foreign gene (excluding a gene conferring viral resistance) is introduced and expresses the foreign gene.   The transgenic mollusk according to claim 1, which is a shellfish.   The transgenic mollusk according to claim 2, which is a pearl oyster.   The transgenic mollusk according to any one of claims 1 to 3, wherein the foreign gene is a gene involved in coloration, and any tissue is colored.   The transgenic mollusk according to claim 4, wherein the gene involved in coloration is a green fluorescent protein protein, and any tissue emits fluorescence.   A recombinant vector in which a desired foreign gene to be introduced or a nucleic acid containing the foreign gene is incorporated into a vector is microinjected into a male and / or female gonad of a mollusk, and these male and female are mated. The method for producing a transgenic mollusk according to any one of claims 1 to 5, comprising producing a single generation and selecting an individual expressing the desired gene.   7. The method of claim 6, further comprising mating the first generation male and female expressing the desired gene to create a second generation and selecting an individual expressing the desired gene. Method. A recombinant vector incorporating a nucleic acid functionally linked to the desired gene downstream of a promoter that exhibits promoter activity in a mollusc to be transformed is applied to an unfertilized egg, a fertilized egg, or an embryo of the mollusk. The trans according to any one of claims 1 to 7, comprising introducing, growing the unfertilized egg, fertilized egg or embryo into an individual, and selecting an individual expressing the desired gene. A method for producing a transgenic mollusk.