JP2005168460A - Method for producing transformed silk worm and method for inoculating recombinant virus to egg of silk worm - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method equipped with a high gene-introducing efficiency and easiness for injection jointly, for transforming silk worms. <P>SOLUTION: This method for producing the transformed silk worms is provided by applying a recombinant AcNPV having a transferase derived from a transpozon on the tip end of a tungsten needle and thrusting the egg of the silk worm with the needle to introduce the recombinant virus into the egg of the silk worm in order to introduce the recombinant AcNPV having an objective gene which is to be introduced into the chromosome of the silk worm together with the objective gene to be introduced into the chromosome of the silk worm in a good efficiency. Thereby, it becomes possible to introduce the objective gene into the chromosome of the silk worm in the good efficiency, and the production of the transformed silk worms becomes easier. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel method for producing a transformed silkworm using recombinant AcNPV for producing a transformed silkworm. More specifically, a recombinant AcNPV having a gene intended to be introduced into the silkworm chromosome and a recombinant AcNPV having a transposon-derived transferase in order to efficiently introduce the target gene to be introduced into the silkworm chromosome. The present invention relates to a method for producing a transformed silkworm by introducing it into an egg. The present invention introduces recombinant AcNPV, which can be used for functional analysis of silkworm genes, improvement of silkworm varieties, and mass production of useful proteins by silkworms, into silkworms by a simpler method than conventional techniques. It is possible to make a conversion.

  Silkworms are used as laboratory animals in basic research fields such as physiology / biochemistry and genetics in addition to being livestock insects for producing silk thread. Furthermore, since it has an excellent protein synthesis ability, it is highly expected as a host animal for producing useful proteins. Therefore, the development of a technique for producing a transformed silkworm by introducing a foreign gene into the silkworm has been conducted at home and abroad.

  Mori et al. Introduced a gene without killing silkworm larvae by infecting silkworm larvae with Autographa californica nuclear polyhedrosis virus (AcNPV). A method capable of transmitting was developed (Bio / Technology 13, 1005-1007, 1995: Non-Patent Document 1). By developing this method, Yamao et al. Constructed an AcNPV incorporating a part of the fibroin L chain gene sequence, infecting it with silkworm larvae, and inserting the foreign gene into the fibroin L chain gene of the silkworm genome by gene targeting. A silkworm was successfully produced (Genes Dev. 13, 511-516, 1999: Non-Patent Document 2).

  On the other hand, Tamura et al. (Nat. Biotechnol. 18, 81-84, 2000) inserted a foreign gene by microinjecting a silkworm egg with a plasmid vector incorporating a piggyBac transposon derived from the lepidopteran insect Trichoplusia ni. Have successfully produced transformed silkworms.

  On the other hand, Mori et al. Focused on the advantages of both AcNPV and piggyBac, and proposed a method to introduce foreign genes into the silkworm genome using the transfer activity of piggyBac using AcNPV and piggyBac together. However, the production efficiency of transformed silkworms by this method is not always satisfactory, and it was necessary to inoculate a very large number of silkworms with the virus. Further, in the next generation, it was necessary to collect genomes from silkworm tissues of a large number of progeny and to select silkworms having a target gene for each individual using a technique such as PCR (Japanese Patent Application No. 2002-306167: Patent) Reference 1).

Japanese Patent Application No. 2002-306167 Bio / Technology 13, 1005-1007, 1995 Nat. Biotechnol. 18, 81-84, 2000

  As described above, as a method for producing a transformed silkworm capable of transmitting a foreign gene throughout the generation, a method of introducing a foreign gene using AcNPV as a vector, and a microinjection of a plasmid vector incorporating piggyBac into a silkworm egg Two methods for introducing foreign genes have been developed.

  The method using AcNPV has the advantage that the vector injection method is easy because the virus is inoculated into silkworm larvae. However, the efficiency of gene integration into the genome is low, and labor and time are required to obtain transformed silkworms.

  The method using a plasmid vector incorporating piggyBac has the advantage of high gene transfer efficiency because it uses the gene transfer action of piggyBac, but it is not technically easy to inject a small amount of plasmid into a silkworm egg. Has drawbacks. Therefore, it has not been established as a daily method.

  Therefore, the invention of the present application has been made in view of the above circumstances, and has solved the problems of the prior art, and by using AcNPV, a novel silkworm transformation having high gene transfer efficiency and easy injection. The challenge is to provide the law.

  In order to solve the above problems, the present invention provides the following inventions (1) to (4). (1) Recombinant AcNPV in which a DNA fragment consisting of transposase, a transposon piggyBac transferase, is incorporated into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV), and a reverse terminal repeat sequence of transposon piggyBac and sandwiched between them. A recombinant AcNPV in which a foreign gene DNA fragment is incorporated into the AcNPV genome is applied to the tip of a tungsten needle, and a silkworm egg is poke with the tungsten needle coated with the above two types of viruses. A method for producing a transformed silkworm.

(2) A transformed silkworm produced by the method of (1) above and having the ability to produce a gene product of the foreign gene in order to have the foreign gene in its genome.

(3) Recombinant AcNPV in which a DNA fragment consisting of transposase piggyBac transposase, transposon piggyBac, is incorporated into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV), and the inverted terminal repeat sequence of transposon piggyBac and sandwiched between them. The recombinant AcNPV in which the foreign gene DNA fragment is incorporated into the AcNPV genome is applied to the tip of a tungsten needle, and the silkworm egg is poke with the tungsten needle coated with the above two types of viruses. A method of inoculating eggs with a recombinant virus.

(4) A transformed silkworm obtained by hatching an egg inoculated with the method of (3) above and having the ability to produce a gene product of the foreign gene in order to have the foreign gene in its genome.

  According to the present invention, a recombinant AcNPV having a gene intended to be introduced into the silkworm chromosome and a recombinant AcNPV having a transposon-derived transferase to efficiently introduce the target gene to be introduced into the silkworm chromosome are converted into tungsten. A method for producing a transformed silkworm was given by applying it to the tip of a needle and introducing it into a silkworm egg by poking with the needle. According to the present invention, the target gene can be efficiently inserted into the silkworm chromosome, and the production of transformed silkworm becomes easier.

  The present invention provides a new method for producing a transformed silkworm using a recombinant baculovirus for producing a transformed silkworm. In the present invention, two recombinant viruses of Autographa californica nuclear polyhedrosis virus, which is an attenuated virus for silkworms, are used. One of the two types of recombinant viruses is a recombinant virus having a gene intended to be introduced into the silkworm chromosome, and other viruses are transposons in order to efficiently introduce the target gene into the silkworm chromosome. It is a recombinant virus with a derived transferase.

  These two kinds of recombinant viruses are applied to the needle tip, and the egg is infected with the virus by piercing the silkworm egg. By the propagation of the virus, a transferase is expressed, the target gene is efficiently inserted into the silkworm chromosome, and a transformed silkworm is obtained.

  The transposon piggyBac is a transposon belonging to Class II isolated from TN-368, which is a cultured cell derived from the lepidopteran insect Trichoplusia ni as described above. It consists of a transposase gene in the center and 13 bp inverted repeats at both ends, and is about 2.5 kb in length. A transposase protein is synthesized from the transposase gene, and by the action of this enzyme, a region sandwiched between reverse sequences (piggyBac itself) is transferred to the target sequence TTAA. In the method of the present invention, a foreign gene can be introduced into a silkworm using AcNPV, and the foreign gene can be stably and highly efficiently recombined into a silkworm genomic DNA using the transfer activity of piggyBac.

  A promoter / enhancer can be used to promote the expression of the transposase gene and / or foreign gene. As such promoter / enhancer, the endogenous promoter / enhancer of each gene may be used as it is, or the endogenous promoter is replaced with an exogenous promoter, or an exogenous enhancer is incorporated upstream of the endogenous promoter. But it ’s okay. Examples of the promoter to be replaced include a silkworm actin promoter and a Drosophila HSP70 promoter.

  In silkworms inoculated with the aforementioned two types of recombinant viruses, the foreign gene DNA sandwiched between a pair of inverted repeats is transferred to the target sequence TTAA of the silkworm genome by the action of the transposase protein. This prevents instability of the foreign gene in the genome by transferring the transposase gene together with the foreign gene into the silkworm genome, and the foreign protein is stably expressed in the silkworm genome.

  Tamura et al. Succeeded in producing a transformed silkworm having a foreign gene inserted by microinjecting a plasmid vector incorporating a piggyBac transposon into a silkworm egg. In this method, a hole is made in the eggshell of a silkworm egg using a tungsten needle under a stereomicroscope, and then a plasmid vector is injected with a glass capillary. This method has the advantage of high gene transfer efficiency because it uses the gene transfer action of piggyBac as described above, but on the other hand, it is not technically easy to microinject a plasmid into a silkworm egg. In addition, special and expensive instruments such as microinjectors and micromanipulators are required for microinjection.

  The present invention provides a method for introducing a recombinant AcNPV into a silkworm egg using a tungsten needle under a stereomicroscope without using a special instrument, and stably and efficiently incorporating a foreign gene into the silkworm genomic DNA. is there.

  There are no particular restrictions on the foreign gene DNA to be introduced, and various useful foreign gene DNAs can be used. Therefore, in the present invention, a genetic structure for performing silkworm breeding, silkworm gene knockdown, silkworm gene knockdown, or silkworm gene knockout by gene targeting method is used. can do.

Using the above-described silkworm AcNPV for gene recombination, a transformed silkworm is prepared as follows.
(1) Recombinant AcNPV into which a foreign gene has been inserted is applied to the tip of a tungsten needle and introduced into a silkworm non-dormant egg by poking with this needle. Individuals expressing a foreign gene are selected from hatched larvae.
(2) Crossing individuals expressing a foreign gene and breeding the next generation. In the next generation, a foreign gene is transferred to silkworm genomic DNA, and silkworm larvae expressing the foreign gene are selected.

  The screening of (1) and (2) can be performed by a method in which genomic DNA is extracted from larval body fluid and a foreign gene is detected by PCR using this as a template. In addition, a marker gene can be simultaneously incorporated into a virus together with a foreign gene, and in this case, screening can be performed using the phenotype of the marker gene as an index. For example, when a fluorescent protein gene containing GFP is incorporated as a marker gene, it can be easily screened by a method in which excitation light is applied to eggs and larvae and the fluorescence is observed.

  Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples. However, the invention of this application is not limited to the following examples.

Example 1: Production of recombinant AcNPV
Using the plasmid p3E1.2 containing the piggyBac gene as a template, the piggyBac transferase gene was primer 1 (5'-AAAAAAGCTTATGGGATGTTCTTTAGACGA-3 ': SEQ ID NO: 1) and primer 2 (5'-AAAAAAGCTTATACTAATAATAAATTCAAC-3': SEQ ID NO: 2) Amplification was performed by PCR using the oligonucleotides. The amplified DNA fragment was digested with restriction enzyme HindIII and then inserted into the HindIII site of baculovirus transfer vector pBlueBacHisC (Invitrogen).

  Next, the Drosophila heat shock protein promoter was inserted into the HindIII and BamHI sites of this transfer vector to construct a recombinant transfer vector pHSPpigTrans. The obtained recombinant transfer vector was transfected into sweet potato-derived cultured cells (Sf21) using lipofectin (Gibco) together with linear baculovirus DNA (Pharmingen).

  Next, the recombinant virus produced in the culture supernatant was purified by the plaque method, and the virus was further infected with Sf21 cells to prepare a high-titer virus solution. The produced virus is a recombinant virus (AcpigTrans) that expresses piggyBac transferase under the control of the Drosophila heat shock protein promoter.

  Next, recombinant AcNPV was prepared in which a fusion DNA fragment consisting of a pair of inverted repeats of piggyBac and a foreign gene DNA sandwiched between them was incorporated into the genomic DNA of AcNPV. First, the above-mentioned p3E1.2 was digested with SacI and PstI to blunt the ends, and then the synthesized multicloning site (XbaI-BamHI-SmaI-KpnI-EcoRI-SacI-XhoI) was inserted.

  From this vector, a region between ITR and SalI and EcoRV was excised and inserted into the EcoRV and XhoI sites of pBacPAK9 (Clontech) to construct pAcpigMCS. A recombinant transfer vector pAcpigEGFP was constructed by inserting a fragment consisting of the chicoactin promoter, EGFP gene, and SV40 polyA addition signal into the SmaI site of pAcpigMCS. Then, recombinant virus AcpigEGFP was prepared in the same manner as AcpigTrans.

  FIG. 1 is a diagram showing two types of recombinant viral vectors prepared for introducing the EGFP gene into the silkworm chromosome. One is a virus AcpigEGFP in which an EGFP gene is inserted between inverted terminal repeats (ITRs) present at both ends of piggyBac. This recombinant virus expresses the EGFP gene under the control of the silkworm actin promoter as a selection marker for transformed silkworms. The other recombinant virus, AcpigTrans, expresses the piggyBac transferase gene under the control of the Drosophila heat shock protein promoter. When these two types of recombinant viruses are inoculated, the EGFP gene is considered to be introduced at random by recognizing the sequence TTAA on the chromosome.

Example 2: Introduction of recombinant virus into silkworm eggs using a tungsten needle AcpigTrans and AcpigEGFP prepared in Example 1 were used as non-dormant eggs for non-dormant eggs (300-400 grains) produced by one silkworm cocoon. The injection was performed as follows.

  The tip of the tungsten needle was sanded with sand, a mixture of AcpigTrans and AcpigEGFP was applied to the tip, and the virus was introduced into the silkworm egg by poking a silkworm non-dormant egg under the stereomicroscope. FIG. 2 shows how a recombinant virus is introduced into a silkworm egg using a tungsten needle. AcpigTrans and AcpigEGFP were injected into non-dormant eggs in silkworm silkworm non-dormant eggs (300-400 grains) as follows. The tip of the tungsten needle was sanded with sand, a mixture of AcpigTrans and AcpigEGFP was applied to the tip, and the virus was introduced into the silkworm egg by poking a silkworm non-dormant egg under the stereomicroscope. The silkworm non-dormant eggs that were inoculated with the virus were those within 2 hours after egg laying. Non-dormant eggs that had been inoculated with the virus were placed in a petri dish with absorbent cotton moistened with water and allowed to blue at 25 ° C.

  Thereafter, screening for producing transformed silkworms was performed (FIG. 3). Screening of G0 larvae hatched from eggs inoculated with the recombinant virus was performed by observing the green fluorescence of EGFP (FIG. 4). When the first-instar larvae immediately after hatching were observed with a fluorescence microscope, green fluorescence was observed from the whole body. When the first instar larvae immediately after hatching were observed with a fluorescence microscope, green fluorescence was observed from the whole body. The appearance rate of silkworms emitting green fluorescence was 2.2% (Table 1). In Table 1, the appearance rate of transformed silkworms when the virus is injected into silkworm eggs is shown.

  G0 larvae in which green fluorescence was observed were raised and G1 generation was collected. Genomic DNA was extracted from this G0 蛾. An attempt was made to amplify the EGFP gene by performing PCR using the obtained genomic DNA as a template. As the primers used for this PCR, oligonucleotides of SEQ ID NOs: 3 and 4 were used for amplifying the sequence of the EGFP gene. A 600 bp amplification product is obtained with this primer. After completion of the reaction, electrophoresis was performed with 1.5% agarose. As a result, amplification products of 600 bp were obtained in two individuals (Table 1).

Example 3: Southern blot analysis of genome extracted from individuals of G1 generation showing green fluorescence The G1 generation in the 2nd section was collected and observed with a fluorescence microscope. In the 1st ward, 30 out of 284 G1 larvae and in the 1st ward, 22 out of 141 G1 larvae emitted green fluorescence (Table 1, FIG. 5). Screening in the G1 generation is shown in FIG. Screening of G1 larvae was performed by a method of observing green fluorescence of EGFP. In FIG. 5, (A) is a bright field and (B) is a photograph taken under fluorescence.

  G1 larvae in which green fluorescence was observed were raised and G2 generation eggs were collected. After seeding G2 generation eggs, genomic DNA was extracted from G1 generation silkworms. Genomic DNA was digested with restriction enzymes BamHI and EcoRI (lanes 1 and 2), EcoRI and PstI (lanes 3 and 4), and then Southern hybridization was performed using the fluorescently labeled EGFP gene as a probe. In FIG. 6A, the size (kbp) of the DNA marker is shown on the right side of the figure. Lanes 1 and 3, and lanes 2 and 4 are genomic DNAs extracted from different G1 cells. Signals corresponding to 1265 bp were detected in lanes 1 and 2, and 5459 bp and 4794 bp were detected in lanes 3 and 4, respectively. The 1265 bp signal detected in lanes 1 and 2 is consistent with the fragment size expected from the AcpigEGFP construct.

  FIG. 6B is a schematic diagram of the AcpigEGFP construct. The restriction enzyme used for digestion of genomic DNA and the expected size (kbp) of the fragment are shown. The black line shows the fragment used as a probe. It is also known that there is no PstI site in the construct. From these results, it became clear that the EGFP gene was integrated into silkworm genomic DNA in G1 larvae that emitted green fluorescence.

  As described above in detail, the present invention provides a novel silkworm transformation method that has both high gene transfer efficiency and ease of injection, and is completely different from conventional methods. According to the present invention, it becomes possible to easily improve the variety of silkworms by transformation and to produce transformed silkworms that produce a large amount of useful proteins, thereby enabling effective utilization of silkworms in a wide range of fields.

  Since silkworms have a very high protein production capacity, it has already been carried out to produce useful proteins such as interferon in silkworms using silkworm nuclear polyhedrosis virus as a vector. It is considered that the present invention can be used in industries such as drug development by producing useful proteins using such silkworms. Furthermore, the properties of silk produced by silkworms according to the present invention can be modified at the gene level, and are considered to be used in the textile industry.

FIG. 1 is a diagram of a viral vector for introducing a foreign gene into a silkworm genome using AcNPV and piggyBac in combination. FIG. 2 is a photograph showing the state of introduction of the recombinant virus into silkworm eggs using a tungsten needle. FIG. 3 is a diagram showing an outline of a screening method for producing transformed silkworms. FIG. 4 is a photograph showing the state of screening in the G0 generation. FIG. 5 is a photograph showing the screening in the G1 generation. (A) is a bright field, and (B) was taken under fluorescence. FIG. 6 is a photograph of a Southern blot analysis of a genome taken from an individual of G1 generation showing green fluorescence, and a schematic diagram of an AcpigEGFP construct.

Claims (4)

  Autographa californica nuclear polyhedrosis virus (AcNPV) genome, recombinant AcNPV in which a transposon piggyBac transferase transposase DNA fragment is incorporated, and transposon piggyBac inverted terminal repeats and foreign elements sandwiched between them Recombinant AcNPV, in which the gene DNA fragment is integrated into the genome of AcNPV, is applied to the tip of a tungsten needle, and the silkworm egg is poke with the tungsten needle coated with the above two types of viruses. Manufacturing method.   A transformed silkworm produced by the method according to claim 1 and having the ability to produce a gene product of the foreign gene in order to have the foreign gene in its genome.   Autographa californica nuclear polyhedrosis virus (AcNPV) genome, a recombinant AcNPV in which a transposon piggyBac transposase transposase DNA fragment is integrated, and a transposon piggyBac inverted terminal repeat sequence and a foreign site sandwiched between them Recombinant silkworm eggs, which are made by applying recombinant AcNPV, in which the gene DNA fragment is integrated into the genome of AcNPV, to the tip of a tungsten needle and then poking the silkworm egg with the tungsten needle coated with the above two types of viruses. How to inoculate the virus.   A transformed silkworm obtained by hatching an egg inoculated with the method according to claim 3 and having the ability to produce a gene product of the foreign gene in order to retain the foreign gene in its genome.

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