JP2004180679A - Method for reconstruction of genomic dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for effectively reconstructing a genomic DNA composing foreign genes segmented and cloned in the genome of a microorganism. <P>SOLUTION: The target DNA of ≥50 kb is introduced into the genome of microorganism at once by using a plasmid having target DNA of ≥50 kb, and transducing a microorganism with a genome into which two homologous regions interleaving target DNA in the plasmid were introduced previously. The genomic DNA composing the foreign genes segmented and cloned in the plasmid is reconstructed in the genome of microorganism, and reconstructed genome genes are appropriately used for preparation, or the like, of genetically modified non-human animals or plants. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a method for efficiently reconstituting genomic DNA constituting a foreign gene which has been divided and cloned into a microorganism genome, and the like.

  Many eukaryotic genes such as animals and plants have gene lengths ranging from several tens to several hundreds kb in the genome, and in some cases, a sequence acting on cis exists at a distance of 100 kb from the gene. In particular, in order to observe genes that are expressed in a development stage-specific or tissue-specific manner, it is desirable that DNA in a region up to about several hundred kb can be handled at a time. After cloning and modifying a giant DNA of several tens of kb or more from the genome of a eukaryote such as an animal or a plant, the modified giant DNA is introduced into the original animal or plant individuals, tissues, cultured cells, and the like. As a result, in a series of operation techniques for producing genetically modified animals and plants, (1) large DNA can be cloned with a small number of operations, (2) DNA modification can be simplified, (3) cloned DNA is stably retained And (4) easy recovery of the cloned target DNA are important issues.

Regarding the above-mentioned problems (1) to (3), the present inventors have proposed a method of inserting a giant DNA into the genome of Bacillus subtilis and modifying the inserted DNA (for example, Non-Patent Document 1). And Patent Document 1). In addition, the present inventors have proposed a simple and high-purity method for recovering a target DNA as a plasmid with respect to the above problem (4) (for example, see Patent Document 2).
However, regarding the problem of the above (1), although a method of fragmenting a large DNA in advance and inserting it into the genome of Bacillus subtilis has been provided, for example, it has been cloned as a plasmid having excellent operability and stability. There is no report on a method for transferring a large DNA of 50 kb or more into the B. subtilis genome by a single transformation. In Bacillus subtilis, it is known that a circular DNA such as a plasmid is also cleaved at an arbitrary location and incorporated into a linear form. This is because it is considered that the probability of occurrence of desired homologous recombination is extremely low when the DNA to be introduced is a huge one of 50 kb or more.

In recent years, BAC (for example, see Non-Patent Document 2) and YAC (for example, see Non-Patent Document 3) have been developed as vectors capable of cloning huge DNA. For example, since BAC can hold DNA of 100 kb or more, and YAC can hold DNA of 1000 kb or more, many genomic libraries using such vectors have been constructed.
Therefore, if a method of transferring giant DNA from a plasmid having a giant DNA of 50 kb or more to a microorganism genome such as Bacillus subtilis is established, a giant DNA derived from the genome can be easily prepared using such a clone in a genomic library. Was expected to be available. Further, by establishing such a method, finally, eukaryotes such as animals or plants as described above can handle genes ranging from several tens of kb to several hundred kb in the genome at once. Was expected to be.
JP 2000-93 A JP 2002-17350 A Mitaniyasu Itaya, "Bacillus subtilis genome vector learned naturally", Bioscience and Industry, vol.57, No.8, 540-543 (1999) Shizuya, H., et al., Proc. Natl. Acad. Sci. USA, 87, 8242-8247 (1990). Burke, DT, et al., Science, 236, 806-811 (1987)

  The present invention has been made to provide a method for efficiently reconstituting genomic DNA constituting a foreign gene which has been divided and cloned into a microorganism genome, and the like.

  The present inventors have conducted intensive studies to achieve the above object, and as a result, have found that a partial fragment of genomic DNA of 50 kb or more inserted in a BAC vector can be introduced into the Bacillus subtilis genome by a single transformation. I found it. Further, they have found that genomic DNA constituting a foreign gene can be reconstituted in the Bacillus subtilis genome by linking the partial fragments by homologous recombination. The present invention has been accomplished based on these findings.

That is, according to the present invention, the following method (1) is provided.
(1) A method for reconstituting genomic DNA constituting a foreign gene into a microorganism genome, the method comprising the following steps:
(A) A plasmid group in which partial sequences having different total lengths of genomic DNA constituting a foreign gene are inserted as the target DNA into the same vector, and each target DNA has an overlapping sequence with any other target DNA. Process,
(B) a step of transforming a microorganism cell having a genome in which two homologous sequences sandwiching the target DNA in the plasmid have been previously inserted with any plasmid containing the target DNA prepared in the above step (a);
(C) selecting another plasmid containing the target DNA having an overlapping sequence with the target DNA in the plasmid prepared in step (a) and used in step (b),
(D) transforming the microbial cells transformed in step (b) with the plasmid selected in step (c);
(E) From the microbial cells transformed in the above step (d), the target DNAs in the two plasmids used in the above steps (b) and (d) are ligated by homologous recombination at the overlapping sequence and the homologous sequence, respectively. Selecting a transformant having the isolated DNA,
(F) repeating the above steps (c) to (e) and introducing all the target DNAs prepared in the above step (a) into the microbial cells, whereby a genome constituting a foreign gene in the microbial genome; Reconstituting the DNA.

According to another aspect of the present invention, there is provided the following method (2).
(2) A method for reconstituting genomic DNA constituting a foreign gene into a microorganism genome, comprising the following steps:
(A) Step of preparing a plasmid group in which partial sequences having different total lengths of genomic DNA constituting a foreign gene are inserted as target DNAs into a vector, and each target DNA has a sequence overlapping with any other target DNA. ,
(B) Two homologous sequences sandwiching the target DNA in each of the plasmids prepared in the above step (a) are previously inserted into microbial cells having the same genome at the same position in the microbial genome, and the microbial cells are each Transforming with the plasmid of
(C) a step of extracting genomic DNA from a microbial cell having any of the target DNAs in the genome transformed in the step (b);
(D) Transforming microbial cells having another target DNA having a sequence overlapping with the target DNA in the genomic DNA extracted in the step (c) in the genome with the genomic DNA obtained in the step (c). Process,
(E) From the microbial cells transformed in the above step (d), the target DNAs in the two microbial genomes used in the above steps (c) and (d) are, respectively, an overlapping sequence and a common sequence in the microbial genome. Selecting a transformant having DNA linked by homologous recombination,
(F) genomic DNA constituting a foreign gene in the genome of a microorganism by repeating the above steps (c) to (e) and introducing all the target DNAs prepared in the above step (a) into the microorganism. The step of reconstructing.

According to these preferred aspects of the present invention,
(3) the microorganism genome has two or more cI repressor genes between two homologous sequences, a spacer inserted between each cI repressor gene, and The method according to the above (1) or (2), wherein a marker DNA placed under the control of a Pr promoter is inserted at another position.
(4) the method according to any one of (1) to (3), wherein the selection of the transformant is performed using a marker DNA;
(5) The method according to any one of (1) to (4) above, wherein the microorganism used as a host is a bacterium belonging to the genus Bacillus or a highly thermophilic bacterium.

According to another aspect of the present invention, there is provided (6) a microorganism produced by the method according to any one of (1) to (5).
Further, according to another aspect of the present invention,
(7) a plasmid comprising the full length of genomic DNA constituting the foreign gene and having a length of 100 kb or more;
(8) A method for producing a genetically modified non-human animal or plant, comprising using the plasmid according to (7),
(9) A method for transferring a target DNA from a plasmid having a target DNA of 50 kb or more to a microorganism genome, wherein the target DNA is inserted into a microorganism having a genome in which two homologous sequences sandwiching the target DNA in the plasmid are inserted in advance. A method of transforming the obtained plasmid,
Is provided.

  According to a preferred embodiment of the above (9), there is provided the method according to the above (9), wherein the (10) plasmid is a BAC vector.

  According to the present invention, a genomic DNA constituting a foreign gene can be easily reconstituted in a microorganism genome using a genomic library using a vector such as BAC, and the full length can be obtained in the form of a plasmid or the like. can do.

Hereinafter, the present invention will be described in more detail.
1. Method for reconstituting genomic DNA constituting foreign genes in microorganism genome (method 1)
A first method of reconstituting genomic DNA constituting a foreign gene into the microorganism genome of the present invention is characterized by comprising the following steps.
(A) A plasmid group in which partial sequences having different total lengths of genomic DNA constituting a foreign gene are inserted as the target DNA into the same vector, and each target DNA has a sequence overlapping with any other target DNA to each other is prepared. Process,
(B) a step of transforming a microorganism cell having a genome in which two homologous sequences sandwiching the target DNA in the plasmid have been previously inserted with any plasmid containing the target DNA prepared in the above step (a);
(C) a step of selecting another plasmid containing the target DNA having an overlapping sequence with the target DNA in the plasmid prepared in step (a) and used in step (b),
(D) transforming the microbial cells transformed in step (b) with the plasmid selected in step (c);
(E) From the microbial cells transformed in the above step (d), the target DNAs in the two plasmids used in the above steps (b) and (d) are ligated by homologous recombination at the overlapping sequence and the homologous sequence, respectively. Selecting a transformant having the isolated DNA,
(F) repeating the above steps (c) to (e) and introducing all the target DNAs prepared in the above step (a) into the microbial cells, whereby a genome constituting a foreign gene in the microbial genome; The step of reconstituting DNA.

FIG. 3 schematically shows the outline of the method 1. Hereinafter, details of each step will be described.
Step (a) of Method 1
The genomic DNA constituting the foreign gene used in the present invention (hereinafter, may be referred to as “genomic gene”) includes all introns and exons present at a certain locus other than the microorganism into which the foreign gene is introduced. Although it means DNA, it does not necessarily need to contain all of the original genomic gene as long as it has the same function when reconstituted and used. In addition, those obtained by genetic engineering modification such as insertion, substitution, deletion and mutation after reconstitution are also included in the full length of the genomic gene of the present invention. The genomic gene may be derived from any species other than the microorganism into which the genomic gene is introduced, or may constitute a gene having any function. The size and the like are not particularly limited, but those having a size of 100 kb or more are preferably used. Specific examples of the biological species include mammals and plants, and examples of the mammals include mammals such as humans and rodents such as mice and rats. Further, for example, genomic DNA constituting a gene related to cancer, immunity, metabolic abnormality, growth, cold resistance, drought resistance, secondary metabolism and the like is preferably used.

  A plasmid group is prepared in which partial sequences having different total lengths of such genomic genes are inserted into the same vector as the target DNA, and each target DNA has an overlapping sequence with any other target DNA. The target DNA is, of any of the partial sequences of the genomic gene, preferably one having a length of 50 kb or more, more preferably 100 kb or more, which have overlapping sequences with each other, and It covers the entire length. In addition, the overlapping sequence only needs to be long enough to cause homologous recombination, and specifically, it is preferably 1 kb or more and 10 kb or less. These target DNAs do not need to have all of their base sequences determined, but are analyzed to the extent that it is possible to recognize which target DNA has an overlapping sequence with which other target DNA and how long. Need to be.

  The vector into which the target DNA is inserted may be any vector as long as it can stably hold the DNA, but a vector having the ability to stably hold a large target DNA of 50 kb or more is preferably used. Specifically, for example, vectors such as BAC (Bacterial artificial chromosome) and YAC (Yeast artificial chromosome) can be mentioned. For YAC, physical damage to DNA during cloning operation, cloning efficiency, stability, etc. In particular, since problems are likely to occur on the surface (Larionov, V., et al., Nucleic Acids Research, 22, 20, 4154 (1994); Neil, DL, et al., Nucleic Acids Research, 18, 6,1421 (1990)), BAC vectors are preferably used. More specifically, for example, from the genomic library such as a BAC library into which a partial fragment of genomic DNA derived from any species is inserted as the plasmid group, the genomic DNA constituting the target gene differs in the total length. A clone group having a partial sequence and each partial sequence having an overlapping sequence with any other partial sequence is selected, and a clone amplified and purified can be used. As such a genomic library, commercially available ones can be preferably used, and genomic DNA extracted from a target biological sample (tissue or cell, etc.) is digested with an appropriate restriction enzyme to obtain an appropriate length. Or by physically dividing it into an appropriate length by using an ultrasonic crusher or the like, adding an adapter having an appropriate restriction enzyme recognition sequence thereto, and recognizing these fragments with the same restriction enzyme. It can also be prepared using a method of constructing a genomic library by ligating a sequence, for example, into a BAC vector, and transforming this into a host.

  As an analysis method for selecting the above plasmid group, a clone containing a target gene is selected from a genomic library using a probe or a primer having a partial nucleotide sequence of the target gene, and then contained in the clone. It can be performed by analyzing which part of the target genomic gene the inserted DNA is a partial sequence (fragment) by a known method such as a genome mapping method.

Step (b) of Method 1
A genome in which two homologous sequences sandwiching the target DNA in the plasmid are inserted in advance by any one of the plasmids prepared in the above step (a) (hereinafter referred to as “homologous sequence inserted genome”) Are transformed. At this time, the plasmid can be arbitrarily selected, and may include any partial sequence as the target DNA. As the two homologous sequences sandwiching the target DNA in the plasmid, sequences adjacent to both ends of the target DNA may be used, and the length thereof is not particularly limited, but is generally 1 kb or more, preferably 2 kb or more. is there. For example, when a sequence of a BAC vector (hereinafter sometimes referred to as a “BAC vector sequence”) is used, it is preferable to insert the full length (about 7 kb) of the BAC vector by about ず つ.

  Here, the term "homologous recombination" as used herein means recombination that occurs in a portion of a pair of double-stranded DNAs having a homologous base sequence. In addition, a homologous sequence referred to in the present specification does not need to have 100% sequence identity as long as the desired homologous recombination can be caused. That is, the two homologous sequences may be substantially homologous, for example, 60% or more, preferably 70% or more, more preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more homology. What is necessary is just to have the property.

  The insertion of the homologous sequence into the microorganism genome is such that, when the homologous sequence-inserted genome is transformed with a plasmid, homologous recombination occurs in the homologous sequence, and as a result, the target DNA flanked by the homologous sequence is integrated into the genome. Need to be done. This will be described in detail with reference to FIG. The selected plasmid (see “plasmid” in FIG. 3) has two homologous sequences indicated by arrows, and has a target DNA (black line indicated by “A” in FIG. 3) sandwiched between the two homologous sequences. When this homologous sequence is inserted into the microorganism genome, each homologous sequence is placed in the same orientation as that existing in the plasmid with the target DNA interposed, as shown in "Microbial genome" in FIG. Insert into the genome. When this is inserted into the microorganism genome in the reverse direction, the microorganism genome is replaced with the target DNA by homologous recombination, and a transformant cannot be obtained.

  As a microorganism into which such a homologous sequence is previously inserted, any microorganism can be used as long as it can retain the genomic gene of the present invention on its own genome and can introduce a modification into the genomic gene. Alternatively, highly thermophilic bacteria are preferably used. The type of Bacillus bacterium is not particularly limited. For example, B. subtilis (Bacillus subtilis), B. megaterium (macrobacillus), B. anthracis (splenic anthrax), B. cereus, B. stearothermophilus (moderately favorable) Heat bacterium) and the like. Preferably, B. subtilis (Bacillus subtilis), which has an excellent ability to take up DNA and recombination ability, is used. Extremely thermophilic bacteria are a general term for bacteria that can grow only under high temperatures.

  Transformation and homologous recombination can be performed by a conventional method. Specifically, for example, a culture solution containing the microorganism cells cultured under ordinary conditions contains any of the plasmids in the above-described plasmid group. What is necessary is just to add a DNA solution. Bacillus subtilis has the property of spontaneously taking up DNA fragments from a DNA solution, and the DNA taken up within the cells undergoes homologous recombination at a homologous sequence previously integrated into the genome. As a result, the target DNA can be introduced into the Bacillus subtilis genome. The position for incorporating the homologous sequence in the microorganism genome may be any position as long as the microorganism is not killed by insertion of the homologous sequence or the target DNA, but in the case of Bacillus subtilis, for example, the proB gene or the like may be used. And the like of the amino acid-requiring gene. Details of transformation and homologous recombination can be found in Mitsuo Ogura, Bioscience and Industry, Vol.57, No.8, 532-535 (1999), Mitsumata Itaya, Journal of the Japanese Society of Agricultural Chemistry, Vol.67, No.4, 703-706 (1993) and Mitaniyasu Itaya, Journal of the Japanese Society of Agricultural Chemistry, Vol. 68, No. 11, 1545-1550 (1994).

  The transformant thus obtained is cultured in a commonly used medium under appropriate conditions for an appropriate time. The medium may be liquid or solid, but a liquid medium is preferred. The cultivation temperature is not particularly limited as long as the bacterium used in the method of the present invention can grow, but is usually in the range of 20 to 80 ° C. The pH during the cultivation may be any value as long as the host bacteria can grow.

  Here, preferably, a transformant in which the target DNA in any one of the plasmids in the plasmid group has been correctly introduced into the genome by homologous recombination at two homologous sequences sandwiching the target DNA in the plasmid is selected. I do. As a selection method, these are inserted in advance so that a marker DNA exists between two homologous sequences in the microorganism genome (the position indicated by “M1” in “microorganism genome” in FIG. 3), and the marker Examples include a method of selecting the loss of DNA expression as an indicator, a method of analyzing the sequence by PCR, and the like to confirm that the DNA has been correctly introduced, and a method using a marker DNA is preferable. Examples of the marker DNA include a drug resistance gene, an auxotrophic gene, and a DNA having a function of controlling them, and a drug resistance gene is preferably used. Specific examples thereof include antibiotic resistance genes such as a neomycin resistance gene, a spectinomycin resistance gene, a tetracycline resistance gene, an erythromycin resistance gene, and a blasticidin S resistance gene.

Furthermore, double selection combining positive selection under conditions in which only microbial cells into which the target DNA has been correctly introduced grows, and negative selection under selection conditions under which a specific trait is expressed to kill the cells. Transformants can also be selected by selection or the like.
In addition, by using a repressor gene, selection combining two or more markers can be performed. Specifically, for example, a microorganism having a neomycin resistance gene (Pr-neo) whose expression is controlled by a Pr promoter at another position in the genome, a spectinomycin resistance gene and cI between two homologous sequences. When the DNA containing the repressor gene is inserted, the strain expressing the cI repressor gene becomes neomycin sensitive because the cI repressor protein binds to the Pr promoter and suppresses the expression of the downstream neomycin resistance gene. In addition, spectinomycin resistance is exhibited by expression of the spectinomycin resistance gene. When this microbial cell is transformed by the method described above, the target DNA is introduced by homologous recombination between two homologous sequences, and the spectinomycin resistance gene and the cI repressor are lost. The strain in which the cI repressor protein is no longer supplied becomes free from expression of the Pr promoter, expresses the neomycin resistance gene, exhibits neomycin resistance, and becomes spectinomycin sensitive. By utilizing this change in drug resistance, a target transformant can be selected (Itaya, M., Biosci. Biotech. Biochem., 63, 602-604 (1999)).

  In addition, two or more repressor genes can be introduced into one microorganism genome and used. That is, two or more promoters can be used for one promoter. In particular, in the case of the cI repressor gene, mutation may occur spontaneously in the course of transformation or the like, and a colony may be formed even though the target DNA has not been correctly introduced. When a large number of such colonies are generated, there is a problem that the probability of obtaining a colony into which the target DNA has been introduced is reduced, or that screening for selecting these is further required. However, if two or more cI repressor genes are used for one Pr promoter, the probability of simultaneous mutation in two or more cI repressor genes is extremely low, so that the above problem can be avoided. . The number of cI repressor genes to be introduced is preferably two or more, and particularly preferably two, for one promoter.

  Here, when two or more cI repressor genes are introduced into one microorganism genome and used, a spacer is preferably inserted between each cI repressor gene. When two or more cI repressor genes are present in close proximity, homologous recombination may occur between them.However, such a homologous recombination can be avoided by inserting an appropriate spacer. can do. As the spacer, it is preferable to use a spacer having no homology with the target DNA or the sequence in the genome of the microorganism. Specifically, those having homology to these sequences of 50% or less, preferably 40% or less are used. The length of the spacer may be any length that can prevent homologous recombination between the two cI repressor genes, but is preferably 50 kb or more, more preferably 100 kb or more. The upper limit is preferably 300 kb or less. Further, it is also preferable to select a DNA having a GC content of about 50% as a DNA used as a spacer.

  Specific examples of such a spacer include, for example, a partial sequence of the Arabidopsis thaliana mitochondrial genome possessed by BAC clone F1O22 (manufactured by Kazusa DNA Research Institute). F1O22 is obtained from the IGF Arabidopsis BAC library (Mozo, T. et al., Mol.Gen. Genet. 258, 562-570 (1998)), and in a BAC vector derived from pBeloBAC11 (pBeloBAC-Kan), A partial sequence of the Arabidopsis mitochondrial genome of about 100 kb is inserted.

  In the method using the cI repressor gene as described above, a marker DNA for positive selection (expressed when the target DNA is correctly introduced and the cI repressor gene is lost) placed under the control of the Pr promoter is used. In addition, it is preferable to use a marker DNA for screening (expressed when the target DNA has not been introduced) in combination. As the marker DNA, those described above are used.

  Regarding the method using two or more cI repressor genes for one Pr promoter described above, two cI repressor genes, a neomycin gene for positive selection controlled by the Pr promoter, and a spectinomycin for screening The case of using a combination of a resistance gene and a blasticidin S resistance gene will be described in more detail below.

  First, a DNA serving as a spacer, for example, a partial sequence of Arabidopsis thaliana mitochondrial genome of BAC clone F1O22 (manufactured by Kazusa DNA Research Institute) is introduced between two homologous sequences in the microorganism genome. This microorganism has a neomycin resistance gene (Pr-neo) whose expression is controlled by the Pr promoter at another position in the genome. Next, a product obtained by linking the cI repressor gene and the spectinomycin resistance gene to one end of this spacer (cI-Spc) is introduced. For example, when this cI-Spc is introduced into the 3 ′ end of the spacer, a product in which the cI repressor gene and the blasticidin S resistance gene are linked to the other 5 ′ end (cI-BS) is introduced. At this time, either cI-Spc or cI-BS may be at the 3 ′ end or 5 ′ end of the spacer, but it is possible to insert the two cI repressor genes so that they are opposite to each other in the genome. Preferred (for example, see “Bacillus subtilis BEST6528 strain” in FIG. 5). For example, when cI-Spc and cI-BS are introduced, they are introduced such that each drug resistance gene is adjacent to the spacer side and the cI repressor gene is adjacent to the homologous sequence side. By introducing in such an order, the probability that homologous recombination occurs between two cI repressor genes can be suppressed, and if homologous recombination occurs between two cI repressor genes, In addition, loss of the spacer and the drug resistance gene can be avoided.

  The microbial cells thus prepared have neomycin sensitivity and are resistant to spectinomycin and blasticidin S because the Pr promoter is suppressed by the expression of the cI repressor protein and the expression of the neomycin resistance gene is suppressed. is there. When this microbial cell is transformed by the method described above, the target DNA is introduced by homologous recombination between the two homologous sequences, and cI-Spc, cI-BS, and the spacer are lost. As a result, the cI repressor protein is not supplied, the suppression of the Pr promoter is released, and the neomycin resistance gene under the control of the promoter is expressed to become neomycin resistance. On the other hand, it becomes sensitive to spectinomycin and blasticidin S. By utilizing this change in drug resistance, a target transformant can be selected.

  When a transformant is selected using a drug resistance gene as a marker DNA, a drug suitable for each drug resistance gene is added to the above-mentioned medium, followed by culturing. Specifically, for example, when a microorganism having a genome into which a neomycin resistance gene is inserted (for example, see the Bacillus subtilis genome into which neo (neomycin resistance gene) is introduced in FIG. 2), 5 What is necessary is just to add 1010 μg / ml neomycin.

Step (c) of Method 1
Next, another plasmid containing the target DNA having an overlapping sequence with the target DNA in the plasmid used for the transformation in the above step (b) is selected from the above plasmid group. The selected plasmid may have an overlapping sequence with the 3 'end of the target DNA in the plasmid previously used for the transformation, or may have an overlapping sequence with the 5' end.

Step (d) of Method 1
The microbial cells transformed in the above step (b) are transformed with the plasmid selected in the above step (c) (see “After insertion” in FIG. 3). Transformation can be performed in the same manner as described above.

Step (e) of method 1
From the microbial cells obtained in the above step (d), the target DNAs of the two plasmids used in the above steps (b) and (d) are genomically ligated to the DNA ligated by homologous recombination at the overlapping sequence and the homologous sequence. Select the transformants contained therein. As a result of the transformation in the above step (d), an overlapping sequence between the target DNA in the plasmid used in this step and the target DNA in the plasmid introduced into the microorganism genome by the transformation in the above step (b) The homologous recombination with the homologous sequence in the plasmid adjacent to the opposite end causes the two target DNAs to be ligated (see “after ligation” in FIG. 3). A transformant having such a DNA in the genome can be selected by a method using a marker DNA, a method of confirming the sequence by a PCR method, or the like, as in the method described in detail in (2) above.

  Here, when a transformant linked by the above homologous recombination is selected using a marker DNA, the marker DNA is located between the overlapping sequence of the target DNA already inserted into the microorganism genome and the homologous sequence (see FIG. 3). (At the position indicated by “M2” of “after insertion”). The marker DNA used at this time may be the same as or different from the marker DNA used in the above (2).

Step (f) of Method 1
By repeating the above steps (c) to (e) and introducing all the target DNAs contained in the plasmid group prepared in the above step (a) into the microorganism, a foreign gene can be introduced into the microorganism genome. The constituent genomic DNA is reconstructed.

  Bacillus bacteria or highly thermophilic bacteria, especially B. subtilis (Bacillus subtilis), have the ability to stably maintain large DNAs in their genomes, and also provide genomically engineered DNAs in their genomes. It is easy to modify by manipulation (Mitsuyasu Itaya, Bacillus subtilis genome vector learned naturally, Bioscience and Industry, 57, 540-543 (1999)). Therefore, for example, modifications such as insertion, substitution, and deletion can be made to the genomic gene reconstituted in the microorganism genome by a known method.

2. Method for reconstituting genomic DNA constituting foreign genes in microorganism genome (method 2)
The second method of the present invention for reconstituting genomic DNA constituting a foreign gene in a microorganism genome is characterized by comprising the following steps.
(A) Step of preparing a plasmid group in which partial sequences having different total lengths of genomic DNA constituting a foreign gene are inserted into a vector as target DNAs, and each target DNA has a sequence overlapping with any other target DNA. ,
(B) Two homologous sequences sandwiching the target DNA in each of the plasmids prepared in the above step (a) are previously inserted into microbial cells having the same genome at the same position on the microbial genome, and each of the microbial cells is Transforming with the plasmid of
(C) a step of extracting genomic DNA from a microbial cell having any of the target DNAs obtained in the step (b) in the genome,
(D) Transforming a microbial cell having in the genome another target DNA having an overlapping sequence with the target DNA in the genomic DNA extracted in the step (c) with the genomic DNA obtained in the step (c). Process,
(E) From the microbial cells transformed in the above step (d), the target DNAs in the two microbial genomes used in the above steps (c) and (d) are respectively the duplicated sequence and the common sequence in the microbial genome. Selecting a transformant having DNA linked by homologous recombination,
(F) genomic DNA constituting a foreign gene in the genome of a microorganism by repeating the above steps (c) to (e) and introducing all the target DNAs prepared in the above step (a) into the microorganism. The step of reconstructing.

An outline of the method 2 is schematically shown in FIG. Hereinafter, details of each step will be described.
Step (a) of Method 2
The plasmid group prepared in this step can be prepared in the same manner as the method described in the step (a) of the above method 1. The same can be used for the partial sequence of genomic DNA constituting the foreign gene used as the target DNA, the vector, and the like. However, in this method, since the homologous recombination occurs in the overlapping sequence of the two target DNAs and the common sequence in the microorganism genome as described in detail in the step (e) described later, the vector into which each target DNA is inserted is used. Not all need to be identical. However, it is preferable that they be inserted into the same vector from the viewpoint of recombination efficiency, simplicity of operation, and the like. The vector is more preferably a BAC vector, and an appropriate vector can be selected from a commercially available BAC library or the like and used in the same manner as in the step (a) of the above-mentioned method 1.

Step (b) of Method 2
Next, two homologous sequences sandwiching the target DNA in each of the plasmids prepared in the above step (a) (see “Plasmids” in FIG. 4) are previously placed at the same position in the microorganism genome in the microorganism cells having the same genome. Insert and transform the microbial cells with each plasmid (see "Microbial Genome" in FIG. 4).

  Microbial cells having the same genome may be selected from those similar to those described in detail in step (b) of Method 1 above. In such a microbial cell, two homologous sequences sandwiching the target DNA in each plasmid prepared in the above step (a) are inserted in advance in the same manner as in the step (b) of the above method 1. Here, when the vector constituting the plasmid is the same, one kind of microbial cell having the homologous sequence inserted therein may be prepared, but when the vector is different, the appropriate sequence in each vector is inserted in advance. Then, a microbial cell for each plasmid is prepared. At this time, the homologous sequence is inserted at the same position in the microorganism genome. In this method, as described in detail in step (e) described below, since homologous recombination occurs at a common sequence in the microorganism genome and the overlapping sequence of the two target DNAs, the overlapping sequence and the common sequence are appropriately It must be located at a position where homologous recombination can occur. That is, the “same position” does not need to be exactly the same position on the sequence, and may be within the range where the above-described homologous recombination can occur.

  The length of two homologous sequences sandwiching the target DNA in the plasmid, the position where the homologous sequence is incorporated in the microorganism genome, the method of transformation and homologous recombination, the method of selecting a transformant into which the target DNA has been correctly introduced, etc. The method can be performed in the same manner as in the step (b) of the method 1.

Step (c) of Method 2
Next, genomic DNA is extracted from a microbial cell having a genome containing any target DNA prepared in the step (b). The extraction of genomic DNA can be performed by a commonly used method known per se, for example, a lysozyme-SDS treatment method (Saito, H. and Miura, K., Biochem. Biophys. Acta., 72, 619-629). (1963)). The genomic DNA extract thus prepared contains fragmented microbial genomes, but has a continuous sequence of the target DNA introduced in step (b) and a common sequence on the microbial genome. What is necessary is just to be included.

Step (d) of method 2
Microbial cells containing another target DNA having an overlapping sequence with the target DNA in the microorganism genome used for extracting the genomic DNA in the step (c) are transformed with the genomic DNA obtained in the step (c). (See “After Insertion” in FIG. 4). Transformation can be performed in the same manner as described above. The target DNA contained in the microbial cells used herein may have an overlapping sequence with the 3 ′ end of the target DNA in the previously transformed microbial cells, or may have an overlapping sequence with the 5 ′ end May be.

Step (e) of method 2
From the microorganism cells obtained in the above step (d), the target DNAs in the two microorganism genomes used in the above steps (c) and (d) are linked by homologous recombination at the overlapping sequence and the common sequence in the microorganism genome. A transformant having the obtained DNA is selected. As a result of the transformation in the step (d), the overlapping sequence of the target DNA contained in the genome of the microbial cell in the target DNA contained in the genomic DNA obtained in the step (c) and the reverse sequence Homologous recombination occurs with a common sequence adjacent to the end of the target DNA, thereby linking the two target DNAs (see "after ligation" in FIG. 4). Selection of a transformant having such a DNA in the genome is performed by a method using a marker DNA, a method of confirming the sequence by a PCR method, or the like, in the same manner as the method described in detail in the step (b) of the above method 1. be able to.

Step (f) of method 2
By repeating each of the above steps (c) to (e) and introducing all the target DNAs prepared in the above step (a) into the microorganism, the genomic DNA constituting the foreign gene in the microorganism genome is reproduced. Be composed.
Bacillus bacteria or highly thermophilic bacteria, especially B. subtilis (Bacillus subtilis), have the ability to stably maintain large DNAs in their genomes, and also have a genetically engineered DNA in their genomes. It is easy to modify by manipulation (Mitsuyasu Itaya, Bacillus subtilis genome vector learned naturally, Bioscience and Industry, 57, 540-543 (1999)). Therefore, for example, modifications such as insertion, substitution, and deletion can be made to the genomic gene reconstituted in the microorganism genome by a known method.

3. According to another embodiment of the present invention, there is provided a plasmid comprising the full length of genomic DNA constituting a foreign gene and having a length of 100 kb or more. Provided.
Such a plasmid can be obtained, for example, from a microorganism prepared by the method described in 1 or 2 above. Specifically, a portion containing the full-length genomic DNA constituting the foreign gene is recovered as a plasmid from the microorganism genome prepared by the method using a method described in JP-A-2002-17350 or the like, and purified. Can be obtained. When a rare cutter recognition sequence has been introduced into the 5 ′ end and 3 ′ end of the genomic gene in advance, the genomic gene can be cut out and recovered by treating the genomic gene with the rare cutter. The rare cutter refers to, for example, an endonuclease that recognizes a base sequence composed of eight or more bases and specifically cleaves the sequence, and examples thereof include I-PpoI and I-SceI.

  The total length of the genomic DNA constituting the foreign gene is 100 kb or more, preferably 200 kb or more. The foreign gene includes, for example, those similar to those described in 1 above. That is, as long as it has a function as a genome, it does not necessarily have to have the full length, and genomic engineering modifications such as insertion, substitution, deletion, mutation, etc. have been added to the original genomic DNA Including things.

  The thus obtained plasmid of the present invention can be suitably used as a transgene for producing a genetically modified non-human animal or plant. In the production of genetically modified non-human animals or plants, it is considered that it is more efficient to use the genomic DNA itself that constitutes the gene, rather than using an artificially produced transgene, The plasmid of the present invention is very useful.

  Also, for example, if the genomic DNA constituting the reconstituted foreign gene is configured to be under the control of an effective expression control region in the bacterium of the genus Bacillus or thermophilic bacterium, the specific protein A transformant with increased productivity, a transformant that produces a protein with improved function, and the like can be easily obtained.

4. Method for producing a genetically modified non-human animal or plant using the plasmid of the present invention A genetically modified non-human animal or plant can be produced using the plasmid of the present invention described in 3 above. In particular, a plasmid obtained from a microorganism prepared by the method described in 1 or 2 above can be easily modified by genome engineering in the genome of the microorganism. It is suitable for production.

The plasmid of the present invention according to 3 above, an unfertilized egg of a non-human animal, a fertilized egg, a germ cell containing a sperm and its progenitor cells, a living organ, a tissue cell, a cultured cell, or a plant individual, organ, By introducing the gene into a tissue, a cultured cell, or the like, a genetically modified non-human animal or a genetically modified tissue or a cultured cell of an animal and a plant can be produced.
The animals or plants on which genetic modification is performed include not only individuals but also living organs, tissues, and cultured cells in animals, and organs, tissues, and cultured cells in plants. The kind of animal is excluding human as an animal individual, but other types are not limited at all, and examples thereof include mouse egg cells and ES cells.

  Non-human animal non-fertilized eggs, fertilized eggs, germ cells containing sperm and their progenitor cells, living organs, tissue cells, cultured cells, or plant individuals, organs, tissues, cultured cells, etc. There is no particular limitation on the form of the DNA to be used in the introduction, and for example, the obtained circular plasmid may be directly provided as a donor DNA, or the obtained plasmid may be cut with a restriction enzyme and separated by electrophoresis or the like. The desired sequence alone may be separated and purified by a means and used as donor DNA.

  The method for introducing the plasmid is not particularly limited, and a commonly used method can be used. For example, a method of direct microinjection of DNA into the pronucleus of a fertilized egg (Palmiter, RD et al., Annu. Rev. Genet., 20, 465-499 (1986)), electro-transfer of embryonic stem cells (ES cells) A method for introducing DNA by poration (Karin, ML, et al., Proc. Natl. Acad. Sci. USA, 81, 7161-7165 (1984)), lipofection in which donor DNA wrapped in lipids is fused with ES cells Method (Strauss, WM et al., R. EMBO J., 11, 417-422 (1992)), a method in which a donor strain having a plasmid is used as a protoplast and fused with spheroplast of ES cells (Davies, NP et al. al., Biotechnology, 11, 911-914 (1993)), a method of injecting intracytoplasmic sperm into unfertilized eggs (Perry, ACF et al., Science, 284, 1180-1183 (1999)). It can be introduced into the above-mentioned cells and the like using a method such as the sperm vector method (Lavitrano, M. et al., Cell, 57, 717-723 (1989)) for introducing DNA into a fertilized egg cell. . In addition, the use of an adenovirus (Chartier, C. et al., J. Virol., 70, 4805-4810 (1996)) as the plasmid of the present invention, or the use of a retrovirus or the like enables the production of oocytes or ES cells. A method of introducing a gene by infecting with a virus can also be used.

In addition, by the above-described DNA introduction method, somatic cells, organs of a living body, a genomic gene reconstructed in tissue cells or the like, or a mutant thereof can be introduced, and cell culture or tissue culture can be performed. The non-human gene mutant animal of the present invention can also be prepared by fusing these cells with the above-mentioned germ cells by a cell fusion method known per se.
In the case of plants, individuals, or organs such as leaves, petals, stems, roots and seeds, or tissues such as epidermis, phloem, xylem, parenchyma, vascular bundles, etc., are performed on animals known per se. DNA can be introduced into the protoplasts by a similar method or electroporation. Selection of the transformant into which the thus obtained reconstructed genomic gene or a mutant thereof has been introduced is carried out by selecting an agent or the like suitable for the marker DNA when marker DNA has been introduced at the same time, and further selecting The analysis can be performed by analyzing DNA introduced by PCR or Southern analysis.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
In the following examples, ampicillin, chloramphenicol, erythromycin, tetracycline, spectinomycin, and neomycin antibiotics were purchased from Sigma. Blasticidin S antibiotics were purchased from Funakoshi. The restriction enzyme NotI was purchased from Takara Shuzo. I-PpoI nuclease was purchased from Promega. Other restriction enzymes, Alkaline phosphatase (E. coli), and T4 DNA ligase were purchased from Toyobo. Gene amplification by PCR was performed using TaKaRa Ex Taq HS from Takara Shuzo Co., Ltd. according to the attached instructions. The reaction conditions of PCR were as follows: After the reaction at 94 ° C. for 5 minutes, the following temperature cycle was performed 30 times. 94 ° C, 1 second: 60 ° C, 1 second: 72 ° C, 2 seconds. As the medium components and the agar of the LB medium, those manufactured by Becton Dickington were used. All other biochemical reagents were from Sigma and Nacalai Tesque.

  Plasmids other than those specified were constructed using Escherichia coli strain JA221 (available from American Type Culture Collection as ATCC37436 strain). For Southern hybridization, Roche DIG DNA Labeling and Detection Kit was used. Procedures such as extraction, assay, and large-scale preparation of the constructed plasmid are described in a standard protocol (Sambrook. J. et al., Molecular Clonoing: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)). Followed. Apparatus and method of use of pulse field electrophoresis Counter clumped gel Electrophoresis (CHEF) have already been reported (Itaya, M. and Tanaka, T., J. Mol. Biol., 220, 631-648 (1991), and JP 2001) -321170). Transformation of competent Escherichia coli JA221 and competent Bacillus subtilis was performed as described previously (Itaya, M. Mol. Gen. Genet., 241, 287-297 (1993)).

Preparation of Bacillus subtilis having BAC vector sequence in its genome Bacillus subtilis was used as a microorganism incorporating the target DNA, and BAC vector was used as a plasmid containing the target DNA. First, a BAC vector sequence was inserted into the genome of Bacillus subtilis as a homologous sequence for introducing the target DNA into B. subtilis.
(1) Preparation of Bacillus subtilis as a base Bacillus subtilis strain RM125 (Itaya, M., Biosci. Biotech. Biochem. 61, 56-64 (1997); and Uozumi, T. et al., Mol. Gen. Genet. , 152, 65-69 (1977)), a proB :: pBRTc (a structure containing a pBR322 sequence and a tetracycline resistance gene at the NotI recognition site in the proB gene) that specifies an integration site; Itaya, M., Mol. Gen. Genet., 241, 287-297 (1993)) and a neomycin resistance gene for positive selection (Itaya, M., Biosci. Biotech. Biochem. 63, 602-604 (1999)). Bacillus subtilis BEST7003 strain was prepared. The Bacillus subtilis RM125 strain is a strain having the property of irrespective of the origin of the DNA to be incorporated.

  First, a neomycin resistance gene (hereinafter, sometimes referred to as “Pr-neo”) whose expression is controlled by a Pr promoter derived from λ phage of Escherichia coli as a neomycin resistance gene for positive selection was incorporated to prepare a BEST6225 strain. This strain is obtained by transforming the pNEXT4-PN2 plasmid for integration of Pr-neo into the RM125 strain and obtaining a neomycin-resistant strain, and at the position of yvfC-yveP (3516.219kb-3522.452kb) in the genome, Pr-neo is obtained. It has a gene.

  E. coli plasmid pNEXT4-PN2 was prepared as follows. After blunt-ending an approximately 5 kb PstI fragment derived from a plasmid pBS52 having a Pr promoter (Breitling, R., et al., Gene, 93, 35-40. (1990)) with T4 DNA polymerase, the neomycin resistance gene was Plasmid pBEST515 having only a structural gene (Itaya, M. and Tanaka, TJ Mol. Biol., 220, 631-648. (1991)) was introduced into the SmaI recognition site, and Pr was inserted upstream of the structural gene of the neomycin resistance gene. A plasmid pBEST515A to which a promoter was linked was prepared. This plasmid was digested with EcoRI and purified by electrophoresis to obtain a Pr-neo fragment. An EcoRI / NotI / BamHI Adapter (manufactured by Takara Shuzo Co., Ltd.) having a phosphorylated 5′-protruding end was prepared in advance using T4 polynucleotide kineae (manufactured by Toyobo) and ligated to both ends of the Pr-neo fragment with T4 DNA Ligase. After that, purification was performed by agarose gel electrophoresis. Next, by cutting the NotI recognition site of the Adapter with NotI, a NotI cassette of a Pr-neo fragment was prepared, and this cassette was pBEST43 (Toda, T., et al. Biosci.Biotech.Biochem., 60, 773-778. (1996)) to obtain pBEST533. Plasmid pNEXT4 (Itaya, M., and Tanaka, TJ Mol. Biol., 220, 631-648. (1991)) having a PstI fragment containing yvfC-yveP in the Bacillus subtilis genome was digested with restriction enzyme NotI, and yvfC After removing the NotI fragment of about 1 kb between -yveP, the Pr-neo / NotI cassette obtained by digesting pBEST533 with NotI was inserted instead to obtain a plasmid pNEXT4-PN2 for integration into Pr-neo.

  The BEST6225 strain incorporating Pr-neo was obtained by transforming the RM125 strain with this plasmid and selecting for neomycin resistance. This BEST6225 strain was transformed using the genomic DNA of the BEST6006 strain (Japanese Patent Application Laid-Open No. 2001-321170), and a tetracycline-resistant strain was obtained as a BEST7003 strain having both Pr-neo and proB :: pBRTc. BEST7003 strain is erythromycin sensitive because it uses pNEXT4-PN2 at the time of Pr-neo insertion.

(2) Insertion of BAC vector sequence into B. subtilis BEST7003 strain In order to insert the BAC vector sequence into the BEST7003 strain prepared in (1) above, E. coli plasmid p310-108 (NHBN) MIMCISP-5 was constructed (FIG. 1). ).
First, the E. coli BAC vector pBAC108L (Shizuya, H. et al., Proc. Natl. Acad. Sci. Then, the endogenous 64-bp NotI fragment was removed to prepare plasmid pBAC108N. Next, a linker having the NotI / BamHI / HindIII / NotI sequence was inserted into the NotI recognition site of this pBAC108N, and the plasmid pBAC108NHBN was obtained in E. coli.

  In order to introduce a MluI recognition site into one of the EcoRV recognition sites of pBAC108NHBN (closer to the parA gene), 335 bp from this EcoRV recognition site to the SSe8387I recognition site in the repE gene were amplified by PCR. That is, PCR was performed using pBAC108NHBN as a template and primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2). A HincII-MluI sequence was previously added to the EcoRV-side primer used at this time, and the fragment amplified by PCR was digested with HincII and SSe8387I to obtain a 335 bp HincII-SSe8387I fragment. A linear fragment (6.5 kb) obtained by digesting pBAC108NHBN with EcoRV and SSe8387I was purified by agarose gel electrophoresis, and the PCR amplified fragment (HincII-SSe8387I fragment) was ligated to this. EcoRV and HincII gave blunt ends, and the EcoRV recognition sequence disappeared after ligation, so that pBAC108NHBN-Mlu having a MluI recognition site was obtained.

  Subsequently, in order to introduce an I-PpoI recognition site, a linker having the sequence of MluI / I-PpoI / MluI was inserted into the MluI recognition site of pBAC108NHBN-Mlu to construct pBAC108NHBN-MIM. The plasmid encoding the cI repressor protein and the cI cassette containing the spectinomycin resistance gene were ligated to the HindIII-BamHI recognition site of this plasmid to prepare pBAC108 (NHBN) MIM-CISP. As the cI cassette, a HindIII-BamHI digested fragment derived from pCISP310B (Itaya, M. et al. J. Biochem., 128, 869-875 (2000)) was used after purification by agarose gel electrophoresis. The plasmid pBAC108 (NHBN) MIM-CISP derived from this BAC vector was completely digested with I-PpoI, and pCISP310B completely digested with I-PpoI was ligated to construct p310-108 (NHBN) MIMCISP-5. And a plasmid for incorporating the BAC vector sequence serving as a scaffold.

  The Bacillus subtilis BEST310 strain into which the BAC vector sequence was incorporated was transformed with the BEST7003 strain prepared in the above (1) using the pBR322-derived sequence of p310-108 (NHBN) MIMCISP-5, and converted as a spectinomycin-resistant strain. I got it. The structure of p310-108 (NHBN) MIMCISP-5 is shown in FIG.

Introduction of partial sequence of mouse genomic DNA into Bacillus subtilis genome (1) Selection of plasmid having target DNA Two BAC clones were used as target DNAs to be introduced into B. subtilis BEST310 strain. One is a BAC clone (pKARUD) in which a mouse genome of about 90 kb of the 129 / SV strain derived from mouse chromosome 13 was inserted into a BAC vector (pBeloBAC11), and the vesl-1 gene (Kato, A et al., FEBS Letters, 412, 183-189 (1997)). The other is a BAC clone pKANEI in which approximately 110 kb of 129 / SV mouse genomic DNA derived from mouse chromosome 13 has been inserted into the same BAC vector (pBeloBAC11), and the jmj gene is contained in the mouse genomic region. (Takeuchi, T. et al., Genes & Development, 9, 1211-1222 (1995)). The former was provided by Dr. Inoguchi of the Mitsubishi Chemical Life Science Research Institute, and the latter was provided by Dr. Takeuchi of the Institute.

  Details of the BAC vector (pBeloBAC11) are disclosed in Kim, UJ. Et al., Genomics, 34, 213-218. (1996), and were incorporated as homologous sequences into the Bacillus subtilis genome in Example 1 above. It has almost the same structure as the BAC vector pBAC108L. The two BAC clones were purified by ultracentrifugation according to a standard protocol (Sambrook. J. et al., Molecular Clonoing: ALaboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)) before use. .

(2) Introduction of a partial sequence of genomic DNA constituting vesl-1 gene into Bacillus subtilis A BAC clone (pKARUD) having about 90 kb mouse genomic DNA was used for the B. subtilis BEST310 strain prepared in Example 1 above. A partial sequence of genomic DNA constituting the vesl-1 gene was incorporated. pKARUD contains the 3 'region of the vesl-1 gene, and the size of the insert DNA was determined by creating a restriction map of NotI, XhoI, and SalI by pulse field electrophoresis (CHEF).

  As shown in FIGS. 1 and 2, the two BAC vector sequences incorporated as homologous sequences into the BEST310 strain each have a half region of about 3.5 kb derived from pBAC108L, and therefore, when transformed using pKARUD, Homologous recombination (double crossing-over) occurs between the BAC vector sequence of pKARUD incorporated in the competent BEST310 strain and the BAC vector sequence in the Bacillus subtilis genome, and the entire insert DNA of pKARUD is in the B. subtilis genome. Be incorporated.

  Selection of the Bacillus subtilis strain into which the insert DNA was incorporated was performed according to the method described in the literature Itaya, M. Biosci. Biochem. Biotech., 63 602-604. (1999), JP-A-2001-321170, and the like. Was. That is, a cI cassette (a gene encoding a cI repressor protein and a spectinomycin resistance gene) is integrated between two homologous sequences (BAC vector sequence) in the BEST310 strain, while the same strain has a yvfC -yveP (3516.219kb-3522.452kb) contains a neomycin resistance gene (Pr-neo) whose expression is controlled by the Pr promoter. The BEST310 strain expressing the cI gene is spectinomycin-resistant, and is neomycin-sensitive because the cI repressor protein binds to the Pr promoter and suppresses the expression of the downstream neomycin resistance gene. Strains that have incorporated pKARUD lose the cI cassette between the homologous sequences, become spectinomycin sensitive by the lack of the spectinomycin resistance gene in the cI cassette, and also lack the cI repressor gene. This strain in which the cI repressor protein is no longer supplied expresses the neomycin resistance gene since the expression of the Pr promoter is released, and becomes a neomycin resistant strain.

  pKARUD was purified as circular DNA by ultracentrifugation. About 5 μg of pKARUD was used for transformation of the BEST310 strain. As a result, 216 neomycin-resistant colonies were selected. Among them, 100 were screened for spectinomycin sensitivity, and as a result, 50 were obtained as spectinomycin-sensitive strains. Genomic DNA was purified by mixing these 50 colonies in groups of 5, and the presence or absence of the insert DNA contained in the BAC clone was confirmed using I-PpoI nuclease. Bacillus subtilis BEST310 strain incorporates two I-PpoI recognition sites immediately outside the BAC vector sequence inserted as a homologous sequence (FIG. 2), digests with I-PpoI, and performs pulse field electrophoresis (CHEF ) Can be separated and confirmed. As a result, all the 10 sets (50 in total) mixed with each other gave an I-PpoI fragment of about 90 kb. Five sets of genomic DNA contained in one set were separately prepared, and subjected to pulse field electrophoresis again to confirm that an I-PpoI fragment of about 90 kb was obtained. For further detailed examination, two of these strains were digested with restriction enzymes (EcoRI, EcoRV, HindIII) that recognize 6 bases, and then subjected to genomic Southern using pKARUD as a probe, and all gave the same signal. These results indicate that a partial sequence of about 90 kb mouse genomic DNA was successfully cloned into B. subtilis genome by a single transformation.

(3) Introduction of Partial Sequence of Genomic DNA Constituting jmj Gene into Bacillus subtilis Next, in the same manner as in (2) above, a BAC clone (pKANEI) containing about 110 kb mouse genomic DNA in Bacillus subtilis BEST310 strain Was used to incorporate the partial sequence of the genomic DNA constituting the jmj gene. pKANEI contains the 3 'region of the jmj gene, and the size of the insert DNA was determined by preparing restriction maps of NotI, XhoI, and SalI by pulse field electrophoresis (CHEF).

  Since pKANEI was also cloned into the pBeloBAC11 vector in the same manner as pKARUD used in (2) above, the BAC vector sequence incorporated into the BEST310 strain was used as a homologous sequence. The selection method and principle of the prepared strain were performed in the same manner as in the above (2). pKANEI was also transformed using about 5 μg of the circular DNA purified by ultracentrifugation, and 248 neomycin-resistant colonies were obtained. Of these, 100 were screened for spectinomycin sensitivity, and 35 were spectinomycin-sensitive strains. Genomic DNA was separately purified for 10 of these, and the presence or absence of the insert DNA contained in the BAC clone was confirmed using I-PpoI nuclease.Six strains gave an I-PpoI fragment of about 110 kb. . Furthermore, when the two strains were examined in detail with restriction enzymes (EcoRI, EcoRV, HindIII) that recognize 6 bases, the same signal was given by genomic Southern using pKANEI as a probe. These results indicate that a partial sequence of about 110 kb mouse genomic DNA was successfully cloned into a B. subtilis genome vector by a single transformation.

Method for introducing a partial sequence of mouse genomic DNA into Bacillus subtilis genome using two cI repressor genes As in Example 2 above, using a single cI repressor gene in Bacillus subtilis BEST310 strain When DNA was introduced, not all of the neomycin-resistant colonies obtained initially were strains having the introduced DNA, and further screening was sometimes required. This was probably due to mutations occurring in the cI repressor gene at a certain frequency. That is, due to mutations in the cI repressor gene at a certain frequency, the cI repressor protein is not expressed, and the colony is cloned despite the absence of the target DNA (hereinafter, the strain thus generated is referred to as " (Sometimes referred to as "background strains").
Therefore, a method for avoiding this problem by introducing two cI repressor genes was examined.

(1) Introduction of a 100 kb spacer DNA sequence into the Bacillus subtilis genome When two cI repressor genes were placed in the B. subtilis genome, it was thought that they would cause homologous recombination with each other. Therefore, in order to suppress the frequency of this homologous recombination, a long spacer DNA sequence was used.
As the spacer DNA sequence, a partial sequence of the mitochondrial genome of Arabidopsis thaliana was used. The introduction of the spacer DNA sequence was performed by transforming the Bacillus subtilis BEST310 strain in the same manner as in Example 2 above, using a BAC clone F1O22 of about 100 kb.

  F1O22 was obtained from the IGF Arabidopsis BAC library (Mozo, T. et al., Mol.Gen. Genet. 258, 562-570 (1998)), and the pBeloBAC11-derived BAC vector (pBeloBAC-Kan) was used for Arabidopsis thaliana. (Arabidopsis thaliana) mitochondrial genome partial sequence is inserted. The BAC clone was provided by Kazusa DNA Research Institute. Details of the BAC vector (pBeloBAC-Kan) are disclosed in Mozo, T. et al., Mol.Gen. Genet. 258, 562-570 (1998), and in the Bacillus subtilis genome in Example 1 above. It has almost the same structure as the BAC vector pBAC108L incorporated as a homologous sequence. F1O22 was also purified by ultracentrifugation and used as a ring.

  Transformation of the BEST310 strain with F1O22 resulted in the selection of 553 neomycin-resistant colonies, 69% of which were screened as spectinomycin-sensitive strains. The obtained colonies were analyzed by I-PpoI digestion and genomic Southern analysis in the same manner as in Example 2 above. As a result, two strains (BEST6487 strain and BEST6488 strain) in which the entire F1O22 was correctly introduced were obtained.

(2) Preparation of Bacillus subtilis strain into which two cI repressor genes have been introduced
As a cI cassette having a cI repressor gene, `` cI-Spc (a gene encoding a cI repressor protein and a spectinomycin resistance gene) '' and `` cI-BS (a gene encoding a cI repressor protein and Blasticidin S resistance gene linked). These were finally used by inserting them into a BAC vector. At this time, for the BAC vector having cI-BS, cI-BS was inserted in the reverse direction to the BAC vector. cI-Spc was inserted in the forward direction with respect to the BAC vector.

  Insertion of both cassettes was performed as follows. First, about 2 kb of each end of the F1O22 insert was amplified by PCR. The sequence of the F1O22 insert has been published on the NCBI website (ACCESSION No. NC_001284; http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NC_001284). Next, these amplified fragments were inserted into a previously prepared BAC vector having cI-Spc and a BAC vector having no pBeloBAC-derived insert. First, one of the amplified fragments (the fragment on the 5 'end side of the insert) is ligated to a BAC vector in which cI-Spc has been inserted in advance, followed by the spectinomycin resistance gene. And the plasmid pBAC-CISP-F1A was constructed. The other fragment (the fragment on the 3 'end side of the insert) was inserted into a BAC vector having no pBeloBAC-derived insert, and then cI-BS was added in the reverse direction to the BAC vector, blasticidin S A resistance gene was inserted so as to be ligated adjacent to the fragment to construct a plasmid pBAC-CIBS-F1B.

  By transforming the BEST6487 strain obtained in the above (1) using the two plasmids thus obtained, a Bacillus subtilis BEST6528 strain having two cI repressor genes as shown in FIG. 5 was prepared. The Bacillus subtilis BEST6528 strain has two cI repressor genes, each connected as a cassette to a drug resistance gene, between two homologous sequences, which are opposite to each other. A spacer is inserted between the two cassettes.

(3) Comparison of background strain frequency
It was examined whether introduction of two cI repressor genes into the Bacillus subtilis genome could reduce the frequency of background strains produced by Bacillus subtilis into which only one cI repressor gene had been introduced. For verification, for each of the Bacillus subtilis BEST310 strain and the BEST6528 strain obtained in (2) above, the same amount of cells as those usually used for transformation were cultured overnight, and how much of each strain was It was examined whether a neomycin resistant colony (background strain) was formed at a frequency of.

As a result, 185 neomycin-resistant colonies were formed from the BEST310 strain. In contrast, no neomycin resistant colonies were formed from the BEST6528 strain.
This indicates that the probability of simultaneous mutation of two cI repressor genes during transformation is low. That is, by using a Bacillus subtilis strain having two cI repressor genes, the appearance of a background strain can be suppressed, and a strain into which the target DNA has been correctly introduced can be obtained with a high probability. It turns out that can be done.

(4) Introduction of partial sequence of mouse genomic DNA into Bacillus subtilis BEST6528 strain Actually, mouse genomic DNA was introduced using the BEST6528 strain obtained in (2) above. As the partial sequence of the mouse genomic DNA, pKARUD used in Example 2 above was used. The structure of the Bacillus subtilis genome in which the BAC clone has been introduced into the BEST6528 strain has exactly the same structure as when the clone is introduced into the BEST310 strain, which has only one cI repressor gene ( See Figure 5).

  As in Example 2, transformation was performed using about 2 micrograms of the circular DNA (pKaruD) purified by ultracentrifugation. BEST310 strains yielded 147 neomycin-resistant colonies, 27% of which were spectinomycin-sensitive strains. When these were further digested with I-PpoI and analyzed for genomic Southern, it was confirmed that a partial sequence (pKaruD) of the target mouse genomic DNA was introduced into 10.8% of the first neomycin-resistant colonies. On the other hand, 12 neomycin-resistant colonies were obtained from the BEST6528 strain, 11 of which were spectinomycin-sensitive. This was a very high probability of 91.7%. These were further confirmed by I-PpoI digestion and genomic Southern analysis. As a result, the partial sequence (pKaruD) was introduced into 36% of the first neomycin-resistant colonies.

  These results confirm that the use of a Bacillus subtilis strain having two cI repressor genes between the homologous sequences in the genome enables more efficient selection of the introduction of large DNA such as mouse genomic DNA into the Bacillus subtilis genome. Was done.

  According to the method for reconstituting genomic DNA of the present invention, it is possible to easily reconstitute genomic DNA constituting a foreign gene in a microorganism genome using a genomic library using a vector such as BAC, In such a state, the entire length can be obtained. In addition, the reconstructed genomic gene may be subjected to genomic engineering modification or the like in the microorganism genome, and this may be obtained as a plasmid. The thus obtained plasmid may be used to produce a genetically modified non-human animal or plant. Can be suitably used. When a bacterium belonging to the genus Bacillus or a highly thermophilic bacterium is used as a microorganism, the stability of the DNA in the incorporated genome is high, and long-term storage and transport are easy by forming the spores.

FIG. 2 shows the structure of plasmid p310-108 (NHBN) MIMCISP-5 used to incorporate a BAC vector sequence as two homologous sequences into the Bacillus subtilis genome. In the figure, “cI” indicates the cI repressor gene, and “spcR” indicates the spectinomycin resistance gene. FIG. 4 is a view showing a step of transforming a Bacillus subtilis cell into which a BAC vector sequence and a marker DNA have been previously inserted using a BAC clone. “CI” indicates a cI repressor gene, “spc” indicates a spectinomycin resistance gene, and “neo” indicates a neomycin resistance gene. FIG. 1 is a view schematically showing a first (method 1) of a method of the present invention for reconstituting genomic DNA constituting a foreign gene in a microorganism genome. In the figure, black arrows and white arrows indicate two homologous sequences, and black squares indicate overlapping sequences. “M1” and “M2” mean marker DNA. FIG. 3 is a diagram schematically showing a second (method 2) of the method of the present invention for reconstituting genomic DNA constituting a foreign gene in a microorganism genome. In the figure, four arrows indicate different homologous sequences, and black squares indicate overlapping sequences. The wavy lines indicate the common arrangement. “M1” and “M2” mean marker DNA. FIG. 4 is a view showing a step of transforming a Bacillus subtilis cell into which a BAC vector sequence and a marker DNA have been previously inserted using a BAC clone. The Bacillus subtilis BEST310 strain, which has only one cI repressor gene, and the Bacillus subtilis BEST6528 strain, which has two cI repressor genes and has a spacer in between, resulted from the transformation with the BAC clone. This shows that the structures of the resulting Bacillus subtilis strains are the same. In the figure, “cI” indicates the cI repressor gene, “Spc” indicates the spectinomycin resistance gene, “BS” indicates the blasticidin S resistance gene, and “neo” indicates the neomycin resistance gene. The triangle connected before neo indicates the Pr promoter.

Claims (10)

A method for reconstituting genomic DNA constituting a foreign gene into a microorganism genome, comprising the following steps:
(A) A plasmid group in which partial sequences having different total lengths of genomic DNA constituting a foreign gene are inserted as the target DNA into the same vector, and each target DNA has an overlapping sequence with any other target DNA. Process,
(B) a step of transforming a microorganism cell having a genome in which two homologous sequences sandwiching the target DNA in the plasmid have been previously inserted with any plasmid containing the target DNA prepared in the above step (a);
(C) selecting another plasmid containing the target DNA having an overlapping sequence with the target DNA in the plasmid prepared in step (a) and used in step (b),
(D) transforming the microbial cells transformed in step (b) with the plasmid selected in step (c);
(E) From the microbial cells transformed in the above step (d), the target DNAs in the two plasmids used in the above steps (b) and (d) are ligated by homologous recombination at the overlapping sequence and the homologous sequence, respectively. Selecting a transformant having the isolated DNA,
(F) repeating the above steps (c) to (e) and introducing all the target DNAs prepared in the above step (a) into the microbial cells, whereby a genome constituting a foreign gene in the microbial genome; Reconstituting the DNA. A method for reconstituting genomic DNA constituting a foreign gene into a microorganism genome, comprising the following steps:
(A) Step of preparing a plasmid group in which partial sequences having different total lengths of genomic DNA constituting a foreign gene are inserted as target DNAs into a vector, and each target DNA has a sequence overlapping with any other target DNA. ,
(B) Two homologous sequences sandwiching the target DNA in each of the plasmids prepared in the above step (a) are previously inserted into microbial cells having the same genome at the same position in the microbial genome, and the microbial cells are each Transforming with the plasmid of
(C) a step of extracting genomic DNA from a microbial cell having any of the target DNAs in the genome transformed in the step (b);
(D) Transforming microbial cells having another target DNA having a sequence overlapping with the target DNA in the genomic DNA extracted in the step (c) in the genome with the genomic DNA obtained in the step (c). Process,
(E) From the microbial cells transformed in the above step (d), the target DNAs in the two microbial genomes used in the above steps (c) and (d) are, respectively, an overlapping sequence and a common sequence in the microbial genome. Selecting a transformant having DNA linked by homologous recombination,
(F) genomic DNA constituting a foreign gene in the genome of a microorganism by repeating the above steps (c) to (e) and introducing all the target DNAs prepared in the above step (a) into the microorganism. The step of reconstructing. The microorganism genome has two or more cI repressor genes between two homologous sequences, a spacer inserted between each cI repressor gene, and another position on the genome. 3. The method according to claim 1, wherein a marker DNA placed under the control of a Pr promoter is inserted into the DNA. The method according to any one of claims 1 to 3, wherein the selection of the transformant is performed using a marker DNA. The method according to any one of claims 1 to 4, wherein the microorganism used as a host is a bacterium belonging to the genus Bacillus or a thermophile. A microorganism produced by the method according to claim 1. A plasmid comprising the entire length of genomic DNA constituting a foreign gene and having a length of 100 kb or more. A method for producing a genetically modified non-human animal or plant, comprising using the plasmid according to claim 7. What is claimed is: 1. A method for transferring a target DNA from a plasmid having a target DNA of 50 kb or more to a genome of a microorganism, comprising the steps of: transferring a microorganism having a genome in which two homologous sequences sandwiching the target DNA in the plasmid have been inserted in advance; Transformation method. The method according to claim 9, wherein the plasmid is a BAC vector.

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