JP2007124915A - Recombineering construct, and vector for making gene targeting construct - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a recombineering construct, to provide a vector for making a gene targeting construct(vector), and to provide a method for making the vector. <P>SOLUTION: The vector is a plasmid vector, which has two lethal selective marker genes(e.g. SacB gene) and four different cleavage sites(e.g. SfiI cleavage site) recognized by the identical restriction enzyme in proximity to the upstream and downstream sides of the two lethal selective marker genes. Further, another version of the plasmid vector has, in addition to the above-mentioned construct, a selective marker gene such as GFPuv gene. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a vector useful in the field of gene manipulation technology and a method for producing the same. More specifically, the present invention relates to a recombinering construct, a vector for producing a gene targeting (gene target) construct (or vector), a production method thereof, and a gene targeting vector produced using the vector.

  Gene targeting methods by homologous recombination have been widely used so far because target genes can be freely modified. This technique is extremely effective not only for knocking out a target gene but also for general gene modification such as knock-in and introduction of complex constructs.

  Gene targeting vector construction is a step that requires a great deal of labor and time for genetic modification. Conventionally, in order to clone homology arms necessary for homologous recombination from a genomic clone, it has been necessary to go through a screening of a genomic library, creation of a restriction enzyme map, and a number of cloning steps. Recently, since the mouse genome sequence has become available, it has become possible to eliminate the trouble of recreating a wide range of restriction maps of the mouse genome. However, the generation of gene targeting constructs by conventional methods still requires complex cloning steps. In particular, in order to incorporate large-sized components (selection markers, homology arms, etc.), it is necessary to set up a restriction enzyme site that cleaves at an appropriate location, and setting up this restriction enzyme is very complicated and difficult. It was.

  Recently constructed ET recombination (Zhang et al., Nat Genet, 20: 123-128,1998) or recombineering (Copeland et al., Nat Rev Genet, 2: 769-779, 2001) is a recombinogenic technique that is a very effective DNA manipulation technique that does not require the setting of restriction enzyme sites. Since this technique enables modification of a DNA molecule having a large size, an insert derived from a BAC (bacterial artificial chromosome) can be used for the preparation of a gene targeting vector. In order to create a gene targeting vector using recombinogenic technology, a linear chain incorporating components necessary for homologous recombination in E. coli, such as part of the target gene and homologous reagions. It is necessary to prepare a DNA substrate.

  When the size of the homologous region to be incorporated into the linear DNA substrate is short (for example, about 50 bp), it is possible to synthesize a DNA portion corresponding to the homologous region as an oligonucleotide, usually amplifying a selection marker gene, etc. It is given as a PCR primer. In this case, a linear DNA substrate is prepared by PCR, then inserted into a BAC clone using recombinogenic techniques, and homologous recombination is performed in E. coli to produce the desired gene targeting vector. be able to. Such a method is often used to construct a relatively simple gene targeting vector. However, when a linear DNA substrate is prepared by the PCR method, mutations may be introduced into the DNA substrate region at a relatively high frequency during the PCR process. In particular, when preparing a DNA substrate having a long and complex structure including IRES, loxP, and a reporter gene, it is difficult to obtain a desired substrate because mutation is likely to occur.

Angland et al. Succeeded in modifying the λ genomic clone as a result of attempts to avoid the risk of mutations by combining the ET recombination method with the conventional cloning method using ligation (Angrand et al., Nucleic Acids Res , 27: e16, 1999). However, since this method requires two-step modification, a somewhat complicated work is required.
Liu et al. (Non-patent Document 1, Patent Document 1, Patent Document 2, and Patent Document 3) and Valenzuela et al. (Non-patent Document 2, Patent Document 4, and Patent Document 5) introduce homologous regions into linear DNA substrates. Therefore, cloning based on the ligation method was used. They used a 200-500 bp homologous region amplified by PCR or a 50-200 bp homologous region provided as an oligonucleotide. The advantage of this approach is that it avoids the possibility of mutations caused by PCR and eliminates unwanted recombination products with increasing recombination frequency due to longer homologous regions. However, in the case of a linear DNA substrate amplified by PCR, most of the background clones can be removed by cleaving the PCR template with DpnI, whereas the method of Liu et al. It was difficult to effectively remove background clones from the vector. Furthermore, complicated work such as setting a plurality of restriction enzymes (EcorI, BamHI, etc.) having a 6-base recognition site is also necessary.

Liu et al., Genome Res, 13: 476-484, 2003. Valenzuela et al., Nat Biotechnol, 21: 652-659, 2003. WO02 / 14495 US2003 / 0224521 US2004 / 0092016 US2002 / 0106628 US2005 / 0144455

As described above, when producing a gene targeting vector, the work efficiency of the stage of modification to BAC by recombinogenic technique or recombining has been remarkably improved, but the stage before that That is, for the production of a construct used in recombinogenic engineering, it is still necessary to use a complicated method by a conventional method.
Accordingly, an object of the present invention is to provide a vector for producing a gene targeting construct (vector) and a method for producing the vector.
Furthermore, the objective of this invention is providing the gene targeting vector produced using this vector.

  As a result of intensive research on how to easily produce a linear DNA substrate incorporating a homology arm, a selectable marker gene, and a recombinase recognition sequence required in recombinogenic engineering, the present inventors have conducted extensive research. Placing four different cleavage sites recognized by the same restriction enzyme (eg SfiI) in the vicinity of the two negative selectable marker genes (eg SacB gene) and upstream and downstream of these two negative selectable marker genes Thus, it was found that a linear DNA substrate can be prepared in one step, and the present invention was completed.

That is, this invention is the following (1)-(12).
(1) The invention according to the first embodiment of the present invention is such that “two negative selection marker genes, and the same restriction enzyme in the 5 ′ upstream and 3 ′ downstream sides of the two negative selection marker genes” A “plasmid vector having four different cleavage sites to be recognized”.
(2) The invention according to the second embodiment of the present invention is “the plasmid vector according to (1) above, wherein the negative selection marker gene is a SacB gene”.
(3) The invention according to the third embodiment of the present invention is “the plasmid vector according to (1) or (2) above, wherein the restriction enzyme is SfiI”.
(4) The invention according to the fourth embodiment of the present invention is “a plasmid vector according to any one of (1) to (3) above, which further comprises a selectable marker gene”.
(5) The invention according to the fifth embodiment of the present invention is “the plasmid vector according to any one of (1) to (4) above, wherein the further selectable marker gene is a GFPuv gene”.
(6) The invention according to the sixth embodiment of the present invention is the above (1) to (1) further comprising a restriction enzyme recognition site for linearizing the plasmid vector incorporating a target region and two homologous regions. (5) A plasmid vector according to any one of (5).
(7) The invention according to the seventh embodiment of the present invention is “a plasmid vector according to any one of (1) to (6) above, wherein the restriction enzyme is I-sceI”.
(8) The invention according to the eighth embodiment of the present invention is “a plasmid vector according to the above (1) selected from pADY, pAEF, and pADW”.
(9) The invention according to the ninth embodiment of the present invention is “the plasmid vector according to any one of (1) to (8) above, wherein the negative selection marker gene is replaced with a homologous region”.
(10) The invention according to the tenth embodiment of the present invention is "a method for producing a gene targeting vector using the plasmid vector according to any one of (1) to (9) above".
(11) The invention according to the eleventh embodiment of the present invention is “a gene targeting vector produced by the method according to (10) above”.
(12) The invention according to the twelfth embodiment of the present invention is “the gene targeting vector according to the above (11) selected from pAGE, pAGF, pAGI, pAGJ, and pAHV”.

  By using the vector of the present invention, a linear DNA substrate retaining components such as a homology arm and a selection marker gene necessary for constructing a gene targeting vector can be prepared in one step.

  Furthermore, in this one-step cloning process, complicated restriction enzyme setting work can be avoided and the appearance of background clones can be suppressed without going through a purification process such as gel filtration. The time and labor required for construction can be reduced.

  Further, by allowing the vector of the present invention to hold a further selection marker gene such as a GFPuv gene, selection of a plasmid carrying a linear DNA substrate and selection of a recombinant in the process of recombinogenic engineering can be easily performed. be able to.

  From the above points, by using the vector of the present invention, high-throughput genetic modification can be performed efficiently and simply. In particular, it is effective for constructing a gene targeting vector having a complicated structure.

The present invention relates to a vector, and when this vector is used, it is possible to construct a homologous region necessary for recombining and gene targeting in one step. Specifically, the vector of the present invention has two negative selectable marker genes and a position so as to sandwich the two negative selectable marker genes (that is, a number base to 5 ′ upstream and 3 ′ downstream of the marker gene). Different recognition sequences that are recognized by the same restriction enzyme are arranged at two locations, a total of four locations, at positions separated by several hundred bases.
Here, the “negative selection marker gene” may be nutrient selective or drug selective, and can distinguish a host cell carrying a plasmid inserted with the marker gene under a specific condition. Any gene can be used as long as it is such a gene. The marker gene can be easily selected by those skilled in the art. For example, a SacB gene, an rpsL gene, a ccdB gene, and the like can be preferably used. Since the SacB gene produces a lethal metabolite from E. coli from sucrose, E. coli carrying the SacB gene cannot grow in the presence of sucrose. Therefore, by culturing the transformant in a sucrose-containing medium, only the target clone in which the SacB gene is replaced with the homologous region can be selected. Further, at least two marker genes are present in one vector according to the present invention. The marker gene is necessary to confirm whether two homologous regions have been cloned. The desired homologous recombination cannot be achieved with only one homologous region. Here, the “homologous region” is a region necessary for homologous recombination in E. coli or ES cells, and is a DNA sequence region that is homologous to a part of the DNA region to be recombined.

In addition, “different recognition sequences recognized by the same restriction enzyme” means a sequence that causes non-identical and non-complementary 3 ′ or 5 ′ overhangs (protruding ends) when cleaved. That is. When such a recognition sequence is cleaved at multiple sites, ligation does not occur between the cleavage sites, and both ends of the homologous region DNA fragment to be cloned into the cleavage sites are designed to be complementary to the cleavage sites. Then, the DNA fragment can be inserted in a predetermined direction. Therefore, when cloning homologous regions, self-ligation of only the vector and only the insert can be avoided, and the cloning can be achieved by a one-step ligation reaction by using only one kind of enzyme.
Furthermore, it is desirable that an enzyme capable of recognizing such a different cleavage sequence is a rare cutter when cloning a homologous region. In particular, when cloning a long homologous region, a rare cutter restriction enzyme that can be excised without cleaving the homologous region itself can be suitably used.
Any restriction enzyme can be used in the present invention as long as it has the above-mentioned properties and is not particularly limited. For example, SfiI, XcmI, BstXI, BbsI, BsaI and the like can be suitably used. SfiI recognizes a palindromic sequence interrupted with 5 bases (5′-GGCCNNNNNGCC-3 ′; SEQ ID NO: 1), and after cleavage, a non-identical and non-complementary 3 nucleotide 3 ′ overhang is generated It is an enzyme. SfiI is a rare cutter and is therefore a preferred enzyme for use in the present invention.

  The vector of the present invention comprises a homologous region, a selectable marker gene, and / or a component necessary for conditional gene targeting (for example, recombinase recognition sites such as loxP and FRT sequences), etc. Can be used in the process of combinogenic engineering. When making it linear, it is desirable to make it linear without cut | disconnecting the said component. Therefore, the vector of the present invention may have a restriction enzyme recognition site for linearization after constructing the constituent elements. As such an enzyme, a rare cutter having a long recognition sequence is desirable. For example, meganuclease I-SceI, I-CeuI, PI-SceI, or NotI which is a rare cutter for 8-base recognition is preferably used. Is possible.

The vector of the present invention may contain an additional selection marker gene. “Further selectable marker gene” means a marker gene that can function as both a “positive selectable marker” and a “negative selectable marker”. That is, in the cloning process, a clone containing “an additional selectable marker gene” is selected in one step, and a clone not containing an “additional selectable marker gene” is selected in another step, thereby achieving the purpose of cloning. It is a gene.
The “further selectable marker gene” is not limited, and for example, galK, lacZ, GFPuv and the like can be used. In particular, GFPuv is preferred. GFPuv is a variant of GFP, but emits maximum fluorescence when excited with uv. The GFPuv gene functions as a positive selection marker when selecting a plasmid immediately after the construction of a homologous region, a target region, etc., but if it is designed so that the GFRuv gene is removed after linearization, When selecting a clone in which homologous recombination was correctly performed in the process of genetic engineering, it can be used as a negative selection marker. That is, in order to select a plasmid immediately after the construction of a homologous region, a target region, etc., it is only necessary to select a clone that emits fluorescence when UV irradiation is applied to a UV illuminator or the like. When selecting a clone in which homologous recombination was correctly performed in the process, a gene targeting vector can be finally obtained by selecting a clone that does not emit fluorescence. Here, the “target region” includes components to be incorporated into the target genomic region to be targeted (for example, any gene to be incorporated, a selection marker gene, a homology arm, a recombinase recognition sequence such as loxP or FRT cassette, etc.). It means DNA.

  Gene targeting vectors produced by the method of the present invention are also included in the scope of the present invention. Here, “gene targeting” is a concept that is most widely recognized by those skilled in the art, and means not only knockout of a specific gene but also general gene modification such as knock-in and conditional gene targeting. The gene targeting vector of the present invention can be easily prepared by those skilled in the art by using the gene targeting construct production vector of the present invention. In the production of a vector for conditional gene targeting, a commercially available template can also be used for the production of components contained in the construct to be introduced.

  The vector of the present invention can be used for normal cloning such as components necessary for efficient replication in E. coli (for example, an origin of replication) and multiple cloning sites required for efficient cloning. And all constituents deemed necessary by those skilled in the art. In preparing the vector of the present invention, a known plasmid vector can be used as the backbone. Any known plasmid vector may be used as long as it can be used in the technical field. For example, pBR322, pBluescript, and pUC plasmids can be used.

  Next, the present invention will be described with reference to specific examples, but the present invention is not limited to these examples.

The gene manipulation technique used in this example is based on a method commonly used by those skilled in the art (Sambrook et al., Molecular cloning: A laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Habor, NY. 1989). .
1. Description of the vector of the present invention The novel cloning vectors constructed in the present invention (FIG. 1, pADY (SEQ ID NO: 53), pAEF (SEQ ID NO: 54), pADW (SEQ ID NO: 55)) can clone homologous regions in one step. Designed. In this example, the SfiI site (5′-GGCCNNNNNGCC-3 ′, SEQ ID NO: 1) was introduced into these vectors based on the following two reasons. The first reason is that when an SfiI recognition site having an interrupted palindromic structure is cleaved by SfiI, a non-identical and non-complementary 3 nucleotide 3 ′ overhang is generated. If the sequences at both ends of the homologous region DNA fragment are designed to be complementary to the SfiI recognition sequence to be inserted, the DNA fragment will be in the desired position and orientation (see the top of FIG. 1 and FIG. 3). Can be cloned. In addition, each end of the four SfiI sites introduced into the vector is generated by cleavage of the same SfiI enzyme, but is not complementary or self-complementary, so self-ligation by only the vector or only the insert does not occur. Accordingly, the two homologous regions can be cloned easily and rapidly by a one-step ligation reaction, and no other enzyme than SfiI is required. The second reason is that since SfiI is a rare cutter restriction enzyme, it is suitable for cloning of a relatively long DNA fragment that can function as a homology region. In this example, four SfiI sites were introduced into a pBluescript-based vector.
In addition, these vectors carry two SacB genes, each sandwiched between two SfiI sites (FIG. 1). The SacB gene product is called levansucrase and converts sucrose to levan which is toxic to host cells (Gay et al., J Bacteriol, 164 (2): 918-921, 1985). Therefore, the SacB gene is useful as a negative selection marker. The SacB gene can be easily replaced with a homologous region during the cloning process (FIG. 3).

1-1. pADY Vector The pADY vector (FIG. 1) was developed for the construction of a linear DNA substrate used in the recombinogenic cloning method. In this vector, a multicloning site (including AscI, XbaI, SpeI and NheI) is arranged. XbaI, SpeI and NheI sites give the same sticky ends (5′-CTAG-3 ′, SEQ ID NO: 2). After the homology region and target fragment were constructed in the vector (eg, pAFD in FIG. 3), the plasmid was cleaved with the meganuclease I-SceI to produce a ready-to-use linear DNA substrate. In early recombinogenic engineering using a PCR-amplified linear DNA substrate, the appearance of background clones derived from PCR template plasmid contamination could be suppressed by cleaving the template with DnpI. However, in this example, since a linear DNA fragment derived from a plasmid was used as a substrate, such an approach was not practical. Therefore, as an alternative, the GFPuv gene was introduced 3 ′ downstream of the lac promoter of pADY into the vector of the present invention (FIG. 1). Only colonies carrying pADY and pAEF with the GFPuv gene fluoresced by UV lamp irradiation (FIG. 2).

1-2. pAEF Vector The pAEF vector (FIG. 1) can be used to construct a gene targeting vector by subcloning BAC clones using the recombinogenic cloning technique. A vector obtained by replacing the SacB gene in the pAEF vector with a homologous region (for example, pAFQ in FIG. 5) was digested with AscI and used for BAC subcloning. The GFPuv cassette contained in pAEF can be used to remove GFPuv negative non-recombinant clones. In addition, a diphtheria toxin fragment A (DT-A) cassette, which is a negative selection marker used for gene targeting of ES cells, was introduced into pAEF. In order to linearize the obtained gene targeting vector, an I-SceI recognition site was arranged.

1-3. pADW vector pADW (FIG. 1) was developed for the generation of gene targeting constructs by conventional PCR-based methods. PCR amplified homology arms were introduced into pADW (eg, pAIP in FIG. 5). In this case, since a longer homology arm promotes efficient homologous recombination in ES cells, the length of the homology arm needs to be sufficiently long (-10 kb). Similar to pADY, AscI, XbaI, SpeI and NheI enzyme sites and DT-A cassette are included in pADW.

1-4. pAEJ and pAEK vectors pAEJ (SEQ ID NO: 56) and pAEK vector (SEQ ID NO: 57) (FIG. 1) are plasmids for introducing SfiI sites at both ends of the homologous region. PCR amplified homologous regions with AscI sites at both ends can be cloned into the MluI sites of these vectors after AscI cleavage. Since AscI is a rare cutter restriction enzyme, longer DNA fragments can be cloned into these vectors and used as homologous regions.

2. Construction of vector of the present invention All restriction enzymes used in the practice of the present invention were purchased from New England BioLab (NEB). To construct pAEJ and pAEK, the multicloning site of pUC119 was replaced with SfiI and MluI sites. Each SfiI site was designed to give non-identical sticky ends. pADY, pADW and pAEF were constructed by multiple steps. The backbone of these vectors is pBluescript, except for multiple cloning sites other than the KpnI site. Multiple cloning sites (AscI, XbaI, SpeI and NheI) are provided as oligonucleotide linkers (SEQ ID NO: 3 and SEQ ID NO: 4). The SacB gene used is derived from pDNR-Dual (Clontech). The SacB gene having SfiI sites on both sides was amplified by PCR using the following primers as a pDNR-Dual template, cut into BamHI / EcoRI, and introduced into the pUC119 vector. The SacB cassette was then used for construction in pADY, pADW and pAEF using BbsI, I-SceI and XhoI.
GE-F2 for pADY (SEQ ID NO: 5)
GE-R3 (SEQ ID NO: 6)
GE-F4 for pAEF (SEQ ID NO: 7)
GE-R2 (SEQ ID NO: 8)
GE-F1 for pADW (SEQ ID NO: 9)
GE-R1-2 (SEQ ID NO: 10)

The GFPuv gene in pAEF was amplified from pGFPuv (Clontech) using the following primer set, cut into AscI, and then introduced into the vector.
GE-F3 (SEQ ID NO: 11)
GE-R5 (SEQ ID NO: 12)
The GFPuv gene in pADY was amplified from pGFPuv (Clontech) using the following primer set, and the PCR amplification product was cleaved with BstI and then introduced into the KpnI site of the vector.
F (SEQ ID NO: 13)
R (SEQ ID NO: 14)
The GFPuv gene in pADY is arranged so that the reading frame fits under the control of the lac operator sequence of pBluescript. The DT-A gene in pADW and pAEF is derived from the HindIII / XhoI fragment of the pMC1DTpA vector (12).

3. 1. One-step cloning of homologous region 3-1. Results A one-step cloning procedure for homologous regions using the vectors disclosed by the present invention is shown in FIG. The homologous region is first inserted into pAEJ and pAEK (FIG. 3-a). The homologous region is also provided as a double-stranded oligo (FIG. 3-b) or a PCR amplified fragment (FIG. 3-c) with a sticky end complementary to the SfiI site of the vector. The SfiI digested vector (pAEY in FIG. 3) can be directly ligated in a single reaction with both SfiI digested HR1 (homology region 1) and HR2 (homology region 2). Using sticky ends generated by non-identical SfiI cleavage yields correctly ligated clones such as pAFD (FIG. 3).

  In order to evaluate whether the SacB gene and the SfiI site present in the vector of the present invention function efficiently in the cloning of the homologous region, the cloning efficiency was examined (Table 1). In this experiment, different concentrations of homology region (HR) supplied as a 50 bp-double stranded oligo were used for ligation with the SfiI cleaved pAEY vector containing the target fragment (FIG. 3). The sequence of the oligo is homologous to that of the H2-M5 locus, which is one of the nonclassical class I MHC molecules. The cloning efficiency of the homologous region was compared between the vector with and without gel filtration, and the LB plate with and without sucrose (Table 1). When higher concentrations of oligo were used as homologous regions, most of the resulting clones were correct clones that retained the homologous region, even without sucrose. Many background clones were generated when a lower concentration of oligo was ligated with a non-gel filtration vector and the transformants were cultured in the absence of sucrose, but were significantly reduced by culturing in the presence of sucrose. did. Under this condition, all selected recombinant clones contained both homologous regions in the correct position and in the correct orientation (Table 1, kan / amp / suc). Even when a 400-600 bp PCR amplified fragment was used as a source for obtaining a homologous region (FIG. 3-c), good results were obtained.

  When the DAN fragment supplying the homologous region was supplied from the insert of pAEJ and pAEK (FIG. 1), the GFPuv gene encoding the mutant green fluorescent protein contained in pADY and pAEF effectively functioned as a positive selection marker. Table 2 shows the effect of using the GFPuv gene as a positive selection. In this experiment, pAFE and pAFF retaining HR1 and HR2 of the H2-M5 locus (Takada et al., Genome Res, 13 (4): 589-600, 2003), respectively, were used in pAEJ and pAEK (Table 3). reference). Gel filtration was not performed on homologous regions prepared by cleaving pAFE and pAFF with SfiI. These fragments were ligated with SfiI digested vectors pAEF (FIG. 1) or pAEY (FIG. 3) that were not gel filtered or dephosphorylated. Therefore, a vector backbone derived from pAFE and pAFF that can cause many background clones to appear was also included in the ligation mix. pAEY derived from pADY contains a complex insert containing neo that confers resistance to kanamycin in E. coli (FIG. 3). When transformants were selected in the presence of ampicillin and sucrose, a number of background clones derived from uncleaved and / or self-flyed pAFE and pAFF vectors were generated. These background clones can be avoided by introducing other selectable markers such as zeocin instead of using the β-lactamase gene for pAEJ and pAEK. Alternatively, the correct clone can be efficiently selected from the background clones by the presence or absence of green fluorescence emitted by the GFPuv gene contained in the vector of the present invention. In this experiment, after GFP fluorescence selection, more than 1/2 clones (GFP +) were found as correct clones (Table 2). This result indicates that the GFPuv gene is useful as a positive selection marker in cloning homologous regions. In particular, background clones derived from the pAEY vector could be completely removed by selection with kanamycin.

    The vector of the present invention could also be used effectively for cloning a long homologous region of 5 kb or more (FIGS. 4 and 5). Therefore, it shows that the vector of the present invention can also be used for integration of a long homologous region that can be directly used for homologous recombination in ES cells. This method makes it possible to construct a gene targeting vector within one week.

3-2. Methods HR1 (homology region 1), HR2 (homology region 2), HR3 (homology region 3) and HR4 (homology region 4) for the construction of H2-T3TV (H2-H3 gene targeting vector) are synthesized using KOD-plus DNA polymerase ( Using TOYOBO, a BAC clone (citb585c7 [AF532116] (Takada et al., Genome Res, 13 (4): 589-600, 2003)) was used as a template for PCR amplification with the following primers.

T3-uHOM-F for HR1 (SEQ ID NO: 15)
T3-uHOM-R (SEQ ID NO: 16)
T3-dHOM-F for HR2 (SEQ ID NO: 17)
T3-dHOM-R (SEQ ID NO: 18)
T3-uHOM-TVET-F for HR3 (SEQ ID NO: 19)
T3-uHOM-TVET-R (SEQ ID NO: 20)
T3-dHOM-TVET-F for HR4 (SEQ ID NO: 21)
T3-dHOM-TVET-R2 (SEQ ID NO: 22)

  The amplified fragment is used to generate pAFB (HR1), pAFC (HR2), pAFI (HR3), pAFO (HR4), and the MluI site (dephosphorylated) of pAEJ (HR1 and HR3) or pAEK (HR2 and HR4). Inserted) using AscI. The DH5α strain (TAKARA) retaining pAEY and pAEF was cultured overnight in LB medium supplemented with ampicillin. The plasmid was isolated from 4 ml of the culture solution by the alkaline method and finally dissolved in 50 μl of TE (10 mM Tris, 0.1 mM EDTA). 3 μl of this lysate was reacted in 100 μl reaction solution with 40 U SfiI at 50 ° C. for 3 hours. After the reaction, pAEY and pAEF cleaved with SfiI were phenol extracted, recovered by ethanol precipitation, and dissolved in 2 μl of water. For pAFB, pAFC, pAFI and pAFO, SfiI cleaved samples were gel-electrophoresed, the inserts were excised, extracted with GeneElute Extraction Kit (Sigma), and finally dissolved in 2 μl of water. To make pAFD, 0.15 μl of pAEY sample cut with SfiI and 0.15 μl of each sample containing inserts derived from pAFB and pAFC cut with SfiI were used with 36 U T4 DNAligase in the presence of 5% PEG8000. And ligated in a total volume of 0.9 μl (FIG. 3). After reacting at 16 ° C. for 30 minutes, DH5α was transformed with 10 μl of the ligated sample. After reacting in SOC medium at 37 ° C for 1 hour, 1/2 of the sample was plated on an LB plate supplemented with 100 µg / ml ampicillin, 25 µg / ml kanamycin, 7% sucrose, and incubated at 37 ° C. Incubated overnight.

  The next day, a large number (> 300) of colonies (> 98%) emitting fluorescence by GFPuv appeared. Eight colonies were selected from these colonies, and it was confirmed by PCR and sequencing that the correct insert was inserted. As a result, all the colonies checked were correct colonies, and one of them (pAFD) was used for the next recombining. PAFQ, a subcloning construct, was ligated with pAEF cleaved with SfiI and pAFI cleaved with SfiI and pAFO cleaved by the same procedure as that for pAFD except that ampicillin and sucrose were present. The cloning efficiency of pAFQ was almost the same as that of pAFD. pAFD and pAFQ were used for production of H2-T3TV (pAFP) (FIG. 5-a).

  Another H2-T3TV (pAIP) was also generated by standard ligation based cloning using pAEX. Long and short homology arms (FIG. 5-c) were PCR amplified from BAC clones with KOD-plus DNA polymerase (TOYOBO) using the following primers.

For long arm T3-uHOM-ADW-F2 (SEQ ID NO: 23)
T3-uHOM-ADW-R2 (SEQ ID NO: 24)
For short arm T3-dHOM-ADW-F2 (SEQ ID NO: 25)
T3-dHOM-ADW-R2 (SEQ ID NO: 26)

  The amplified fragment was cleaved with AscI and cloned into pAEJ and pAEK to give pAIH and pAIG. From these plasmids, the homology arm region was excised with SfiI and purified on an agarose gel. Both homology arms were ligated in one step with SfiI cleaved pAEX (without gel filtration and dephosphorylation) to give pAIP (FIG. 5-b).

The procedure of the experiment shown in Table 1 is shown below.
A 15 bp oligo homologous to the H2-M5 locus was used as a source of homologous regions. The sequence of the oligo is as follows.
M5-u-1 for HR1 (SEQ ID NO: 27)
M5-u-2 (SEQ ID NO: 28)
M5-d-1 for HR2 (SEQ ID NO: 29)
M5-d-2 (SEQ ID NO: 30)

  Each oligo was dissolved in TE (10 mM Tris, 0.1 mM EDTA) to a concentration of 300 μM. 8 μl M5-u-1 and M5-u-2 (HR1) or M5-d-1 and M5-d-2 (HR2) were mixed with 2 μl water, 2 μl 10 × T4 polynucleotide kinase buffer (NEB). The total volume was 20 μl. These oligo solutions were denatured at 96 ° C. for 5 minutes, cooled to room temperature, and diluted with water (1: 100, 1: 1000, 1: 10000). Two samples (about 100 ng each) corresponding to 3/50 of pAEY isolated from 2 ml of the overnight culture sample were cut with 40 U SfiI at 50 ° C. for 3 hours. After phenol extraction and ethanol precipitation, one sample was dissolved in 2 μl of water and used for ligation (see “No gel filtration” in Table 1). In contrast, other samples were electrophoresed on a low temperature dissolved agarose gel using 1 × TAE. The SfiI fragment having the vector sequence and the 4.3 kb region was excised from the gel and purified with GeneElute Kit (Sigma). The SfiI fragment (1.9 kb) containing the SacB gene was not excised from the gel. The purified DNA was ethanol precipitated, dissolved in 2 μl of water, and used for ligation (gel filtration in Table 1).

  SfiI digested vector (0.3 μl) was used in a total volume of 1.8 μl using 72 U T4 DNAligase in the presence of 0.3 μl diluted HR1, 0.3 μl diluted HR2 and 5% PEG8000. Ligated. The concentration of HR (oligo) in the ligation mixture was 200, 20, and 2 nM, respectively. A sample containing only a vector to which no fragment of the homologous region was added also reacted as a negative control. Ligation was performed at 16 ° C. for 30 minutes. 20 μl of DH5α (TaKaRa) was transformed with each ligated sample. After incubation at 37 ° C. in LB fold, 1/5 samples were plated on LB plates containing ampicillin (100 μg / ml), kanamycin (25 μg / ml) and sucrose (7%). After counting the number of colonies, 8 colonies were selected from each plate and PCR analysis was performed.

The correctly inserted homologous region was confirmed by PCR using the following primers.
M5-dHOM-F (SEQ ID NO: 31)
M13F (SEQ ID NO: 32)
as well as,
PKG-R2 (SEQ ID NO: 33)
M13R (SEQ ID NO: 34)
as well as,
Globin-F2 (SEQ ID NO: 35)
M13F (SEQ ID NO: 32)

The procedure of the experiment shown in Table 2 is shown below.
HR1 and HR2 (corresponding to pAFE and pAFF, respectively) at the H2-M5 locus were used. In order to prepare these homologous regions, a BAC clone (citb544e14 [AF532114]) was used as a template, and KOD-plus PCR was performed with the following primers.
M5-uHOM-F for pAFE (SEQ ID NO: 36)
M5-uHOM-R (SEQ ID NO: 37)
M5-dHOM-F for pAFF (SEQ ID NO: 31)
M5-dHOM-R (SEQ ID NO: 38)

  The amplified fragment was cut with AscI, precipitated with ethanol, gel-filtered, and ligated with pAEJ or pAEK to finally produce pAFE and pAFF. A 1/25 volume of plasmid DNA (pAEF, pAEY, pAFF and pAFF) from each 4 ml overnight culture was cleaved with 40 U SfiI at 50 ° C. for 3 hours for a total volume of 100 μl. After ethanol precipitation, all samples were dissolved in 2 μl of water. Prior to ligation, all samples were not gel filtered and dephosphorylated. PAEF or pAEY digested with SfiI (0.15 μl each) was prepared using 36 U T4 DNAligase in the presence of 0.15 μl SfiI digested pAEF and 0.15 μl SfiI digested pAFF and 5% PEG8000. Ligated in 9 μl.

After ligation at 16 ° C. for 30 minutes, 10 μl of DH5α (TaKaRa) was transformed with each ligated sample. Each sample was diluted with SOC medium, and a part of them was plated on an LB plate containing antibiotics shown in Table 2. After counting the number of colonies, the expected number of colonies obtained from the total amount of transformed samples was calculated and shown in Table 2. The fluorescence of GFPuv was confirmed using a long wavelength UV lamp, and the correctly inserted homologous region was confirmed by colony PCR using the following primers.
Globin-F2 (SEQ ID NO: 35)
M13F (SEQ ID NO: 32)
as well as,
GFPuv-F (SEQ ID NO: 39)
M5-uHOM-R (SEQ ID NO: 37)
as well as,
DT-seq (SEQ ID NO: 40)
M13R (SEQ ID NO: 34)

4). Application example of vector of the present invention for gene targeting vector construction 4-1. Results One of the simplest methods for introducing a homology arm into a gene targeting vector is to clone a PCR amplified fragment derived from genomic DNA. pADW is a vector designed for such a method. Using pAEX produced from pADW (FIG. 5-b), a gene targeting vector having a homology arm corresponding to the H2-T3 locus was constructed (FIG. 4, 5-b, c). This method has been found to be useful for cloning longer homologous regions, including a 6.5 kb long arm and a 1.4 kb short arm using the SfiI site. However, the homology arms obtained by PCR are often mutated and do not cause unexpected phenotypes in the resulting knockout mice. In addition, when performing long PCR with high accuracy on genomic DNA, there is also a problem that a target region of 5 kb or more cannot often be amplified. Therefore, in the following experiment, a mouse BAC clone capable of obtaining a homology arm without mutation was used.

  The conventional method for constructing a gene targeting vector using a restriction enzyme without using the PCR method largely depends on whether or not an appropriate restriction enzyme site exists on the genomic DNA. On the other hand, recombinogenic engineering is based on homologous recombination in E. coli and enables efficient and simple modification of BAC efficiently. Since it is not necessary to examine an appropriate restriction enzyme site on genomic DNA, it seems to be an effective method for constructing a gene targeting vector. By combining the vectors of the present invention, pADY and pAEF, in a recombinogenic engineering system, a rapid and simple gene targeting vector could be constructed (FIG. 5).

In this experiment, first, a 4.3 kb construct (containing a recombinase recognition site such as ECFP as a selection marker and several site-specific mutant loxP sites) was introduced into the cloning site of pADY, and pAEY was introduced. It produced (FIG. 3). pAEY was prepared to induce not only knockout but also a large deletion or insertion mutation in the H2-T3 genomic region.
In the recombinogenic engineering method, DY380 E. coli strain was used as a host, and pAFD and pAFQ were used for the construction of gene targeting vectors. In this experiment, two-step recombining as shown in FIG. Either of the two steps can be performed first, but if step 1 is performed first as in this example, the pattern of the genomic Southern is checked in the early stage of the experiment using wild type and modified BAC. It is extremely effective when the complete sequence of the BAC clone is unknown.

In this method, in constructing the gene targeting vector, the GFPuv gene was used as a negative selection marker in order to remove the background caused by the vector from which the linear DNA substrate was derived (FIGS. 5-a and c). Whether the obtained recombinant clone and H2-T3TV were the target was confirmed by PCR, sequencing (sequencing) and restriction enzyme analysis (FIG. 6).
By using the vector of the present invention, all method steps including one-step cloning of homologous regions and construction of gene targeting vectors could be completed in about 2 weeks.

4-2. Method A 4.3-kb fragment containing three site-specific recombination sites introduced into the H2-T3 locus, the neomycin resistance gene (neo), IRES and ECFP, was generated by ligation based cloning through multiple steps. did. Three site-specific recombination sites, FRT, mutant lox (mlox: JT15 (Thomson et al., Genesis, 36 (3): 162-167, 2003)), and lox2272 were generated by ligation-based cloning using oligos. . The generated fragment was cloned into pUC119 and used as a cassette. The neomycin resistance gene expressed by the PGK promoter is derived from pPGK-neo-loxP (Gene Bridges). The pCAGGS-FLPe and IRES sequences were amplified by a pair of D1F primer (SEQ ID NO: 41) and D1R primer (SEQ ID NO: 42), cloned into the pUC119 vector, and used as an IRES cassette. The ECFP cassette was prepared from the pECFP-nuc vector (Clontech) by cloning a PCR fragment amplified by a pair of B1F primer (SEQ ID NO: 43) and EYFP-Nuc-R1 primer (SEQ ID NO: 44). The poly A sequence (rabbit β globin poly A) is amplified from the pCGN2Δ2 vector with a Globin-F primer (SEQ ID NO: 45) and Globin-R primer (SEQ ID NO: 46) pair, introduced into pBluescript (SK +), Used as a cassette. All nucleotide cassettes were sequenced by sequencing. These cloning cassettes were used for the complex 4.3 kb construct contained in pAEY (FIG. 2). The 4.3 kb fragment was also inserted into pADW; the resulting plasmid is called pAEX.

The recombining method based mainly on the method of Liu et al. (Liu et al., Genome Res, 13 (3): 476-484, 2003) was applied to construct H2-T3 TV.
First, the citb585c Bac clone (CITB library derived from the 129 / SV mouse strain, Research Genetics) was introduced into the DY380 E. coli host strain (received by Dr. Neal G. Copeland) by electroporation and transformed. It was confirmed by restriction enzyme analysis that the prepared BAC had the correct insert.
DY380 cells containing citb585c Bac were inoculated into 5 ml of LB medium and cultured at 30 ° C. overnight. On the next day, 1 ml was collected from the overnight culture, added to 20 ml of SOB, and cultured with shaking until the value of OD600 reached 0.6. Next, 10 ml of the cells were transferred to a new tube, shaken at 42 ° C. for 15 minutes, and then cooled in ice water for 15 minutes. The cells were centrifuged at 2500 rpm for 10 minutes at 0 ° C. The resulting cell pellet was suspended in 888 μl of ice-cold 10% glycerol and transferred to a 1.5 ml Eppendorf tube. After washing 3 times with 10% glycerol, the precipitate was resuspended in 100 μl ice-cold 10% glycerol and used for electroporation of the linear DNA substrate. To make a linear DNA substrate, 3/50 amount of pAFD or pAFQ obtained from 4 ml overnight culture was cut with I-SceI or AscI, extracted with agarose gel, and finally 2 μl of water Dissolved in. 1 μl of the sample dissolved in water was mixed with 50 μl of competent cells, and transformation was performed by electroporation (GenePulser; BioRad, 1.75 kV, 25 μF, using a 200Ω controller). Thereafter, 1.0 ml of SOB was added to each cuvette and incubated at 30 ° C. for 1 hour. Cells were spread on plates in the presence of appropriate antibiotics.

Correctly recombined clones were screened by PCR using the following primers.
Step 1
T3-uHOM-check-F (SEQ ID NO: 47)
PKG-R2 (SEQ ID NO: 33)
as well as,
T3-dHOM-check-R2 (SEQ ID NO: 48)
Globin-F2 (SEQ ID NO: 35)
Step 2
DT-seq (SEQ ID NO: 40)
Globin-F2 (SEQ ID NO: 35)
And M13R (SEQ ID NO: 34)
T3-dHOM-check-R (SEQ ID NO: 49)

  Regarding the modified BAC clone prepared in Step 1, it was confirmed that the correct recombination was performed by the BglI cleavage pattern and Southern blot analysis using the probe used for detection of the ES target clone described below. The resulting gene targeting vector (pAFP) was confirmed by sequencing (sequencing).

5. Gene targeting in mouse ES cells 5-1. Results The obtained H2-T3TV was cleaved at the unique site I-SceI site and electroporated into E14.1 and E14tg2aES cells. Seven homologous recombinant clones were screened by PCR from 278 G418 resistant colonies. Furthermore, it was confirmed by genomic Southern hybridization analysis whether the obtained clone was the target recombinant (FIG. 6). Using these clones, we have created chimeric mice and confirmed their transmission to the germline.

5-2. Method pAFP, an H2-T3TV prepared by recombinogenic engineering, was linearized with I-SceI. Twenty μg of linear gene targeting vector was applied to 1 × 10 ⁷ E14.1 (Kuhn et al., Science, 254 (5032): 707-710, 1991) and 1 × 10 ⁷ E14tg2a (Hooper et al., Nature, 326 (6110 ): 292-295, 1987) It was introduced into ES cells by electroporation. First, G418 resistant ES cells were screened by PCR using the following primers.

Globin-F2 (SEQ ID NO: 35)
T3-dHOM-TVET-R (SEQ ID NO: 50)
Correctly targeted clones were confirmed by Southern blot analysis using a probe prepared by labeling a fragment amplified from BAC DNA using the following primers with 32P by the random priming method.
T3-short-probe-F (SEQ ID NO: 51)
T3-short-probe-R (SEQ ID NO: 52)

  Two clones derived from E14tg2aES cells were microinjected into (C57BL / 6JxBDF1) F1 mouse blastocysts. A germline chimera was obtained from one of the targeted ES clones (3D2, FIG. 6).

6). Vectors useful for normal gene targeting and conditional gene targeting 6-1. Gene Targeting Vector of the Present Invention From the results shown in detail above, it was revealed that the vectors of the present invention, pADY and pADW, are useful plasmids when constructing a gene targeting vector.
The above-mentioned H2-T3TV contains neo, ECFP gene, and three site-specific recombination sites, and is designed for a specific purpose. Therefore, other gene targeting vectors can be used for more general purposes, such as the production of a normal knockout mouse or a conditional knockout mouse. Other vectors can also be produced simply and quickly by using the vector of the present invention. The vectors derived from pADY useful for the construction of general gene targeting vectors are shown in Table 3 (pAGE (SEQ ID NO: 58), pAGF (SEQ ID NO: 59), pAGI (SEQ ID NO: 60), pAGJ (SEQ ID NO: 61), pAHV (SEQ ID NO: 62)). These vectors can be used for normal gene targeting or conditional gene targeting.

6-2. Production of gene targeting vector of the present invention 6-2-1. Preparation of pAFZ In preparation of pAGE, pAGF, pAGI, pAGJ, and pAHV, PCR was performed using PGK-neo-loxP (GeneBridges) as a template with the following primers, and the resulting amplification product was cleaved with XbaI and EcoRI. XBaI of pACS (a pBluescript-based vector from which MCS other than KpnI has been removed and a linker containing a restriction enzyme site such as I-SceI, SfiI, NheI, StyI, SpeI, XbaI, etc. has been introduced) And inserted into the NheI site (pAFZ).
PGK-neo-F (SEQ ID NO: 63)
PGK-neo-R (SEQ ID NO: 64)

6-2-2. Preparation of loxP cassette (pAFY) loxP-F1 (SEQ ID NO: 65)
loxP-R1 (SEQ ID NO: 66)
loxP-F2 (SEQ ID NO: 67)
loxP-R2 (SEQ ID NO: 68)
PCR is performed without template using loxP-F1 and R1, loxP-F2 and R2 to create primer dimers. As a result, DNA fragments having the following sequences were prepared.
loxP-1 (SEQ ID NO: 69)
loxP-2 (SEQ ID NO: 70)

  Next, loxP-1 was cleaved with XbaI and BbsI, loxP-2 was cleaved with EcoRI and BbsI, and introduced into the XbaI / EcoRI site of pBluescript by 3-fragment ligation. The sequence of the created insert is XbaI_loxP_StyI_loxP_NheI_EcoRI (SEQ ID NO: 71), and the resulting vector is called pAFY.

6-2-3. Preparation of FRT cassette (pA166) Two types of double-stranded oligonucleotides were prepared by PCR using the following two primer sets (no template; allowing primer dimers to be made), then cleaved with restriction enzymes, respectively. -Fragment ligation introduced into the vector at once.
082-FRT (SEQ ID NO: 72)
083-FRT (SEQ ID NO: 73)
as well as,
084-FRT (SEQ ID NO: 72)
085-FRT (SEQ ID NO: 73)

First, PCR was performed without a template using 082-FRT and 083-FRT, 084-FRT and 085-FRT. As a result, DNA fragments having the following sequences were prepared.
FRT1 (SEQ ID NO: 76)
FRT2 (SEQ ID NO: 77)
Next, FRT1 was cleaved with BamHI and BbsI, FRT2 was cleaved with EcoRI and BbsI, and introduced into the BamHI / EcoRI site of the pUC119 vector by 3-fragment ligation. The sequence of the created insert is BamHI_SpeI_FRT_StyI_FRT_NheI_EcoRI (SEQ ID NO: 78), and the resulting vector is called pA166.

6-2-4. Preparation of pAGE and pAGF After pAFZ was cleaved with XbaI / NheI and a fragment containing a 1776 bp neo-resistant gene was recovered, pAFY was cleaved with StyI, and the recovered fragment was introduced into the cleavage site. As a result, pAGB and pAGD having different directions were produced. Next, a fragment containing the neo resistance gene obtained by cleaving pAGB or pAGD with XbaI / NheI was inserted into the XbaI cleavage site of pADY (FIG. 1). The resulting vectors are pAGE (SEQ ID NO: 58) and pAGF (SEQ ID NO: 59), respectively.

6-2-5. Construction of pAGI, pAGJ and pAHV A 1776 bp fragment obtained by cleaving pAFZ with XbaI / NheI was inserted into the StyI cleavage site of pA166. The resulting vector is called pAGG.
On the other hand, pAGF was introduced into EL350 E. coli (sold by Dr. Neal G. Copeland: Genome Res, 13: 476-484, 2003). EL350 is an E. coli host that enables recombining as in DY380, but Cre protein is expressed by adding arabinose to the medium, and as a result, Cre-loxP site-specific recombination can be induced. . Arabinose was added to the culture solution of EL350 into which pAGF was introduced to induce Cre-loxP recombination, and the neo resistance gene region sandwiched between two loxPs in pAGF was deleted. The resulting plasmid is called pAGH.
Next, pAGG was cut with NheI / SpeI. At this time, SpeI was partially decomposed. The obtained 1860 bp fragment was inserted into the XbaI cleavage site of pAGH to insert pAGI (SEQ ID NO: 60) and into the NheI cleavage site of pAGH to prepare pAGJ (SEQ ID NO: 61) and pAHV (SEQ ID NO: 62). (For pAGJ and pAHV, the direction of FRT-neo-FRT is reversed).

Plasmid Name Accession Number Deposit Date pADY FERM AP-20701 October 28, 2005 pADW FERM AP-20702 October 28, 2005 pAEF FERM AP-20703 October 28, 2005

The schematic diagram of the cloning vector developed by this invention is shown. The schematic diagram of each vector shows the necessary components and restriction enzyme sites. All cleavage sites of SfiI are set to different sequences. In addition, pAEJ and pAEK SfiI sites for supplying homologous regions all show different sequences (uppermost). pADY is a vector for constructing a linear DNA substrate used in recombining. pAEF was developed to construct a gene targeting construct by subcloning a BAC clone. pADW was developed to construct a gene targeting construct by conventional methods. SacB represents the SacB gene, DT-A represents the diphtheria toxin fragment A expression cassette, and GFPuv represents the GFPuv gene. The colonies of Escherichia coli carrying pADY and pAEF that emit GFPuv-derived green fluorescence are shown. The left figure shows a colony obtained as a result of culturing Escherichia coli carrying the vector shown in FIG. 1 on a plate to which ampicillin has been added. The right figure shows a colony whose fluorescence was confirmed by a long wavelength UV lamp. The cloning procedure for preparing a linear DNA substrate that can be used for H2-T3 TV in one step is shown. The homologous region can be supplied by a cloned fragment (a), a double-stranded oligo (b), a PCR amplified fragment (c) or the like. HR means homologous region. An electrophoretic photograph of the homologous region cleaved with SfiI, a cloning vector, and its induction vector is shown. A procedure for producing a gene targeting vector using the vector of the present invention will be described. (A) shows the preparation procedure of the gene targeting vector by the recombinering method. (B) shows the procedure for producing a gene targeting construct from pAEX derived from pADW using the homology arm obtained by PCR amplification. (C) shows the procedure of performing homologous recombination in ES cells using H2-T3 TV prepared by the procedure of (a) or (b). The results of restriction enzyme analysis and Southern blot analysis of BAC clones and targeted ES cells are shown. BAC (lane 2) modified in the wild type (lane 1) and recombinogenic engineering method (step 1 in Fig. 5-a) was cleaved with BglI (left), and hybridized with the probe after transcription on the membrane. (Middle figure). The hybridized fragment is 9.4 kb for the wild type and 4.3 kb for the targeted allele (arrow). As a result of Southern blot analysis, a band of 9.4 kb was confirmed from the wild type derived from E14.1, and bands of 9.4 kb and 4.3 kb were confirmed from the targeted ES cell clones (3D2 and 4A2) (right figure). It was.

Claims (12)

  A plasmid vector comprising two negative selection marker genes and four different cleavage sites recognized by the same restriction enzyme in the vicinity of the 5 'upstream and 3' downstream sides of the two negative selection marker genes.   The plasmid vector according to claim 1, wherein the negative selection marker gene is a SacB gene.   The plasmid vector according to claim 1 or 2, wherein the restriction enzyme is SfiI.   The plasmid vector according to any one of claims 1 to 3, further comprising a selectable marker gene.   The plasmid vector according to any one of claims 1 to 4, wherein the further selectable marker gene is a GFPuv gene.   The plasmid vector according to any one of claims 1 to 5, further comprising a restriction enzyme recognition site for linearizing the plasmid vector incorporating a target region and two homologous regions.   The plasmid vector according to any one of claims 1 to 6, wherein the restriction enzyme is I-sceI.   The plasmid vector according to claim 1, which is selected from pADY, pAEF, and pADW.   The plasmid vector according to any one of claims 1 to 8, wherein the negative selection marker gene is replaced with a homologous region.   A method for producing a gene targeting vector using the plasmid vector according to any one of claims 1 to 9.   A gene targeting vector produced by the method according to claim 10. The gene targeting vector according to claim 11, selected from pAGE, pAGF, pAGI, pAGJ, and pAHV.