JP2018007589A - Method for screening cell strain capable of in vivo cloning, method for producing cell strain capable of in vivo cloning, cell strain, in vivo cloning method, and kit for performing in vivo cloning - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening cell strains capable of in vivo cloning with high efficiency.SOLUTION: The present invention provides a method for screening cell strains capable of in vivo cloning, the method including a first selection step for evaluating cell strains for the presence or absence of xthA gene, and selecting a cell strain having xthA gene.SELECTED DRAWING: None

Description

  The present invention relates to a method for screening a cell line capable of in vivo cloning, a method for producing a cell line capable of in vivo cloning, a cell line, a method for in vivo cloning, and a kit for performing in vivo cloning.

  In conventional DNA cloning technology, the target DNA is incorporated into an expression vector using a restriction enzyme and DNA ligase, etc., and the expression vector into which the target DNA is inserted is introduced into a host cell such as Escherichia coli for expression (transformation). Therefore, it has been common to clone the DNA of interest.

In recent years, a method of cloning a target DNA into a vector using a recombination reaction between homologous sequences, so-called seamless cloning has been utilized (for example, see Patent Document 1). In seamless cloning, first, a primer to which a homologous sequence of about 20 bp is added is synthesized, and the target DNA and expression vector are each amplified by PCR. Subsequently, the target DNA can be cloned by introducing and expressing the target DNA and expression vector having homologous sequences in a budding yeast or an Escherichia coli strain in which the RecET system is constructed. Thus, cloning of a target DNA in cells such as yeast and E. coli is called in vivo cloning.
Since budding yeast has very high activity of homologous recombination in cells, seamless cloning can be easily performed (see, for example, Non-Patent Document 1).
In addition, in the E. coli strain in which the RecET system has been constructed, RecE and RecT recombinases in the Rac prophage region are overexpressed, and this RecE and RecT recombinase enhances the efficiency of homologous recombination in E. coli cells and performs seamless cloning. (See, for example, Patent Document 2, Patent Document 3, and Non-Patent Document 2.)

  Recently, it has been reported that a target DNA can be cloned by directly introducing and expressing a plurality of target DNAs having homologous sequences and expression vectors in E. coli DH5α strain (see, for example, Non-Patent Document 3).

US Patent Application Publication No. 2002/0165175 US Patent Application Publication No. 2010/0317061 US Patent Application Publication No. 2013/0210681

Ma, H., et al., “Plasmid construction by homologous recombination in yeast”, GENE, vol.58, p201-216, 1987. Oliner, J. D., et al., “In vivo cloning of PCR products in E. coli”, NUCLEIC. ACIDS. RES., Vol.21, no.22, p5192-5197, 1993. Kostylev, M., et al., “Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies”, PLOS ONE, 2015, DOI: 10.1371 / journal.pone.0137466.

In the method described in Non-Patent Document 1, since the vector into which the cloned target DNA is inserted is unstable in the budding yeast cell, it must be further introduced into a cell line such as Escherichia coli and cloned. .
In addition, in the methods described in Patent Document 2, Patent Document 3, and Non-Patent Document 2, it is necessary to use an E. coli strain having a RecET system. Furthermore, homologous recombination occurs between plasmid DNAs, and dimers or multimers are obtained. It has the problem of being formed.
Furthermore, in the method described in Non-Patent Document 3, it has been reported that the target DNA can be directly cloned using Escherichia coli DH5α strain, but the mechanism has not been clarified. In the method described in Non-Patent Document 3, a cell line with low cloning efficiency and higher cloning efficiency has been demanded.

  The present invention has been made in view of the above circumstances, and provides a screening method for cell lines that can be cloned in vivo with high efficiency.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have found that cell lines such as E. coli have lost the function of the hsdR gene, and having the xthA gene results in highly efficient homologous recombination. The inventors have found that in vivo cloning can be performed efficiently, and have completed the present invention.

That is, the present invention includes the following aspects.
[1] A method for screening a cell line that can be cloned in vivo, comprising the first selection step of evaluating the presence or absence of an xthA gene in the cell line and selecting a cell line having the xthA gene. And how to.
[2] Further, after the first selection step, a second selection step of evaluating the presence or absence of the hsdR gene and selecting a cell line in which all or a part of the function of the hsdR gene has been lost is provided [1]. The method described in 1.
[3] The method according to [1] or [2], wherein the cell line is derived from Escherichia coli or a mammalian cell.
[4] A method for producing a cell line capable of in vivo cloning, comprising a step of inserting an xthA gene into a chromosome of the cell line and highly expressing the xthA gene.
[5] The production method according to [4], further comprising a deletion step in which all or a part of the function of the hsdR gene is lost after or simultaneously with the insertion step when the cell line has the hsdR gene. .
[6] The production method according to [4] or [5], wherein the cell line is derived from Escherichia coli or a mammalian cell.
[7] A cell line having an xthA gene and having lost all or part of the function of the hsdR gene.
[8] The cell line according to [7], wherein the cell line is derived from Escherichia coli or a mammalian cell.
[9] Screening of cell lines capable of in vivo cloning using the method according to any one of [1] to [3] or the production method according to any one of [4] to [6] Or after the production, to the obtained cell line, an expression vector and a target double-stranded DNA having a base sequence homologous to the site to be inserted on the expression vector in the 5 ′ terminal region and the 3 ′ terminal region; In vivo cloning, comprising: an introduction step of introducing a target cell; and an insertion step of culturing the cell line after the introduction step and inserting the target double-stranded DNA into the expression vector by homologous recombination Method.
[10] A kit for performing in vivo cloning, comprising the cell line according to [7] or [8].

  According to the present invention, it is possible to provide a screening method for cell lines that can be cloned in vivo with high efficiency.

It is a figure which shows typically the mechanism of the homologous recombination in the cell which has xthA gene. It is a graph which shows the number of colonies per plate of LB agar medium containing chloramphenicol and ampicillin in each E. coli strain in Example 1. It is a graph which shows the number of colonies per plate of LB agar medium containing chloramphenicol and ampicillin in each Escherichia coli strain in Example 2. It is a graph which shows the number of colonies per plate of LB agar medium containing chloramphenicol and ampicillin in each Escherichia coli strain in Example 2.

<< Method for screening cell lines capable of in vivo cloning >>
<First embodiment>
In one embodiment, the present invention is a method for screening a cell line that can be cloned in vivo, wherein the cell line is evaluated for the presence of the xthA gene and a cell line having the xthA gene is selected. A method comprising the steps is provided.

  According to the screening method of this embodiment, a cell line capable of in vivo cloning with high efficiency can be obtained.

The cell line to be screened in this embodiment is not particularly limited as long as it is usually used as a host cell as a DNA cloning and protein expression system. Examples of the biological species from which the cell line is derived include, but are not limited to, Escherichia coli, Bacillus subtilis, yeast, insect cells, plant cells, animal cells (particularly mammalian cells), and the like. Among them, the biological species from which the cell line is derived is preferably Escherichia coli or a mammalian cell, more preferably Escherichia coli, from the viewpoint of ease of handling.
In the present specification, “in vivo cloning” means a technique for cloning a target DNA in a cell.
Details of the screening method of this embodiment will be described below.

[First selection step]
First, the presence or absence of the xthA gene is evaluated for the cell line, and a cell line having the xthA gene is selected.

  In general, the xthA gene is a gene encoding 3 ′ → 5 ′ exonuclease that liberates 5′-mononucleotide from the 3′-OH end of double-stranded DNA, and the gene product 3 ′ → 5 ′ exonuclease. Has a function of repairing DNA that has been damaged and has a base removed by the activity of DNA glycosylase, or a DNA that is naturally deficient in base.

In the cell line to be screened in the present embodiment, the 3 ′ → 5 ′ exonuclease can be expressed by having the xthA gene.
FIG. 1 is a diagram schematically showing the mechanism of homologous recombination in a cell having an xthA gene. FIG. 1 shows the mechanism of homologous recombination at the site 3b having the homologous sequence among the sites 3a and 3b having the homologous sequence. Further, when the homologous sequence in the site 3b having a homologous sequence is the sequence X, in the target double-stranded DNA 1 (hereinafter sometimes referred to as “dsDNA”) and the vector, the site 3b having the homologous sequence has the sequence It consists of site 3b-1 having X and site 3b-2 having a sequence complementary to sequence X.
First, the expressed 3 ′ → 5 ′ exonuclease 4 degrades the site 3b-1 having the sequence X inward from the 3 ′ end of the target dsDNA1 in the cell into which the target dsDNA1 and the vector 2 have been introduced. Then, a single-stranded region is generated at the end including the site 3b-2 having a sequence complementary to the sequence X. In addition, 3 ′ → 5 ′ exonuclease 4 includes site 3b-1 having sequence X by decomposing site 3b-2 having a sequence complementary to sequence X inward from the 3 ′ end of vector 2. A single-stranded region is generated at the end. As a result, the terminal part including the site 3b-2 having a sequence complementary to the sequence X of the target dsDNA1 and the terminal part including the site 3b-1 having the sequence X of the vector 2 are annealed. Next, the nick is filled with DNA polymerase 5 present in the cell and bound by DNA ligase 6. Since the homologous recombination is also performed by the same mechanism at the terminal portion including the site 3a having the opposite homologous sequence, the target double-stranded DNA is inserted into the vector as a result.
Therefore, by selecting a cell line having the xthA gene, homologous recombination can be performed with high efficiency, and a vector into which the target double-stranded DNA has been inserted can be obtained. In vivo cloning can be performed with high efficiency. it can.

As a method for evaluating the presence or absence of the xthA gene, a known method may be used. For example, DNA is extracted from a cell line, the xthA gene is amplified by a PCR method using a primer set for the xthA gene, and agarose is used. A method of separating by gel, performing staining with an intercalator such as ethidium bromide, and irradiating with ultraviolet rays may be used.
Alternatively, for example, by extracting DNA from a cell line, adding a fluorescent probe or fluorescent dye to the reaction solution in advance by real-time PCR using a primer set for the xthA gene, and quantitatively monitoring the amplification of the xthA gene in real time A detection method may be used.

  Alternatively, for example, RNA is extracted from a cell line, mRNA transcribed from the xthA gene is amplified by RT-PCR using a reverse transcriptase and a primer set for the xthA gene, and the cDNA converted from the mRNA is amplified as described above. A detection method may be used by a detection method (a staining method using an intercalator after agarose gel separation, or a real-time detection method using a fluorescent probe or fluorescent dye).

[Second selection step]
Next, it is preferable that the method further comprises a second selection step after the first selection step for evaluating the presence or absence of the hsdR gene and selecting a cell line that has lost all or part of the function of the hsdR gene.

In general, the hsdR gene is a gene encoding a restriction enzyme that cleaves a specific base sequence, and includes a methylation-modifying enzyme encoded by the hsdM gene and a methylation recognition protein encoded by the hsdS gene. Bear. In this restriction-modification system, when only one side of a specific nucleotide sequence is methylated in a cell (only one side is methylated as occurs during chromosomal DNA replication in the host cell), Methylation-modifying enzymes in the restriction-modification system work to protect specific base sequences from cleavage. On the other hand, when the specific base sequence is not methylated in any chain, the restriction enzyme in the restriction-modification system works to cut the specific base sequence.
Usually, when the target double-stranded DNA and vector that are foreign DNA have a specific base sequence, the sequence is not methylated in any strand, and thus is cleaved by the restriction-modification system.
Therefore, by selecting a cell line in which the function of the hsdR gene is disrupted or deleted, the target double-stranded DNA and vector, which are foreign DNA, are not cleaved by the restriction-modification system. Recombination is performed to obtain a vector into which the target double-stranded DNA has been inserted, and in vivo cloning can be performed with high efficiency.

  In the present specification, “all or part of the function is lost” means that the target gene (in the present embodiment, the hsdR gene) is altered, deleted, replaced, inserted, or the like, or homologously recombined. As a result, the original target gene (hsdR gene in this embodiment) is deleted, deleted or changed, and all or part of the target gene (hsdR gene in this embodiment) cannot be expressed. It means a state in which all or part of the physiological function of the gene (in this embodiment, the hsdR gene) cannot be performed. In a state where “all or part of the function has been lost”, one of the target genes (hsdR gene in the present embodiment) on the homologous chromosome may be deleted, deleted or changed, and both may be deleted. It may be deleted or changed.

In the present specification, changes, deletions, substitutions, insertions, or the like, or homologous recombination may be referred to as “modifying a gene”.
In the present specification, the means for genetic modification such as the terms “change”, “deletion”, “insertion” and “replacement” are not used in a limited sense. Moreover, it is not necessary to use it with the intention of strictly distinguishing it from any of the genetic modification means, and as a result, the hsdR gene is modified by any of the genetic modification means to suppress the expression of the hsdR gene, and all of its functions or Used to mean that some is disabled. However, it can be understood that the above-described means are generally used as described below.

That is, in general, “modification” of genomic DNA means that one or more bases of a genomic DNA fragment of a target gene are altered so that all or part of the expression product of the target gene does not function. It means to do.
“Deletion” of genomic DNA means that all or part of the target gene expression product does not function or does not exist by deleting all or part of the genomic DNA fragment of the target gene. To do.
“Substitution” of genomic DNA means that all or part of the genomic DNA fragment of the target gene is replaced by a separate sequence not related to the target gene, and all or part of the target gene expression product does not function or exists. It means not to.
“Insertion” of genomic DNA means that by inserting a DNA having a sequence other than that into the target gene, all or part of the expression product of the target gene into which DNA other than the target gene is inserted does not function. Or it means not to exist.
In addition, gene modification means such as gene alteration, deletion, insertion or replacement may be used simultaneously on the target gene by combining two or more methods.

  Examples of the method for evaluating the presence / absence of the hsdR gene include the same methods as those exemplified as the method for evaluating the presence / absence of the xthA gene in [First selection step] described above.

<Second embodiment>
In another embodiment, the present invention is a method for screening a cell line capable of in vivo cloning, wherein the cell line is assessed for the presence or absence of the hsdR gene and all or part of the function of the hsdR gene is lost. A method comprising a first selection step of selecting a selected cell line is provided.
Next, it is preferable to further include a second selection step of evaluating the presence or absence of the xthA gene and selecting a cell line having the xthA gene after the first selection step.

<< Method for producing cell line capable of in vivo cloning >>
In one embodiment, the present invention provides a method for producing a cell line that can be cloned in vivo, the method comprising a step of inserting an xthA gene into a chromosome of the cell line and highly expressing the xthA gene.

  According to the production method of the present embodiment, a cell line that can be cloned in vivo with high efficiency can be obtained.

The cell line used in the present embodiment is not particularly limited as long as it is normally used as a host cell as a DNA cloning and protein expression system. Examples of the biological species from which the cell line is derived include those similar to those exemplified in the above << Method for screening a cell line capable of in vivo cloning >>. Among them, the biological species from which the cell line is derived is preferably Escherichia coli or a mammalian cell, more preferably Escherichia coli, from the viewpoint of ease of handling.
Details of the manufacturing method of this embodiment will be described below.

[Insertion process]
First, the xthA gene is inserted into the chromosome of the cell line so that the xthA gene is highly expressed. Even in a cell line already having an xthA gene, the xthA gene can be highly expressed by further inserting the xthA gene.

  The gene region to be inserted on the chromosome of the xthA gene is a gene region that is constantly and stably expressed, even if the gene originally encoded in the region is deleted or modified Any area that can sustain life.

(Insert method)
The xthA gene may be inserted into the chromosome using any known genome editing technique that induces homologous recombination repair (HDR), such as CRISPR-Cas9 system, TALEN system, Zn finger nuclease system. However, it is not limited to this. Among them, the CRISPR-Cas9 system is preferably used as a known genome editing technique because the operation is simple and the target gene can be inserted into the target site with high selectivity.

  The TALEN system is a system using Transcription activator-like effector nuclease (TALEN), which is a fusion of a customized binding domain and a non-specific FokI nuclease domain. The DNA-binding domain consists of conserved repeats derived from transcription activator-like effectors (TALE), a protein that is secreted by Xanthomonas proteobacteria and changes the gene transcription of the host plant. In order to insert the xthA gene on the chromosome by the TALEN system, a TALEN for the target site on the chromosome is prepared, and a donor vector having homology around the insertion site and including the xthA gene is prepared. By co-introducing a vector containing DNA encoding TALEN and a donor vector containing the xthA gene, the target site is cleaved, and the xthA gene is subsequently introduced by HDR using the donor vector. By providing the xthA gene with a drug selection marker, cells in which xthA gene introduction has occurred due to drug selection can be efficiently selected.

On the other hand, the CRISPR-Cas9 system is a system that utilizes a locus that functions as a kind of acquired immunity against nucleic acid (viral DNA, viral RNA, plasmid DNA) invaded from the outside, possessed by bacteria and archaea. In order to insert the xthA gene onto the chromosome by the CRISPR-Cas9 system, a CRISPR-Cas9 vector for cutting the insertion site on the chromosome is prepared. For this purpose, an expression vector encoding a guide RNA that induces Cas9 at a target site and a Cas9 expression vector are required.
By co-introducing these vectors and a donor vector containing the xthA gene, the target site is cleaved, and the xthA gene is subsequently introduced by HDR using the donor vector. By providing the xthA gene with a drug selection marker, cells in which xthA gene introduction has occurred due to drug selection can be efficiently selected.

  The Zn finger nuclease (ZFN) system is a genome editing using an artificial restriction enzyme consisting of a Zn finger domain and a DNA cleavage domain. One Zn finger domain recognizes 3 bases, but a DNA binding protein that recognizes a specific 9 to 15 base sequence can be designed by linking 3 to 5 Zn finger domains. An artificial nuclease ZFN that cleaves a target site is designed by fusing a FokI nuclease domain to this DNA binding protein. In order to insert the xthA gene into a target site on a chromosome by the ZFN system, a ZFN for the target site on the chromosome is prepared, and a donor vector having homology around the insertion site and including the xthA gene is prepared. By co-introducing these vectors, the target site is cleaved, and the xthA gene is subsequently introduced by HDR using a donor vector. By providing the xthA gene with a drug selection marker, cells in which xthA gene introduction has occurred due to drug selection can be efficiently selected.

  The sequence homologous to the site to be inserted on the chromosome may be operably linked upstream (5 ′ side) and downstream (3 ′ side) of the xthA gene, and its length is, for example, 10 bp to 30 bp. For example, it may be 15 bp or more and 25 bp or less.

For the xthA gene, a donor vector inserted into a vector corresponding to the type of cell line to be introduced is prepared, and gene introduction is performed by any of the above insertion methods.
The vector is preferably an expression vector. The expression vector is not particularly limited. For example, plasmids derived from E. coli such as pBR322, pBR325, pUC12, pUC13, and pUC57; plasmids derived from Bacillus subtilis such as pUB110, pTP5, and pC194; plasmids derived from yeast such as pSH19 and pSH15; Bacteriophages such as phages; viruses such as adenoviruses, adeno-associated viruses, lentiviruses, vaccinia viruses, baculoviruses; and modified vectors thereof can be used.

  The xthA gene may be introduced together with a tag sequence, a promoter sequence, a transcription termination sequence, a drug selection marker gene, and combinations thereof. Here, examples of the tag include a fluorescent tag (such as GFP), an affinity tag, a degron tag, and a localization tag.

-Drug selection marker gene The drug selection marker gene may be operably linked upstream (5 'side) or downstream (3' side) of the xthA gene. Examples of the drug selection marker gene include neomycin resistance gene, histidinol resistance gene, puromycin resistance gene, hygromycin resistance gene, blasticidin resistance gene, ampicillin resistance gene, chloramphenicol resistance gene and the like. It is not limited to these. By inserting a drug selection marker gene, cells that have been introduced with the xthA gene can be selected by culturing the cells using a medium containing the drug.

-Promoter In the insertion step of this embodiment, a promoter sequence that controls transcription of the xthA gene is preferably operably linked upstream (5 ′ side) to the xthA gene.
In the present specification, “operably linked” means between a gene expression control sequence (for example, a promoter or a series of transcription factor binding sites) and a gene to be expressed (in this embodiment, the xthA gene). This means functional linkage. Here, the “expression control sequence” means a sequence that directs transcription of a gene to be expressed (in this embodiment, the xthA gene).

  The promoter is not particularly limited and can be appropriately determined according to, for example, the type of cell. Specific examples of the promoter include, for example, an inducible promoter, a viral promoter, a housekeeping gene promoter, a tissue-specific promoter, and the like.

  The inducible promoter is not particularly limited, and examples thereof include a chemically inducible promoter, a heat shock inducible promoter, an electromagnetic inducible promoter, a nuclear receptor inducible promoter, and a hormone inducible promoter.

  The chemically inducible promoter is not particularly limited, and examples thereof include a salicylic acid inducible promoter (see WO 95/19433), a tetracycline inducible promoter (Gatz et al. (1992) Plant J. 2, 397- 404)), an ethanol-inducible promoter, a zinc-inducible metallothionein promoter, and the like.

  The heat shock inducible promoter is not particularly limited. For example, see a heat inducible promoter (see US Pat. No. 5,187,287), a cold inducible promoter (see US Pat. No. 05847102). ) And the like.

  Examples of the electromagnetically inducible promoter include early growth region-1 (EGR-1) promoter (see US Patent Application No. 5206152), c-Jun promoter, and the like.

  The nuclear receptor-inducible promoter is not particularly limited, and examples thereof include an orphan nuclear receptor chicken ovalbumin upstream promoter.

  The hormone-inducible promoter is not particularly limited, and examples thereof include a glucocorticoid-inducible promoter (see Lu and Federoff, Hum Gene Ther 6,419-28, 1995.), an MMTV promoter, a growth hormone promoter, and the like.

  The viral promoter is not particularly limited, and examples thereof include cytomegalovirus (CMV) promoter (CMV immediate early promoter (see, for example, US Pat. No. 5,168,062), human immunodeficiency virus (HIV)). Promoter (for example, HIV long terminal repeat), Rous sarcoma virus (RSV) promoter (for example, RSV long terminal repeat), mouse mammary tumor virus (MMTV) promoter, HSV promoter (Lap2 promoter or herpes thymidine kinase promoter, etc. (Wagner) et al., Proc. Natl. Acad. Sci., 78, 144-145, 1981.), SV40 or Epstein-Barr virus-derived promoter, adeno-associated virus Promoter (e.g., p5 promoter) and the like.

  The housekeeping gene promoter is not particularly limited. For example, GAPDH (glyceraldehyde-3 phosphate dehydrogenase) gene promoter, β-actin gene promoter, β2-microglobulin gene promoter, HPRT1 (hypoxanthine phosphotransferase gene, etc.) Is mentioned.

  The tissue-specific promoter is not particularly limited. For example, in the case of melanoma cells or melanocytes, the tyrosinase promoter or TRP2 promoter; in the case of breast cells or breast cancer, the MMTV promoter or WAP promoter; intestinal cells or intestinal cancer A villin promoter or FABP promoter; for pancreatic cells, PDX promoter; for pancreatic beta cells, RIP promoter; for keratinocytes; keratin promoter; for prostate epithelium; probasin promoter; In the case of (CNS) cells or CNS cancer, nestin promoter or GFAP promoter; for neurons, tyrosine hydroxylase, S100 promoter or neurofilament promoter; Edlund et al., Science, 230: Examples include a pancreas-specific promoter described in 912-916 (1985); in the case of lung cancer, the Clara cell secretory protein promoter; in the case of cardiac cells, the α myosin promoter.

Other configuration In the present embodiment, the xthA gene may be operably linked with a polyadenylation signal necessary for polyadenylation of the 3 ′ end of mRNA downstream (3 ′ side) of the xthA gene. Examples of polyadenylation signals include polyadenylation signals contained in each gene derived from viruses exemplified by the above viral promoters, various human or non-human animals, such as SV40 late gene or early gene, rabbit β globin gene, bovine Examples thereof include polyadenylation signals such as growth hormone genes and human A3 adenosine receptor genes. In addition, in order to further express the xthA gene, splicing signals, enhancer regions, and introns of each gene may be linked 5 ′ upstream of the promoter region, between the promoter region and the translation region, or 3 ′ downstream of the translation region. Good.

  In the present embodiment, the xthA gene may be operably linked to one or more nuclear localization sequences (NLS) upstream (5 ′ side) or downstream (3 ′ side) of the xthA gene. Good.

  Examples of NLS include NLS of SV40 virus large T antigen having amino acid sequence PKKKRVV (SEQ ID NO: 1); NLS derived from nucleoplasmin (eg, nucleoplasmin binary NLS having sequence KRPAATKKAGQAKKKK (SEQ ID NO: 2)); amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP c-myc NLS (SEQ ID NO: 4); SEQ NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY hRNPA1 (SEQ ID NO: 5) M9 NLS; importin - sequence of IBB domains from alpha AruemuaruaizuiefukeienukeijikeiditieiieruaruRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6); Myoma T protein sequence VSRKRPRP (arrangement 7) and PPKKARED (SEQ ID NO: 8); human P53 sequence PQPKKKKPL (SEQ ID NO: 9); mouse c-abl IV sequence SALIKKKKKKMAP (SEQ ID NO: 10); influenza virus NS1 sequences DRLRR (SEQ ID NO: 11) and PKQKKR ( SEQ ID NO: 12); sequence RKLKKKIKKL (SEQ ID NO: 13) of the hepatitis virus delta antigen; sequence REKKFLKRR (SEQ ID NO: 14) of mouse Mx1 protein; Examples include, but are not limited to, the human glucocorticoid sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 16).

  In the present embodiment, the xthA gene may be operably linked to one or a plurality of marker genes such as fluorescent proteins upstream (5 ′ side) or downstream (3 ′ side) of the xthA gene. Examples of the fluorescent protein include green fluorescent protein (Green Fluorescent Protein: GFP), yellow fluorescent protein (Yellow Fluorescent Protein: YFP), blue fluorescent protein (Blue Fluorescent Protein: BFP), cyan fluorescent protein (Cyan Fluorescent Protein, red). Examples thereof include a fluorescent protein (Red Fluorescent Protein).

(Gene transfer method)
As a method for introducing a vector containing the xthA gene into a cell line, a known gene introduction method (for example, calcium phosphate method, electroporation method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc.) ) Can be used.

(Confirmation method)
As a method for confirming whether or not the xthA gene has been inserted, a known method may be used. For example, by inserting a drug selection marker gene together with the xthA gene, the cell line is cultured in a medium containing the drug, A method for selecting viable cells may be used.

  Or the method similar to the method illustrated as a method of evaluating the presence or absence of an xthA gene in the above-mentioned [1st selection process] is mentioned.

[Deficit process]
Next, when the cell line has an hsdR gene, it is preferable that all or a part of the function of the hsdR gene is lost after or simultaneously with the insertion step.

As a method for losing the function of all or part of the hsdR gene, any method using a known genome editing technique for inducing knockout of the hsdR gene, knockin to the hsdR gene, or knockdown of the hsdR gene may be used.
Examples of known genome editing techniques for inducing knock-out of the hsdR gene or knock-in to the hsdR gene include, but are not limited to, the CRISPR-Cas9 system, the TALEN system, and the Zn finger nuclease system.
In addition, examples of known genome editing techniques for inducing knockdown of the hsdR gene include, but are not limited to, methods using RNAi-inducible nucleic acids (eg, siRNA, miRNA, etc.).
More specifically, for example, siRNA (small interfering RNA) for a hsdR gene or a gene having a sequence complementary to the hsdR gene is designed and synthesized, and this is incorporated into a retrovirus vector or an adenovirus vector to produce a cell. Introducing into a strain can cause RNAi.

  In general, “knockdown” means to suppress the expression level of a specific gene. Suppression of the gene expression level can be achieved, for example, by reducing the transcription level of the gene or inhibiting translation. In the production method of the present embodiment, the knockdown cell line refers to a cell line in which the expression level of the hsdR gene in the cell line is reduced compared to the wild type.

  In general, “RNAi” is a phenomenon in which dsRNA (double-strand RNA) specifically and selectively binds to a target gene and efficiently cleaves the target gene by cleaving the target gene. For example, when dsRNA is introduced into a cell line, expression of a gene having a sequence homologous to that RNA is suppressed (knocked down).

  In the present embodiment, a method for producing a cell line using an RNAi-inducible nucleic acid will be described in detail below.

(Design of siRNA)
First, siRNA for a target gene is designed. The siRNA is not particularly limited as long as it is an hsdR gene or a gene having a sequence complementary to the hsdR gene, and any region can be used as a candidate. Further, from the selected region, the sequence starts with AA and has a length of 18 to 30 bases, or when AA is added to the 5 ′ side, the length is 18 to 30 bases, preferably A sequence of 19 bases to 23 bases is selected. The GC content of the sequence may be selected, for example, from 30% to 70%, preferably around 50%. Further, it is preferable to select a GC distribution that has no bias. The siRNA may further include a 2-residue overhang of dT or U on the 3 ′ side of the selected sequence. In addition, it is preferable to perform a BLAST search on the amino acid sequence corresponding to the designed siRNA base sequence to confirm that there is no protein having a sequence similar to the selected sequence.

  Furthermore, by introducing several types of siRNA into a cell line at the same time, gene expression may be more effectively suppressed. Moreover, the expression of two or more target genes can be suppressed by simultaneously introducing several types of siRNA into a cell line.

  Examples of the siRNA synthesis method include the Dicer method. The siRNA synthesis method by the Dicer method is as follows. First, a gene sequence of 500 bp to 1 kbp from the start codon of the base sequence of the hsdR gene or a gene having a sequence complementary to the hsdR gene is incorporated into a vector or the like, and sense RNA and antisense RNA of the gene sequence are transcribed. And annealing to produce dsRNA. Next, by using Dicer Enzyme belonging to the RNase III family, this ds-RNA can be processed into siRNA having 21 to 23 bases having 2 base protruding ends on the 3 ′ end side.

  Moreover, shRNA can also be used instead of siRNA. shRNA is called short hairpin RNA and is an RNA molecule having a stem-loop structure so that a part of a single strand forms a complementary strand with another region. The RNA molecule having this stem-loop structure is processed by Dicer or the like in vivo to produce siRNA.

  shRNA can be designed so that a part thereof forms a stem-loop structure. For example, assuming that the sequence of a certain region is “sequence A” and the complementary strand to sequence A is “sequence B”, these sequences are present in a single RNA strand in the order of sequence A, spacer, and sequence B. Thus, the length is designed to be 42 to 120 bases as a whole. Sequence A is a base sequence of a partial region of a gene having a target hsdR gene or a gene having a sequence complementary to the hsdR gene, and the target region is not particularly limited, and any region is selected as a candidate. Is possible. The length of sequence A is 18 to 50 bases, preferably 21 to 30 bases.

  Moreover, instead of siRNA, microRNA (miRNA) can also be used. miRNA is a small RNA, and can inhibit the expression of a specific gene by inhibiting translation of mRNA having a complementary sequence to protein into a protein.

  miRNA can be designed to recognize mRNA of a specific gene. The length of the base sequence of the miRNA designed as such is, for example, 18 bases or more and 25 bases or less (for example, about 22 bases).

(Introduction of siRNA)
Subsequently, the designed siRNA is introduced into the cells of the cell line. Examples of the introduction method include known gene introduction methods (for example, calcium phosphate method, electroporation method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc.).

Instead of siRNA, a vector containing DNA encoding siRNA may be introduced. As the vector, an expression vector is preferable. Examples of the expression vector include those exemplified in the above [Insertion step].
The DNA encoding siRNA may further be operably linked to the above promoter, polyadenylation signal, NLS, fluorescent protein marker gene, or the like at the 5 ′ end or 3 ′ end of the siRNA encoding DNA. .

  Examples of the method for confirming whether or not all or part of the function of the hsdR gene have been lost include the same methods as those exemplified in the above [Insertion step].

<< cell line >>
In one embodiment, the present invention provides a cell line having the xthA gene and having lost all or part of the function of the hsdR gene.

Since the cell line of this embodiment has the xthA gene, homologous recombination is performed with high efficiency as described in the above << Method for screening cell lines capable of in vivo cloning >>. A vector into which the double-stranded DNA has been inserted can be obtained, and in vivo cloning can be performed with high efficiency.
In addition, since the cell line of this embodiment has lost all or part of the function of the hsdR gene, as described in the above << Method for screening cell lines capable of in vivo cloning >> The target double-stranded DNA and vector, which are DNA, are not cleaved by the restriction-modification system, can perform homologous recombination with high efficiency, and can obtain a vector into which the target double-stranded DNA is inserted In vivo cloning can be performed with high efficiency.

  The cell line of the present embodiment is not particularly limited as long as it can be used as a host cell as a DNA cloning system and a protein expression system. Examples of the biological species from which the cell line is derived include those similar to those exemplified in the above << Method for screening a cell line capable of in vivo cloning >>. Among them, the biological species from which the cell line is derived is preferably Escherichia coli or a mammalian cell, more preferably Escherichia coli, from the viewpoint of ease of handling.

<< In vivo cloning method >>
In one embodiment, the present invention uses the above-described screening method or the above-described production method to screen or produce a cell line that can be cloned in vivo. An introduction step of introducing a target double-stranded DNA having a base sequence homologous to a site to be inserted on the expression vector into the terminal region and the 3 ′ end region; and after the introduction step, culturing the cell line, An in vivo cloning method comprising: an insertion step of inserting the target double-stranded DNA into the expression vector by homologous recombination.

According to the in vivo cloning method of this embodiment, in vivo cloning can be performed with high efficiency, and the target double-stranded DNA or the gene product of the DNA can be obtained in high yield.
In the present specification, the “gene product” means mRNA transcribed from a target gene and a protein translated from the mRNA.

The cell line used in the in vivo cloning method of the present embodiment is a cell line that can be cloned in vivo obtained by the above-described screening method or the above-described production method, and has an xthA gene. Further, the cell line may lose all or part of the function of the hsdR gene. Moreover, the biological species from which the cell line is derived is not limited. Examples of the biological species from which the cell line is derived include those similar to those exemplified in the above << Method for screening a cell line capable of in vivo cloning >>. Among them, the biological species from which the cell line is derived is preferably Escherichia coli or a mammalian cell, more preferably Escherichia coli, from the viewpoint of ease of handling.
The cell line used in the in vivo cloning method of this embodiment may be cultured in advance so that the number of cells suitable for use in the introduction step is obtained.
The in vivo cloning method of this embodiment will be described in detail below.

[Introduction process]
First, the cell line obtained by the above-described screening method or the above-described production method has an expression vector and a base sequence that is homologous to the site for insertion on the expression vector in the 5 ′ end region and the 3 ′ end region. The desired double-stranded DNA is introduced.

The expression vector used in the introduction step is not particularly limited, and examples thereof include those similar to those exemplified in the [Insertion step] in the above << Method for producing a cell line capable of in vivo cloning >>.
The expression vector may include a tag sequence, a promoter sequence, a transcription termination sequence, a drug selection marker gene, and combinations thereof. Here, examples of the tag include a fluorescent tag (such as GFP), an affinity tag, a degron tag, and a localization tag.
Examples of the tag sequence, promoter sequence, transcription termination sequence, and drug selection marker gene are the same as those exemplified in [Insertion step] in the above << Method for producing in vivo cloneable cell line >>. .

The target double-stranded DNA used in the introduction step has a base sequence that is homologous to the site for insertion on the expression vector in the 5 ′ end region and the 3 ′ end region, and the length of the homologous base sequence is: For example, it may be 10 bp or more and 30 bp or less, for example, 15 bp or more and 25 bp or less.
The target double-stranded DNA may contain a tag sequence, a promoter sequence, a transcription termination sequence, a drug selection marker gene, and combinations thereof. Here, examples of the tag include a fluorescent tag (such as GFP), an affinity tag, a degron tag, and a localization tag.
Examples of the tag sequence, promoter sequence, transcription termination sequence, and drug selection marker gene are the same as those exemplified in [Insertion step] in the above << Method for producing in vivo cloneable cell line >>. .

  As a method for introducing an expression vector into a cell line with a foreign DNA having a base sequence homologous to the site to be inserted on the expression vector in the 5 ′ end region and the 3 ′ end region, a known gene introduction method (for example, , Calcium phosphate method, electroporation method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc.).

[Insertion process]
Next, the cell line after the introduction step is cultured, and the target double-stranded DNA is inserted into the expression vector by homologous recombination.

Cell culture conditions can be appropriately selected depending on the type of cells to be cultured.
The culture temperature may be, for example, from 25 ° C. to 40 ° C., for example, from 30 ° C. to 39 ° C., for example, from 35 ° C. to 39 ° C.
The culture environment may be, for example, about 5% CO ₂ condition.
The culture time can be appropriately selected depending on the cell type, the number of cells, etc., and may be, for example, from 1 day to 15 days, for example, from 1 day to 10 days, for example, 1 day or more. It may be 7 days or less.

  In addition, the cell culture medium to be used may be a basic medium containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins), etc. necessary for viable cell growth. It can be selected appropriately. Examples of the cell culture medium include E. coli culture media such as LB medium, E. coli minimal medium (Davis medium), E. coli minimal salt medium (MS); Bacillus subtilis minimal medium (Spizen minimal medium), Bacillus subtilis, and the like. Bacillus subtilis culture medium such as Bacillus subtilis transformation medium I and Bacillus subtilis transformation medium II; yeast culture such as yeast minimum medium (YPD medium) and yeast complete medium (YPAD medium) Medium: Murashige and Skoog medium (MS medium), B5 medium, Hyponex medium and other plant culture medium; Grace insect medium, Schneider insect medium and other insect cell culture medium; DMEM, Minimum Essential Medium (MEM), RPMI- 1640, Basal Medium Eagle (BME), Dulbecco's Mo Examples include, but are not limited to, a medium for animal cell culture such as differentiated Eagle's Medium: Nutrient Mixture F-12 (DMEM / F-12) and Glassgow Minimum Essential Medium (Glasgow MEM).

  As described in the above << Method for screening cell lines capable of in vivo cloning >>, the target double-stranded DNA and expression vector are highly efficient and homologous in the cell by the mechanism shown in FIG. Recombination is performed to form a vector into which the target double-stranded DNA has been inserted. Subsequently, the vector into which the target double-stranded DNA is inserted is cloned in the cell, and the target double-stranded DNA or a gene product of the DNA can be obtained in high yield.

[Confirmation process]
Furthermore, you may have the confirmation process which confirms whether the vector in which the target double stranded DNA was inserted is formed after the insertion process.
In the confirmation step, as a method for confirming whether or not a vector into which the target double-stranded DNA is inserted is formed, a known method may be used. For example, a drug selection marker gene is used as the target double-stranded DNA. And a method of culturing a cell line in a medium containing a drug and selecting viable cells.
Examples of the drug selection marker gene include those similar to those exemplified in [Insertion step] in the above << Method for producing cell line capable of in vivo cloning >>. When the expression vector also has a drug selection marker gene, the drug selection marker gene inserted together with the target double-stranded DNA and the drug selection marker gene of the expression vector are preferably different types.

  Alternatively, as a method for confirming whether or not a vector into which the target double-stranded DNA has been inserted is formed, for example, the above-mentioned << Method for screening a cell line capable of in vivo cloning >> The same method as the exemplified method for evaluating the presence or absence of the xthA gene in 1 selection step] can be mentioned.

<< Kit >>
In one embodiment, the present invention provides a kit for performing in vivo cloning, comprising a cell line having an xthA gene and having lost all or part of the function of the hsdR gene.

  According to the kit of this embodiment, in vivo cloning can be performed with high efficiency, and a target double-stranded DNA or a gene product of the DNA can be obtained in high yield.

  The cell line provided in the kit of the present embodiment is a cell line that has the xthA gene and loses all or part of the function of the hsdR gene. What is necessary is just to be used as a system | strain, and the biological species used as the origin is not limited. Examples of the biological species from which the cell line is derived include those similar to those exemplified in the above << Method for screening a cell line capable of in vivo cloning >>. Among them, the biological species from which the cell line is derived is preferably Escherichia coli or a mammalian cell, more preferably Escherichia coli, from the viewpoint of ease of handling.

The kit of this embodiment may further include an expression vector.
The expression vector provided in the kit of the present embodiment is not particularly limited, and the same expression vectors as those exemplified in [Insertion step] in the above << Method for producing a cell line capable of in vivo cloning >> Can be mentioned.
The expression vector may include a tag sequence, a promoter sequence, a transcription termination sequence, a drug selection marker gene, and combinations thereof. Here, examples of the tag include a fluorescent tag (such as GFP), an affinity tag, a degron tag, and a localization tag.
Examples of the tag sequence, promoter sequence, transcription termination sequence, and drug selection marker gene are the same as those exemplified in [Insertion step] in the above << Method for producing in vivo cloneable cell line >>. .

  The kit of this embodiment prepares a target double-stranded DNA having a base sequence homologous to the site to be inserted on the expression vector in the 5 ′ end region and the 3 ′ end region, thereby obtaining the target double strand. DNA can be easily and efficiently cloned in vivo.

  The kit of this embodiment may further include a cell culture medium for culturing cell lines. The culture medium for cell culture can be appropriately selected according to the type of cell line provided in the kit, and includes the same ones as exemplified in the above << In vivo cloning method >>.

  The kit of this embodiment may further include a transfection reagent for introducing the target double-stranded DNA and expression vector into the cell line. The transfection reagent is selected from the group consisting of, for example, a cationic polymer, a cationic lipid, a polyamine reagent, a polyimine reagent, and calcium phosphate. Such transfection reagents include, for example, Effectene Transfection Reagent (cat. Transfection Reagent (301305, Qiagen, CA), PolyFect Transfection Reagent (301105, Qiagen, CA), LipofectAMINE 2000 Reagent (11668-019, Invitrogen corporation, CA), JetPEI (X ) Conc. (101-30, Polyplus-translation, France), ExGen 500 (R0511, Fermentas Inc., MD), and the like, but are not limited thereto.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to a following example.

[Example 1] Search for E. coli strains that can be cloned in vivo (1) Preparation of insert DNA and vector As insert DNA, it has a chloramphenicol resistance gene derived from pACYC184, and 5 'end side and 3' end side, respectively DNA (992 bp) having a sequence homologous to the vector (20 bp) was used (the base sequence of the insert DNA is shown in SEQ ID NO: 17).
As a vector, a pUC19 vector (2.6 kb) having an ampicillin resistance gene was used (the base sequence of the pUC19 vector is shown in SEQ ID NO: 18).
First, PCR was performed on the insert DNA and the vector using the base sequences of the primers for amplification in PCR shown in Table 1 below to obtain a reaction solution.
The DNA amount per 1 μL of the PCR reaction solution of the insert DNA after the PCR reaction was 100 ng (0.15 pmol), and the DNA amount per 1 μL of the PCR reaction solution of the vector DNA was 80 ng (0.05 pmol). 1 μL of each PCR reaction solution was used for transformation without purification.

(2) Culture of Escherichia coli strain Subsequently, the BW25113 strain, the MG1655 strain, and the W3110 strain were inoculated into 3 mL of LB medium, cultured at 37 ° C. for 16 hours, and the number of bacteria was increased for use in transformation.

(3) Preparation of hsdR gene knockout Escherichia coli strain Next, the chromosomal DNA of ΔhsdR :: kan strain of Keio collection (reference: Baba et al., (2006) Molecular Systems Biology, doi: 10.1038 / msb4100050) was used as a template. The ΔhsdR :: kan region was amplified using the primers shown in Table 2. Then, using the obtained PCR product, a method using λRed recombinase (reference: Datsenko and Wanner, “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products”, PNAS, vol.97, no.12, p6640-6645, 2000.) and introduced into Escherichia coli MG1655 strain and W3110 strain, and the hsdR gene was knocked out. Next, each Escherichia coli strain was inoculated into 3 mL of LB medium, cultured at 37 ° C. for 16 hours, and the number of bacteria was increased for use in transformation.

(4) Introduction of insert DNA and vector into E. coli strain (transformation of E. coli strain)
Next, 1 ml each of the BW25113 strain, MG1655 strain, and W3110 strain cultured in (2) and the MG1655 strain and W3110 strain in which the hsdR gene was knocked out in (3) were warmed to 37 ° C., respectively. The LB medium was inoculated into 60 mL and cultured at 37 ° C. for 90 minutes (usually OD600 = bacterial concentration of about 0.4 to 0.5).
Subsequently, the culture was stopped and the culture medium was cooled in ice. Moreover, all subsequent operations were performed in a cold state in ice. Subsequently, the mixture was centrifuged at 5,000 g and 4 ° C. for 5 minutes, and the supernatant was discarded. Next, each E. coli strain pellet was suspended in 2 mL of ice-cold LB medium. Next, 1.6 mL of 2 × TSS solution having the composition shown in Table 2 below, which was ice-cooled, was added and mixed.

※上記混合後、オートクレーブ１２０℃で１５分後、４℃で長期保存可能。
使用前によく混ぜた。

* After the above mixing, the autoclave can be stored at 120 ° C for 15 minutes and then at 4 ° C for a long time.
Mix well before use.

  Next, 0.4 mL of room temperature DMSO was added and mixed. Next, 0.1 mL of each E. coli strain-containing solution was dispensed on ice, frozen with liquid nitrogen, and stored at −80 ° C. Next, each frozen E. coli strain was thawed on ice, and 1 μL each of the insert DNA and vector PCR reaction solution prepared in (1) was added to the solution containing each E. coli strain and gently pipetted to mix. Subsequently, it left still on ice for 30 minutes. Next, 1 mL each of LB medium was added and cultured at 37 ° C. for 45 minutes. Subsequently, it was centrifuged at 5,000 g and 4 ° C. for 5 minutes, most of the medium was discarded, and about 100 μL of the medium was left. Next, each E. coli strain pellet was suspended and spread on an LB agar medium containing chloramphenicol and ampicillin. Next, the LB agar medium inoculated with E. coli was cultured at 37 ° C. for 16 hours, and the number of colonies that appeared on one LB agar medium plate was counted. The results are shown in FIG. In FIG. 2, “MG1655ΔhsdR :: kan” indicates the MG1655 strain in which the hsdR gene is knocked out, and “W3110ΔhsdR :: kan” indicates the W3110 strain in which the hsdR gene is knocked out.

From FIG. 2, among the BW25113 strain, MG1655 strain, and W3110 strain, the number of colonies of the BW25113 strain was large. In the BW25113 strain, it is known that the hsdR gene has a mutation and the function is lost. Therefore, in the BW25113 strain, the insert DNA and the vector were not cleaved by the restriction enzyme encoded by the hsdR gene, and the insert DNA was inserted into the vector and the chloramphenicol resistance gene and the ampicillin resistance gene were expressed. It was guessed that this was because it was made.
In addition, the MG1655 and W3110 strains in which the hsdR gene was knocked out had a significantly increased number of colonies compared to the wild-type MG1655 and W3110 strains. This is because by knocking out the hsdR gene, the insert DNA and vector were not cleaved by the restriction enzyme encoded by the hsdR gene, and the insert DNA was inserted into the vector, and the chloramphenicol resistance gene and the ampicillin resistance gene were expressed. It was inferred that it was able to grow.

[Example 2] Elucidation of mechanism of in vivo cloning in BW25113 strain (1) Preparation of insert DNA and vector Using the same method as (1) of Example 1, an insert DNA and a vector were prepared, and a PCR reaction solution 1 μL of each was used for transformation without purification.

(2) Cultivation of Escherichia coli strain Subsequently, the BW25113 strain was inoculated into 3 mL of LB medium, cultured at 37 ° C. for 16 hours, and the number of bacteria was increased for use in transformation.

(3) Preparation of various gene knockout Escherichia coli strains Next, BW25113 ΔxthA :: kan strain, ΔrecE :: kan strain, ΔrecT :: kan strain were prepared by Keio collection (reference: Baba et al., (2006) Molecular Systems Biology, doi: 10.1038 / msb4100050) was used.
The BW25113 ΔrecET :: kan strain was prepared using plasmid pKD4 (Reference: Datsenko and Wanner, “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products”, using primers shown in Table 4 below. PNAS, vol.97, no.12, p6640-6645, 2000.) was amplified. Next, using the obtained PCR product, it was introduced into the BW25113 strain using the same method as in Example 1, (3). Next, each E. coli strain was inoculated into 3 mL of LB medium, cultured at 37 ° C. for 16 hours, and the number of bacteria was increased for use in transformation.

(4) Introduction of insert DNA and vector into E. coli strain (transformation of E. coli strain)
Next, instead of the BW25113 strain, MG1655 strain, and W3110 strain cultured in (2), and the MG1655 strain and W3110 strain in which the hsdR gene was knocked out in (3), the BW25113 strain cultured in (2), and It was prepared in (1) using the same method as in (4) of Example 1 except that the BW25113 strain in which the recE gene, recT gene, recE gene and recT gene, or xthA gene was knocked out in (3) was used. The insert DNA and vector were introduced into each E. coli strain, transformed, and spread on an LB agar medium containing chloramphenicol and ampicillin. Next, the LB agar medium inoculated with E. coli was cultured at 37 ° C. for 16 hours, and the number of colonies that appeared on one LB agar medium plate was counted. The results are shown in FIGS.
In FIG. 3, “BW25113ΔrecE :: kan” indicates the BW25113 strain in which the recE gene is knocked out, “BW25113ΔrecT :: kan” indicates the BW25113 strain in which the recT gene is knocked out, and “BW25113ΔrecET :: kan”. And BW25113 strain in which the recE gene and the recT gene are knocked out.
In FIG. 4, “BW25113ΔxthA :: kan” indicates the BW25113 strain in which the xthA gene is knocked out.

FIG. 3 reveals that the recE gene and the recT gene are not involved in homologous recombination and in vivo cloning of the insert DNA and vector.
FIG. 4 also revealed that knockout of the xthA gene significantly reduces the in vivo cloning activity.
From this, it was speculated that the homologous recombination of the insert DNA and the vector was carried out in the cell by the mechanism shown in FIG. 1, and the in vivo cloning could be performed with high efficiency.

  From the above results, it was suggested that the function of the hsdR gene is lost in a cell line capable of in vivo cloning, and it is important to have the xthA gene.

  According to the present invention, it is possible to provide a screening method for cell lines that can be cloned in vivo with high efficiency. Furthermore, according to the cell line obtained by the screening method of the present invention, the target double-stranded DNA or the gene product of the DNA can be obtained in high yield.

  DESCRIPTION OF SYMBOLS 1 ... Target double strand DNA (dsDNA), 1a, 1b ... Target single strand DNA (ssDNA), 2 ... Vector, 3a, 3b ... Site | part which has a homologous sequence, 3b-1 ... Site | part which has sequence X, 3b-2: a portion having a sequence complementary to the sequence X,

Claims (10)

A method for screening a cell line capable of in vivo cloning comprising:
A method comprising evaluating a cell line for the presence or absence of an xthA gene and selecting a cell line having an xthA gene.   Furthermore, the said 1st selection process is equipped with the 2nd selection process of evaluating the presence or absence of an hsdR gene, and selecting the cell strain from which all or some functions of the hsdR gene were lost. Method.   The method according to claim 1 or 2, wherein the cell line is derived from Escherichia coli or a mammalian cell. A method for producing a cell line capable of in vivo cloning comprising:
A production method comprising an insertion step of inserting an xthA gene into a chromosome of a cell line and highly expressing the xthA gene.   Furthermore, the manufacturing method of Claim 4 provided with the defect | deletion process which loses all or one part of functions of the said hsdR gene after the said insertion process when the said cell strain has an hsdR gene.   The method according to claim 4 or 5, wherein the cell line is derived from Escherichia coli or a mammalian cell.   A cell line having an xthA gene, wherein all or part of the function of the hsdR gene has been lost.   The cell line according to claim 7, wherein the cell line is derived from Escherichia coli or a mammalian cell. Obtained after screening or producing a cell line capable of in vivo cloning using the method according to any one of claims 1 to 3 or the production method according to any one of claims 4 to 6. Introducing an expression vector and a target double-stranded DNA having a base sequence homologous to a site to be inserted into the expression vector into the 5 ′ terminal region and the 3 ′ terminal region into the obtained cell line;
After the introduction step, culturing the cell line, and inserting the target double-stranded DNA into the expression vector by homologous recombination;
An in vivo cloning method comprising:   A kit for performing in vivo cloning, comprising the cell line according to claim 7 or 8.

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