WO2011105429A1 - Procédé et kit de recombinaison spécifique à un site - Google Patents

Procédé et kit de recombinaison spécifique à un site Download PDF

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WO2011105429A1
WO2011105429A1 PCT/JP2011/053990 JP2011053990W WO2011105429A1 WO 2011105429 A1 WO2011105429 A1 WO 2011105429A1 JP 2011053990 W JP2011053990 W JP 2011053990W WO 2011105429 A1 WO2011105429 A1 WO 2011105429A1
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attp
attb
integrase
dna
gene
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高橋　秀夫
森田　健太郎
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学校法人日本大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10331—Uses of virus other than therapeutic or vaccine, e.g. disinfectant

Definitions

  • the present invention relates to a site-specific recombination method and kit using integrase.
  • the so-called genome engineering technology that modifies the genome structure of eukaryotes and manipulates their gene expression control is an important method not only for gene therapy but also for breeding various eukaryotes.
  • targeted integration of foreign genes into the eukaryotic genome is particularly important.
  • the integration site is difficult to control, and there is a problem that abnormal gene expression due to random foreign gene incorporation and disruption of important genes are by-produced.
  • Cre recombinase of Escherichia coli phage P1, FLP recombinase of yeast, etc. have been confirmed to cause site-specific DNA recombination and function in eukaryotic cells, but also catalyze excision of the incorporated gene. Therefore, the gene transfer frequency is low as a result, and it is more widely used for creating deletion mutations than gene transfer.
  • integrase of E. coli phage lambda catalyzes site-specific gene integration, but cannot be directly applied to eukaryotic genome modification such as requiring host E. coli factors. Therefore, a recombinase effective for the integration of a foreign gene into the genome is preferably one that catalyzes the integration of a site-specific gene with an enzyme alone, and that cannot excise the incorporated gene with an enzyme alone.
  • a recombinase effective for the gene cloning method is preferably a recombinase that catalyzes site-specific gene incorporation with the enzyme alone, and that the enzyme alone cannot catalyze the excision of the incorporated gene.
  • Serine-type integrase was first discovered by Actinophage R4 by Matsuura et al. And was found to cause specific recombination reactions in Escherichia coli, yeast cells, animal cells, etc. (Matsuura M, Noguchi T, Yamaguchi D, Aida T, Asayama M, Takahashi H, Shirai M. J Bacteriol., 1996 Jun; 178 (11): 3374-6.).
  • Non-Patent Document 11 the integrase produced by actinophage ⁇ C31 is also serine type. Serine-type integrase, unlike tyrosine type, does not require the presence of host factors, and both attB and attP cause site-specific recombination between relatively short sequences of about 40 bp. Use as a tool is expected (Non-Patent Document 11).
  • Patent Document 2 An integrase gene derived from actinophage TG1 (For, F., et al., Gene, 39, 11-16 (1985)) (Patent Document 2).
  • the gene cloning method is generally as follows. 1. Amplify DNA with the gene of interest using PCR. 2. Treat the amplified DNA fragment and the plasmid vector to be inserted with restriction enzyme treatment and modification enzyme treatment, respectively. 3. Mix each DNA fragment and perform a ligase binding reaction (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • the method using lambda phage integrase has the advantage that the above-mentioned enzyme treatment can be performed at once by site-specific recombination reaction. (Invitrogen http://www.invitrogen.co.jp/gateway/index.shtml).
  • the present inventor has constructed a site-specific recombination system in vitro for several types of actinophage integrase. As a result, it was found that the actinophage TG1 integrase exhibits strong and efficient recombination activity in vitro and in vitro against the corresponding attB / attP sequences.
  • the present inventor provides the following site-specific recombination method and kit for site-specific recombination.
  • Item 1 Recombination between the first DNA sandwiched between two attBs and the second DNA sandwiched between two attPs using integrase, or between the first DNA sandwiched between two attPs and the second DNA sandwiched between two attBs A site-specific recombination method, wherein the integrase is a TG1 phage integrase.
  • Item 2. Item 2. The item 1, wherein the attB and attP are sites including a minimum base sequence that functions as attB (TT), attP (TT), attB (TA), and attP (TA) shown below.
  • AttB 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 ' attP
  • TT 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 ' attB
  • TA 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 ' attP
  • TA 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 '.
  • Item 4. 1) a polynucleotide having a recognition sequence (attP), 2) a polynucleotide having a recognition sequence (attB), and 3) A kit for site-specific recombination containing a polypeptide of TG1 phage integrase or a gene encoding TG1 phage integrase.
  • the present invention has made it possible to carry out site-specific gene recombination and cloning with very high efficiency.
  • the method of the present invention has a recombination efficiency much higher than that of the conventional method, and was able to develop a highly efficient and simple cloning technique.
  • FIG. 1 Schematic diagram of cloning method using TG1 integrase
  • a Construction of a plasmid vector (Acceptor vector) with a lethal gene (ccdB gene, etc.) sandwiched between attB sites of two TG1 phages or by cutting between attB and modifying the ends so that self-ligation does not occur To do.
  • b A DNA fragment (Donor DNA) having the target gene sandwiched between the attP sites of the TG1 phage is prepared using GSP (Gene-Specific Primer) -attP primer and AdaptorAdPCR using attP primer.
  • GSP Gene-Specific Primer
  • the target gene is inserted into AcceptorAcvector by site-specific recombination reaction.
  • b Schematic diagram of Donor DNA construction.
  • EGFP was amplified using an EGFP primer to which 48 bp attP was added, and the EGFP gene sandwiched between 48 bp attP was designated as Donor DNA.
  • c Schematic diagram of site-specific recombination reaction using pAVOTT and Donr DNA.
  • TG1 integrase causes a site-specific recombination reaction between attB and attP, and the EGFP gene in Donor DNA is incorporated into pAVOTT. Since Escherichia coli having pAVOTT before recombination has a LacZa gene, it becomes a blue colony on a medium containing X-gal.
  • the site-specific recombination efficiency by TG1 integrase was calculated as the number of white colonies / total number of colonies * 100.
  • Results of recombination efficiency of TG1 integrase and GATEWAY system The results of recombination efficiency of TG1 integrase and GATEWAY system are shown.
  • the vertical axis represents the number of colonies into which the target gene has been incorporated / the total number of colonies * 100 as the recombination efficiency.
  • the horizontal axis represents the amount of insert DNA.
  • Electrophoresis photograph of plasmid extracted from recombinant The plasmid extracted from the recombinant was cleaved with EcoRI and HindIII and confirmed by agarose gel electrophoresis. M. 1 kb plus ladder (Invitrogen), 1. pAVOTT, 2. Plasmid extracted from blue colony, 3-5. Plasmid extracted from white colony. The left represents the length (bp) indicated by the marker. A DNA fragment containing LacZa can be confirmed in 1 and 2, and a DNA fragment containing EGFP can be confirmed in 3 to 5. Electrophoresis photograph.
  • the upper figure is an electrophoresis photograph of 1.2% SeakemGTG, each lane from the left, markers, pUCattP (TT) and 50bp attB (TT), pUCattP (TT) and 50bp attB (TA), pUCattB (TT) and 50bp attP (TT), pUCattB (TT) and 50bp attP (TA), pUCattP (TA) and 50bp attB (TT), pUCattP (TA) and 50bp attB (TA), pUCattP (TA) and 50bp attB (TA), pUCattB (TA) and 50bp attP (TT), pUCattB ( TA) and 50bp attP (TT), pUCattB ( TA) and 50bp ⁇ ⁇ attP (TA), respectively, are recombination reactions and markers.
  • Each of-and + indicates the absence or addition of TG1 Integr
  • the integrase of the present invention is a TG1 integrase and is registered in the database under Accession No. AB251920.
  • each of the first DNA and the second DNA may be any DNA, for example, one may be a marker gene and the other may be any gene intended for introduction.
  • the marker gene include a drug resistance gene, a fluorescent protein (eg, GFP, YFP, etc.) gene, a luminescent enzyme (eg, luciferase) gene, a colored protein (phycocyanin, phycoerythrin, etc.), and target genes include enzymes, antibodies, Examples include genes that encode proteins that constitute intracellular organs such as receptors, hormones, cytoskeletons, and organelles.
  • the integrase of the present invention is obtained by cloning a gene encoding an enzyme having the amino acid sequence shown in SEQ ID NO: 1 from Actinophage TG1, specifically an integrase gene consisting of the base sequence shown in SEQ ID NO: 2, and using this. Prepare.
  • AttB and attP are sites containing the minimum base sequences that function as attB (TT), attP (TT), attB (TA), and attP (TA) shown below: attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 ' attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 ' attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 ' attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 '. Furthermore, it is possible to use a gene further comprising an SD sequence (Shine-Dalgarno Sequence) upstream of the gene encoding integrase, and by using such a gene, the amount of enzyme produced is
  • AttB (TT) and attP (TT) have the core sequence TT
  • attB (TA) and attP (TA) have the core sequence replaced with TA.
  • the core sequences correspond to the two attB or attP sandwiching the first DNA and the two attP or attB sandwiching the second DNA. That is, for example, when two attB sandwiching the first DNA is the attB (TT), it is preferable that the two attP sandwiching the second DNA is the attP (TT), When two attB sandwiching the first DNA is the attB (TA), the two attP sandwiching the second DNA is preferably the attP (TA), When two attB sandwiching the first DNA is one of the attB (TT) and attB (TA), the two attP sandwiching the second DNA is one of the attP (TT) and one of the attP (TA).
  • the two attB sandwiching the second DNA are preferably the attB (TT)
  • the two attB sandwiching the second DNA are preferably the attB (TA)
  • two attP sandwiching the first DNA is one of the attP (TT) and attP (TA)
  • two attB sandwiching the second DNA is one of the attB (TT) and one of the attB (TA).
  • One of the two attB or attP sandwiching the first DNA attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 'or attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 '
  • the other is attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 'or attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 'and one of the two attP or attB sandwiching the second DNA is attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 'or attB (TT): 5'-TCGATCAGCTCCGC
  • Gene cloning, preparation of an expression vector using a cloned DNA fragment, preparation of an integrase using an expression vector, etc. are well-known techniques for engineers belonging to the field of molecular biology. For example, it can be performed according to the method described in “Molecular Cloning” (edited by Maniatis et al., Cold Spring Harbor Laboratories, Cold Spring Harbor, New York (1982)).
  • the TG1 integrase gene was inserted into an appropriate vector and injected into a eukaryotic cell, or mRNA of the integrase gene was injected into a eukaryotic cell and sandwiched between two attB and two attP. Site-specific recombination can be induced between DNA.
  • references Thiagarajan, B., et al., Mol. Cell Biol., 21, 3926-3934 (2001), Gtoth, A. C., et al., Genetics, 166, 1775-1782 ( 2004)).
  • a vector for producing integrase in eukaryotic cells is prepared.
  • an expression control signal used for producing integrase in a eukaryotic cell it is desirable to use a transcription initiation signal and a translation initiation signal that can be artificially controlled and can control the productivity of integrase.
  • vectors that cannot replicate in eukaryotic cells such as various plasmids can be used.
  • a non-replicating plasmid containing a first DNA sequence between two attB (or attP) is prepared, introduced into a target eukaryotic cell, and integrated into a chromosome.
  • Many methods have already been reported for transforming eukaryotic cells, and may be appropriately selected according to the organism used as a host.
  • plasmid that contains a gene (second DNA) intended to be introduced between two attP (or attB) and cannot replicate in eukaryotes is prepared.
  • this vector it is desirable for this vector to have a selection marker different from the previous attB (or attP) -containing vector.
  • the attP (or attB) -containing plasmid and the integrase gene-containing vector are simultaneously injected into the prepared attB (or attP) -containing cells.
  • the integrase is temporarily expressed by the introduced integrase gene-containing vector, and site-specific recombination occurs between attB (or attP) on the chromosome and the introduced attP (or attB) DNA sequence, and attP
  • the (or attB) containing plasmid can be integrated into the attB (or attP) site on the chromosome.
  • integrase mRNA is synthesized in vitro using the integrase gene as a template according to a conventional method, and the integrase is transiently synthesized by injecting the mRNA into the cell instead of the integrase gene-containing vector.
  • the attP (or attB) -containing plasmid injected at the same time can be integrated into the chromosome.
  • the DNA concentration at the time of transformation can be limited, and the integration of a non-site-specific attP (or attB) -containing plasmid can be suppressed. .
  • a plasmid vector that has a lethal gene (for example, ccdB gene) sandwiched between attB sites of two TG1 phages or that has been cleaved between attB and modified so that self-ligation does not occur
  • a DNA fragment having a target gene sandwiched between attP sites of TG1 phage is prepared using PCR.
  • PCR reaction is performed using a primer (GSP-attP primer) of a target gene to which a full length attP sequence is added.
  • Donor DNA is created (FIG. 1b).
  • a site-specific recombination reaction occurs between the attB site on Acceptor vector and the attP site on Donor DNA, and the target gene on Donor DNA is Acceptor vector
  • the above lethal gene is replaced, and the target gene is inserted on the Acceptor vector (Fig. 1c).
  • transformation into E. coli results in replication of the acceptor vector into which the target gene has been inserted.
  • Escherichia coli having an unreacted Acceptor vector has a lethal gene and cannot grow, and only positive clones can be selected.
  • Example 1 Subcloning method using TG1 integrase First, a vector DNA into which a target gene was inserted was constructed. A LacZa gene sandwiched between 48 bp attB (TT) sequences was synthesized downstream of the lac promoter and inserted into the NdeI-HindIII site of pUC57 to construct pAVOTT (Genscript). Next, Donor DNA containing the target gene was prepared.
  • TT bp attB
  • Primers were designed to amplify the EGFP gene sandwiched between 48 bp attP (TT) sequences, and PCR reaction was performed using KOD plus ver.2 (TOYOBO) (the primers used were attP-pLac- full_5: 5'-TTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATCGCAACGCAATTAATGTGAG-3 'and attP-EGFP-full_3: 5'-ATCCCGCCCAACTGGGTAAGAGCAAAGACTAACACTGTTGGGCTGGAATTACTTGTACAGCTCGTCCATGC-3'). The conditions were 95 degrees 2 minutes, (98 degrees 10 seconds, 68 degrees 1 minute) ⁇ 30 cycles.
  • PCR products were purified using the Wizard (R) SV Gel and PCR Clean-Up System (Promega). Cloning reaction was performed using the prepared Donor DNA and vector DNA. 15 pmol purified TG1 integral and 300 ng ( ⁇ 0.15 pmol) pAVOTT, 0 pmol, 0.15 pmol, 0.45 pmol Donor DNA and Reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 25 mM NaCl, 10 mM Spermidine, 1 mM DTT, 0.1 mg / ml BSA) was mixed to a total volume of 20 microliters and reacted at 37 ° C. for 1 hour.
  • Primers were designed so that the EGFP gene sandwiched between attB sequences recognized by BP Clonase of the GATEWAY system was amplified, and PCR reaction was performed using KOD plus ver.2 (TOYOBO) (the primer used was attB1 -pLacEGFP-5: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCGCAACGCAATTAATGTGAG-3 'and attB2-EGFP-3: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTGTACAGCTCGTCCA-3'). The conditions were 95 degrees 2 minutes (98 degrees 10 seconds, 68 degrees 1 minute) ⁇ 30 cycles.
  • PCR products were purified using the Wizard (R) SV Gel and PCR Clean-Up System (Promega). Cloning reaction was performed using the prepared Donor DNA and vector DNA. Mix 4 microliters of BP Clonase Mix (Invitrogen) with 300 ng of pDONR (-), 0 pmol, 0.15 pmol, 0.45 pmol of Donor DNA and 5x Reaction buffer to a total volume of 20 microliters, and react at 37 ° C for 1 hour. went. After the reaction, 2 microliters of 2 mg / ml proteinase K was added and reacted at 37 ° C. for 10 minutes to stop the recombination reaction.
  • pDONR pDONR
  • reaction solution was added to 50 microliters of Competent Quick DH5a (TOYOBO), and the reaction was performed at 42 ° C. for 30 seconds. Thereafter, 450 microliters of LB medium was added and reacted at 37 ° C. for 1 hour. 20 microliters in the reaction solution was smeared on an LB selective medium containing 0.1 mM IPTG, 0.04 mg / ml X-gal, 30 microgram / ml Kanamycin, and cultured at 37 ° C. overnight. Thereafter, the number of colonies was counted, and the number of fluorescent colonies / total number of colonies * 100 was calculated as the recombination efficiency.
  • TG1 integration using an attachment site with a substituted core sequence In order to verify whether the site-specific recombination reaction by TG1 integrase occurs between sequences different from the original attachment site, a plasmid was prepared by substituting the core sequences of attB and attP with TA. The presence or absence of integration activity was measured by the combination of attP (TT) and attB (TT), attP (TA) and attB (TA), attP (TT) and attB (TA), and attP (TA) and attB (TA).
  • the composition of the reaction solution used for the recombination reaction was sterile ultrapure water, 1 ⁇ reaction buffer, 0.06 pmol plasmid, 0.6 pmol 50 bp attsite fragment, 12 pmol TG1 integrase.For dilution of TG1 integrase, 0.5 ⁇ PreScission buffer Using 50% Glycerol, the final concentration in the reaction solution was adjusted to 10%, and the total amount of the reaction solution was adjusted to 20 microliters. This reaction solution was reacted at 30 ° C. for 2 hours, and then subjected to heat shock at 75 ° C. for 10 minutes to stop the enzyme reaction.
  • a plasmid was extracted from the obtained colonies, subjected to restriction enzyme treatment and confirmed, and as a result, a band of the desired size could be confirmed.
  • FIG. 5 as a result of substituting the core sequence of att TG site recognized by TG1 integrase, recombination reactions were observed with pUCattP (TT) and attB (TT), pUCattB (TT) And attP (TT), pUCattP (TA) and attB (TA), pUCattB (TA) and attP (TA), and the core sequence is TT or TA. Confirmed and suggested that unidirectional cloning is possible.
  • TG1 integrase gene cloning can be performed more simply and efficiently than the conventional cloning method.

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Abstract

La présente invention concerne une nouvelle intégrase qui peut catalyser une recombinaison spécifique à un site rapidement à de faibles concentrations de substrat. Le procédé de recombinaison spécifique à un site comprend l'utilisation d'une intégrase pour recombiner un premier ADN flanqué par deux attB et un deuxième ADN flanqué par deux attP, ou un premier ADN flanqué par deux attP et un deuxième ADN flanqué par deux attB, où l'intégrase est une intégrase de phage TG1.
PCT/JP2011/053990 2010-02-24 2011-02-23 Procédé et kit de recombinaison spécifique à un site WO2011105429A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002500861A (ja) * 1997-10-24 2002-01-15 ライフ テクノロジーズ，インコーポレイテッド 組換え部位を有する核酸を使用する組換えクローニング
WO2006041096A1 (fr) * 2004-10-12 2006-04-20 Techno Network Shikoku Co., Ltd. Integrase provenant de bacteriophage staphylococcus aureus φmr11 et procede de recombinaison de gene specifique au site utilisant la sequence de reconnaissance de celui-ci
JP2007512838A (ja) * 2003-12-01 2007-05-24 インヴィトロジェン コーポレーション 組換え部位を含む核酸分子およびその使用方法
JP2007228920A (ja) * 2006-03-02 2007-09-13 Yamasa Shoyu Co Ltd 新規なインテグラーゼおよびその遺伝子

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002500861A (ja) * 1997-10-24 2002-01-15 ライフ テクノロジーズ，インコーポレイテッド 組換え部位を有する核酸を使用する組換えクローニング
JP2007512838A (ja) * 2003-12-01 2007-05-24 インヴィトロジェン コーポレーション 組換え部位を含む核酸分子およびその使用方法
WO2006041096A1 (fr) * 2004-10-12 2006-04-20 Techno Network Shikoku Co., Ltd. Integrase provenant de bacteriophage staphylococcus aureus φmr11 et procede de recombinaison de gene specifique au site utilisant la sequence de reconnaissance de celui-ci
JP2007228920A (ja) * 2006-03-02 2007-09-13 Yamasa Shoyu Co Ltd 新規なインテグラーゼおよびその遺伝子

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ESPOSITO, DOMINIC ET AL.: "Gateway Cloning for Protein Expression.", METHODS. MOL. BIOL., vol. 498, 2009, pages 31 - 54 *
MORITA, KENTARO ET AL.: "In vitro characterization of the site-specific recombination system based on actinophage TG1 integrase.", MOL. GENET. GENOMICS, vol. 282, 2009, pages 607 - 616, XP019760742, DOI: doi:10.1007/s00438-009-0490-2 *
NAKAYAMA, GAKU ET AL.: "Site-specific gene integration in cultured silkworm cells mediated by phiC31 integrase.", MOL. GENET. GENOMICS, vol. 275, 2006, pages 1 - 8, XP019346013, DOI: doi:10.1007/s00438-005-0026-3 *

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