WO2013089123A1 - 遺伝子ターゲティングベクター及びその利用方法 - Google Patents
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Definitions
- the present invention relates to a gene targeting vector and a method for using the same.
- Non-patent Documents 1 and 2 Using the homologous recombination ability of cells, only specific genes on the genome can be destroyed or replaced with artificially introduced DNA fragments (Non-patent Documents 1 and 2). This is called gene targeting. This technique has not only demonstrated tremendous power in individual gene function analysis, but is also expected as an ideal gene therapy method and breed improvement method (Non-patent Document 3). However, the efficiency of gene targeting in general higher animal and plant cells is extremely low, and development of an improved method is desired. It has been found that the use of an exon trap type targeting vector that does not have a promoter in the marker gene reduces the frequency of appearance of random inserts and can increase gene targeting efficiency (Non-Patent Documents 4 and 5). However, since expression of a marker gene can also occur by random insertion into a non-target gene, development of an improved method for further efficiency has been demanded.
- An object of the present invention is to provide a gene targeting vector that enables highly efficient gene targeting.
- Another object of the present invention is to provide a method for producing gene knockout cells using a gene targeting vector that enables highly efficient gene targeting.
- the inventor of the present application adds a DNA sequence enabling bicistronic expression such as an IRES sequence or 2A sequence to the gene targeting vector (for example, exon trap type targeting vector) 5 ′ upstream of the selection marker, Developed technology to improve gene targeting efficiency (Japanese Patent Application 2011-118564, filed May 27, 2011).
- the inventor of the present application caused random insertion by adding a negative selection marker such as a suicide gene having a splice acceptor sequence upstream of the 5 ′ homology region of the target site in the gene targeting vector of Japanese Patent Application No. 2011-118564.
- a negative selection marker such as a suicide gene having a splice acceptor sequence upstream of the 5 ′ homology region of the target site in the gene targeting vector of Japanese Patent Application No. 2011-118564.
- the gist of the present invention is as follows.
- a gene targeting vector having a structure in which a positive selection marker is sandwiched between DNA homologous to the 5 ′ upstream region of the target site and DNA homologous to the 3 ′ downstream region of the target site, the 5 ′ of the positive selection marker A splice acceptor site and a DNA sequence enabling bicistronic expression are added upstream, and a splice acceptor site is also added 5 'upstream of the DNA homologous to the 5' upstream region of the target site.
- Said vector Said vector.
- gene targeting can be performed with higher efficiency than before.
- the method of the present invention is particularly effective for gene targeting using an exon trap type targeting vector.
- the present invention is effective for generalization and efficiency of gene knockout / knock-in in the fields of basic biology, medicine, and agriculture and livestock. This specification includes the contents described in the specification and / or drawings of the Japanese patent application, Japanese Patent Application No. 2011-220772 which is the basis of the priority of the present application.
- Targeting vector structure (A) Structure of a general substitution targeting vector. When a targeting vector is introduced into a cell and colony formation is performed in the presence of a selected drug, a homologous recombinant in which the target site and the drug resistance gene are recombined and a non-targeted insertion of the targeting vector at random positions on the chromosome. Homologous recombinants are obtained, but the latter accounts for the overwhelming majority. That is, since both homologous recombinants and non-homologous recombinants have drug resistance genes, it is difficult to obtain homologous recombinants only by this drug selection.
- a suicide gene such as DT-A
- the non-homologous recombinant is killed by the expression of the suicide gene integrated on the chromosome.
- the ellipses in the figure represent cells, and the rod-shaped squares therein represent chromosomes.
- the dark gray region in the chromosome represents the target site, the light gray region in the chromosome and the targeting vector represents the homologous region.
- the region sandwiched between the arms of the targeting vector represents the drug resistance gene, and the black square region represents DT-A.
- B An example of the structure of a promoterless targeting vector. Unlike replacement targeting vectors, genes used as positive selection markers do not have their own promoters.
- Hyg represents a hygromycin resistance gene
- DTA represents a diphtheria toxin A fragment gene
- Km r represents a kanamycin resistance gene
- Amp r represents an ampicillin resistance gene.
- the underlined portion in the primer sequence represents each att sequence
- N represents the template-specific sequence
- the portion surrounded by a frame represents the recognition sequence for I-SceI.
- the template-specific sequence should be about 25 nt long.
- the positive selectable marker has a splice acceptor site 5 'upstream and a DNA sequence that allows bicistronic expression. A poly A addition signal is added 3 'downstream.
- B Problem of gene targeting by exon trap type targeting vector of Japanese Patent Application No. 2011-118564. Positive selection marker gene expression can occur not only in clones inserted into the target gene by targeting by homologous recombination (upper figure), but also in clones in which random insertion into non-target genes occurred (lower figure) . In particular, clones inserted into genes with high expression levels can acquire stronger drug resistance than clones inserted into target genes.
- a splice acceptor sequence is added upstream of the 5 'arm (the vector shown in the lower part of Fig. 6).
- a fluorescent protein gene here, GFP gene
- a splice acceptor sequence may simply be added upstream of the 5 'arm (bottom).
- Gene targeting is a technology that uses a homologous recombination mechanism to introduce a mutation at an arbitrary position on a chromosome.
- the frequency of homologous recombination in higher organisms is low, and in general, the frequency of random insertion at different sites is more than 100 times higher than the frequency at which targeting vectors introduced into cells are inserted into target sites. Therefore, it is necessary to devise targeting vectors so that homologous recombinants can be efficiently selected and obtained.
- the most commonly used replacement targeting vectors are DNA fragments homologous to the 5 ′ upstream region and 3 ′ downstream region of the target site (region to be deleted) (hereinafter referred to as “5 ′ arm” and “3 ′ arm”, respectively).
- Positive selection markers include drug resistance genes such as Hyg (hygromycin resistance gene), Puro (puromycin resistance gene), ⁇ -geo (a fusion gene of ⁇ -galactosidase gene and neomycin resistance gene), and fluorescent protein genes such as GFP gene And luciferase gene.
- drug resistance genes such as Hyg (hygromycin resistance gene), Puro (puromycin resistance gene), ⁇ -geo (a fusion gene of ⁇ -galactosidase gene and neomycin resistance gene
- fluorescent protein genes such as GFP gene And luciferase gene.
- negative selection genes include suicide genes such as HSV-TK and DT-A.
- negative selection genes include suicide genes such as HSV-TK and DT-A.
- a promoterless method including the “exon trap method” using a drug resistance gene (positive selection marker) without a promoter (FIG. 1B).
- the expression of the positive selectable marker gene is turned on when homologous recombination occurs.
- the expression of the positive selection marker gene may be turned on also by random insertion by non-homologous recombination.
- ExTraPNS method a technique for reducing recombinants due to the latter reaction has been devised.
- Multisite Gateway technology (Iiizumi, S, Nomura, Y, So, S, et al. (2006) Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines.
- Biotechniques 41 A method for producing a replacement targeting vector using 311-316) will be described as an example (Fig. 2).
- a 5 ′ arm having attB4 and attB1 sequences at both ends (corresponding to “a DNA fragment homologous to the 5 ′ upstream region of the target site” in the present invention), pDONR P4-P1R and attB2 sequences 5 'entry by performing BP recombination between the 3' arm with both ends of the attB3 sequence (corresponding to the "DNA fragment homologous to the 3 'downstream region of the target site” in the present invention) and pDONR P2R-P3 Clone and 3 'entry clone are created respectively.
- the 5 'arm and 3' arm are amplified and obtained by genomic PCR.
- the reverse primer for 5 'arm amplification should be designed on the exon of the target gene.
- a SA site splice acceptor site
- the SA site is not limited to the splice acceptor sequence (SA sequence) of the target gene, and other SA sites may be used.
- 5 'upstream of the hygromycin resistance gene (which may be another selectable marker) is a DNA sequence that enables bicistronic expression (eg, IRES sequence (internal ribosomal entry site); ribosomal entry site in mRNA; encephalomyocarditis virus (EMCV), etc.), 2A peptide sequence (2A “self-cleaving” peptide; Those asigna virus (TaV), etc.), IRES2, etc.) are added. Since a DNA sequence allowing bicistronic expression exists 5 ′ upstream of the selection marker, gene expression of the selection marker occurs depending on the promoter of the target gene when gene targeting occurs.
- IRES sequence internal ribosomal entry site
- ribosomal entry site in mRNA e.g., ribosomal entry site in mRNA
- EMCV encephalomyocarditis virus
- 2A peptide sequence 2A “self-cleaving” peptide; Those as
- the hygromycin resistance gene should be sandwiched between lox71 and loxP.
- the marker for selection can be removed from the genome by transient expression of Cre after gene targeting.
- the technique for marker removal is not limited to this. That is, target sequences of other site-specific recombinant enzymes such as lox sequences and FRT sequences can also be used.
- a splice acceptor site may be introduced into the entry clone pENTR IRES-Hyg.
- a reverse primer for 5 'arm amplification can be set in the intron (not the exon).
- DNA sequences that enable bicistronic expression such as IRES and 2A peptide sequences when negative selection markers such as suicide genes such as the DTA gene and fluorescent protein genes such as the GFP gene are inserted into the cassette for negative selection
- negative selection markers such as suicide genes such as the DTA gene and fluorescent protein genes such as the GFP gene
- the preparation of the targeting vector (specifically, the step of linking the 5 ′ arm, the selection marker and the 3 ′ arm) has been described by taking the case of using the Invitrogen MultiSite Gateway system as an example. It is not limited to. That is, production and use by other molecular biological methods (for example, general methods using restriction enzymes and ligases, In-Fusion PCR PCR Cloning, etc.) are also possible.
- a vector that can be used as a base for creating a targeting vector by a normal method without using an entry clone that is, the Gateway system
- a vector in which a DNA fragment and a site for incorporating a DNA fragment homologous to the 3 ′ downstream region of the target site and a linearization site are incorporated may be used.
- a gene knockout cell can be prepared by introducing a gene mutation into a cell using the gene targeting vector of the present invention.
- Gene knockout cells can be obtained by known methods (eg, Adachi, N, So, S, Iiizumi, S, et al. (2006) The human pre-B cell line Nalm-6 is highly proficient in gene targeting by homologous recombination. DNA Cell Biol. 25: 19-24; Adachi, N, Nishijima, H, Shibahara, K (2008) Gene targeting using the human Nalm-6 pre-B cell line. BioScience Trends. 2: 169-180; Toyoda, E, Kagaya, S, Cowell, IG, et al.
- the marker for selection can be removed by introducing a site-specific recombinase expression vector (for example, plasmid pBS185 which is a Cre recombinase expression vector) into cells. Another mutation can be introduced into the cells from which the selectable marker has been removed.
- a site-specific recombinase expression vector for example, plasmid pBS185 which is a Cre recombinase expression vector
- Another mutation can be introduced into the cells from which the selectable marker has been removed.
- cells suitable for gene targeting include, but are not limited to, human Nalm-6 cells, chicken DT40 cells, mouse ES cells, and the like. Rather, the present invention facilitates gene targeting even in general human cell lines.
- pENTR IRES-Hyg is a pENTR loxP plasmid (Iiizumi, S, Nomura, Y, So, S, et al. (2006) Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines.Biotechniques 41: 311-316) was digested with NotI and then added with lox71, IRES, Hyg, pA, and loxP sequentially. lox71 and loxP were added using synthetic linker DNA.
- Hyg is pENTR lox-Hyg (Iiizumi, S, Nomura, Y, So, S, et al. (2006) Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines. Biotechniques 41: 311 -316). 5.
- Destination vector pDEST R4-R3 (Invitrogen) 6.
- pDEST R4-R3 SA-IRES-DTA-pA Destination vector (pDEST R4-R3 SA-IRES-DTA-pA)
- pDEST R4-R3 SA-IRES-DTA-pA was prepared by the following procedure. 1. Perform PCR using primers DTA-Sal Fw (5'-GTCGACATGGATCCTGATGATGTTGTTGAT-3 ') (SEQ ID NO: 7) and DTA-Not Rv (GCGGCCGCTTAGAGCTTTAAATCTCTGTAGGTA) (SEQ ID NO: 8), and obtain the resulting DTA gene fragment as a pIRES vector. (Clontech, Cat. No. 631605) was subcloned into the NotI site.
- PCR was performed using KOD-Plus-DNA polymerase (TOYOBO) under the following conditions. 94 °C 2 min 94 °C 30 sec 65 °C 30 sec x 35 cycles 68 °C 40 sec 68 °C 7 min 2. After digesting pSA ⁇ geo plasmid (Friedrich, G. & Soriano P. Genes Dev. 5, 1513-1523, 1991) with BamHI and blunting the ends with Klenow fragment, the SA site (adenovirus type 2 major late A fragment of about 200 bp containing the transcript splice acceptor) was added upstream of the IRES sequence of the vector prepared in 1. above (XhoI site; blunted with Klenow fragment). 3.
- SA site adenovirus type 2 major late A fragment of about 200 bp containing the transcript splice acceptor
- LB agar medium containing antibiotics LB agar medium containing 50 ⁇ g / ml kanamycin or 50 ⁇ g / ml ampicillin
- Protocol 1 PCR was performed using human genomic DNA as a template under the following conditions to obtain an HPRT genomic fragment sandwiched between att sequences. Primers (1) and (2) were used for amplification of the 5 ′ arm, and primers (3) and (4) were used for amplification of the 3 ′ arm.
- PCR product was purified with a commercially available kit and quantified.
- 3′-entry clone were prepared by BP recombination reaction (FIG. 2A). The following samples were mixed in a 0.5 ml tube.
- pDONR P4-P1R or pDONR P2R-P3 50 fmoles PCR amplified 5'-arm or 3'-arm 50 fmoles Total 8 ⁇ l (combine with TE solution) 4.
- BP clonase II enzyme mix was added and mixed well. 5. Incubate at 25 ° C for 4-5 hours. 6.
- a targeting vector (pHPRT-IRES-Hyg) was prepared by LR recombination reaction (FIG. 2B). Each sample was mixed in a 0.5 ml tube as described below.
- Protocol 1 The targeting vector was digested with restriction enzyme I-SceI. Each reagent was mixed as follows and incubated at 37 ° C. for 4 hours or more.
- Targeting vector 50 ⁇ g 10x I-SceI buffer 40 ⁇ l 100x BSA (10 mg / ml) 4 ⁇ l I-SceI 15 unit total volume 400 ⁇ l (combine with sterile water) 2.
- 40 ⁇ l of 3 M sodium acetate and 0.9 ml of ethanol were added and mixed well. 3.
- TE solution was added to dissolve the DNA (DNA concentration was adjusted to 2-4 ⁇ g / ⁇ l). 7. Incubate at 65 ° C for 15 minutes.
- Growth medium ES medium (Nissui Pharmaceutical) plus 10% calf serum (HyClone). Keep at 37 ° C in a hot water bath. 2. Linearized targeting vector 3. Cell Line Nucleofector (registered trademark) Kit T (Lonza) 4. Selected drug (100 mg / ml hygromycin B): 1 g hygromycin B dissolved in 10 ml of pure water and sterilized by filtration (stored at 4 ° C)
- Protocol 1 Human HT1080 cells (2 ⁇ 10 6 or more) in the logarithmic growth phase were collected in a 50 ml centrifuge tube. 2. Centrifuge at 1,100 rpm for 5 minutes, and gently remove the supernatant. 3. 100 ⁇ l of Solution T was added to the cell mass, and the number of cells was counted. 4. 2 ⁇ 10 6 cells and 2 ⁇ g of the linearized targeting vector were mixed, adjusted to 100 ⁇ l with Solution T, and transferred to a dedicated cuvette. 5. The cuvette was set in an electroporation apparatus (Nucleofector II, Lonza). 6. Program L-005 was executed. 7. The entire volume was immediately transferred to a 60 mm dish containing 4 ml of growth medium. 8.
- Selection medium medium containing hygromycin B: Growth medium with hygromycin B added to a concentration of 0.25 to 0.4 mg / ml 2.
- 0.1% trypsin solution 0.1% trypsin /0.02%EDTA/PBS - 3.
- Lysis buffer 20 mM Tris-HCl buffer (pH 8.0), 250 mM sodium chloride, 1% SDS 4.
- 10 mg / ml proteinase K 100 mg proteinase K dissolved in 10 ml pure water and sterilized by filtration (stored at -20 ° C) 5.
- Saturated NaCl solution 6.
- PCR primers for confirmation of gene targeting; Iiizumi et al. Nucleic Acids Res. 2008 Nov; 36 (19): 6333-6342.)
- HPRT-F 5'-TGAGGGCAAAGGATGTGTTACGTG-3 '(SEQ ID NO: 5)
- HPRT-R 5'-TTGATGTAATCCAGCAGGTCAGCA-3 '(SEQ ID NO: 6)
- Protocol 1 1.5 ml each of selective medium and 0.75 ml of growth medium were dispensed into 24-well plates. 2. After treatment with 0.1% trypsin solution, the solution was transferred to a well containing 1.5 ml of the selective medium while gently pipetting (for genome extraction). 3. From the 24-well plate to which the cells were transferred, 0.25 ml cell solution was taken and transferred to a well containing 0.75 ml of growth medium (for subculture). 4. Incubate at 37 ° C for 2-3 days. 5. After removing each culture solution from the 24-well plate for genome extraction, 250 ⁇ l of lysis buffer and 1 ⁇ l of 10 mg / ml proteinase K were added and transferred to a 1.5 ml tube while pipetting well. 6.
- a drug resistance gene instead of the hygromycin resistance gene, a puromycin resistance gene or a fusion gene of an eGFP gene and a puromycin resistance gene was used, and the above operation was repeated to prepare a targeting vector.
- an IRES2 sequence or a 2A peptide sequence was used instead of the IRES sequence, and the above operation was repeated to prepare a targeting vector.
- the present invention is effective for generalization and efficiency of gene knockouts and gene traps in the fields of basic biology, medicine, and agriculture and livestock.
- SEQ ID NO: 1 shows the DNA sequence of a forward primer for 5 ′ arm amplification targeting the human HPRT gene.
- SEQ ID NO: 2 shows the DNA sequence of the reverse primer for 5 ′ arm amplification targeting the human HPRT gene.
- SEQ ID NO: 3 shows the DNA sequence of the forward primer for 3 ′ arm amplification targeting the human HPRT gene. 5'- GGGG ACAGCTTTCTTGTACAAAGTGG CCTGCAGGATCACATTGTAGCCCTCTGTGTGC -3 ' (Underlined is attB2 array) ⁇ SEQ ID NO: 4> SEQ ID NO: 4 shows the DNA sequence of the reverse primer for 3 ′ arm amplification targeting the human HPRT gene.
- SEQ ID NO: 5 shows the DNA sequence of a PCR primer (HPRT-F) for confirmation of gene targeting.
- HPRT-F 5'-TGAGGGCAAAGGATGTGTTACGTG-3 '
- SEQ ID NO: 6 shows the DNA sequence of PCR primer (HPRT-R) for confirmation of gene targeting.
- SEQ ID NO: 7 shows the DNA sequence of primer DTA-Sal Fw (5′-GTCGACATGGATCCTGATGATGTTGTTGAT-3 ′).
- SEQ ID NO: 8 The DNA sequence of SEQ ID NO: 8, primer DTA-Not Rv (GCGGCCGCTTAGAGCTTTAAATCTCTGTAGGTA) is shown.
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Abstract
Description
(1)標的部位の5’上流領域に相同なDNAと標的部位の3’下流領域に相同なDNAとでポジティブ選択マーカーを挟む構造を持つ遺伝子ターゲティングベクターであって、前記ポジティブ選択マーカーの5’上流にスプライスアクセプター部位とバイシストロン性発現を可能にするDNA配列とが付加されており、さらに、前記標的部位の5’上流領域に相同なDNAの5’上流にもスプライスアクセプター部位が付加されている前記ベクター。
(2)ポジティブ選択マーカーにポリA配列が付加されているが、プロモーターは付加されていない(1)記載のベクター。
(3)標的部位の5’上流領域に相同なDNAの5’上流に付加されたスプライスアクセプター部位の3’下流にポリA配列が付加されている(1)又は(2)記載のベクター。
(4)標的部位の5’上流領域に相同なDNAの5’上流に付加されたスプライスアクセプター部位と、その3’下流に付加されたポリA配列との間に、ネガティブ選択マーカーが導入されている(3)記載のベクター。
(5)さらにリニア化部位が導入されている(1)~(4)のいずれかに記載のベクター。
(6)(1)~(5)のいずれかに記載の遺伝子ターゲティングベクターを用いて、細胞に遺伝子変異を導入することを含む、遺伝子ノックアウト細胞を作製する方法。
本明細書は、本願の優先権の基礎である日本国特許出願、特願2011‐272072の明細書および/または図面に記載される内容を包含する。
5'アーム増幅用のリバースプライマーは、標的遺伝子のエクソン上に設計しておくとよい。これにより、標的遺伝子上において上流のエクソンからポジティブ選択マーカー遺伝子(が挿入されたエクソン)へのナチュラルなスプライシングを可能にするSA部位(splice acceptor site)を5'アーム中に含めることができる。SA部位は、標的遺伝子のスプライスアクセプター配列(SA配列)に限定されるわけではなく、他のSA部位を用いてもよい。
(材料及び方法)
準備するもの
1. ExTaqTM ポリメラーゼ(タカラバイオ)
2. PCRプライマー: HPRT遺伝子の増幅を例にとる。
5'アーム増幅用
(1) HPRT 5'Fw, 5’- GGGGACAACTTTGTATAGAAAAGTTGCACATCACAGGTACCATATCAGTG -3’(配列番号1);
(2) HPRT 5'Rv (エクソン上に設定する), 5’- GGGGACTGCTTTTTTGTACAAACTTGCACATCTCGAGCAAGACGTTCAGT -3’(配列番号2);
3'アーム用
(3) HPRT 3'Fw, 5’- GGGGACAGCTTTCTTGTACAAAGTGGCCTGCAGGATCACATTGTAGCCCTCTGTGTGC -3’ (配列番号3);
(4) HPRT 3'Rv (リニア化部位となるI-SceIサイトを付加しておく), 5’- GGGGACAACTTTGTATAATAAAGTTGCTATATTACCCTGTTATCCCTAGCGTAACTCAGGGTAGAAATGCTACTTCAGGC -3’ (配列番号4)
3. MultiSite Gateway(登録商標) Three Fragment Vector Construction Kit(Invitrogen)
4. 薬剤耐性遺伝子が組み込まれたエントリークローン(pENTR IRES-Hyg)
pENTR IRES-Hygは、pENTR loxPプラスミド(Iiizumi, S, Nomura, Y, So, S, et al. (2006) Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines. Biotechniques 41: 311-316)をNotIで消化した後、lox71, IRES, Hyg, pA, loxPを順次付加して作製した。lox71とloxPは合成リンカーDNAを用いて付加した。IRESとpAはpIRESベクター(タカラバイオ;http://catalog.takara-bio.co.jp/product/basic_info.asp?unitid=U100004407)に由来する。HygはpENTR lox-Hyg(Iiizumi, S, Nomura, Y, So, S, et al. (2006) Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines. Biotechniques 41: 311-316)に由来する。
5. デスティネーションベクター (pDEST R4-R3) (Invitrogen)
6. デスティネーションベクター (pDEST R4-R3 SA-IRES-DTA-pA)
pDEST R4-R3 SA-IRES-DTA-pAは、以下の手順で作製した。
1. プライマー DTA-Sal Fw (5'-GTCGACATGGATCCTGATGATGTTGTTGAT-3')(配列番号7) と DTA-Not Rv (GCGGCCGCTTAGAGCTTTAAATCTCTGTAGGTA) (配列番号8) を用いてPCRを行い、得られたDTA遺伝子断片をpIRESベクター (Clontech, Cat. No. 631605) のNotIサイトにサブクローニングした。PCRは KOD-Plus- DNAポリメラーゼ (TOYOBO) を使用し、下記の条件で行った。
94℃ 2 min
94℃ 30 sec
65℃ 30 sec ×35サイクル
68℃ 40 sec
68℃ 7 min
2. pSAβgeoプラスミド (Friedrich, G. & Soriano P. Genes Dev. 5, 1513-1523, 1991) をBamHIで消化し、Klenow断片を用いて末端を平滑化したのち、SA部位(adenovirus type 2 major late transcript splice acceptorに由来)を含む約200 bpの断片を上記1.で作製したベクターのIRES配列の上流(XhoIサイト; Klenow断片により平滑末端化)に付加した。
3. 上記2.で作製したプラスミドをBglIIとPvuIで消化し、平滑末端化したのち、SA-IRES-DTA-pAを含むDNA断片を pDEST R4-R3プラスミド (Invitrogen) のAflIIIサイト(Klenow断片により平滑末端化)に付加した。
7. 抗生物質含有LB寒天培地:50 μg/mlカナマイシンまたは50 μg/mlアンピシリンを含むLB寒天培地
1. ヒトゲノムDNAを鋳型として下記の条件でPCRを行い,att配列で挟まれたHPRTゲノム断片を取得した。5’アームの増幅にはプライマー(1)と(2)を、3’アームの増幅にはプライマー(3)と(4)を使用した。
3. BP組換え反応により、5’-エントリークローンおよび3’-エントリークローンを作製した(図2A)。下記のサンプルを0.5 mlチューブ内で混合した。
pDONR P4-P1R または pDONR P2R-P3 50 fmoles
PCRで増幅した5’-アームまたは3’-アーム 50 fmoles
全量 8 μl(TE溶液であわせる)
4. 上記反応液4 μl に1 μlのBPクロナーゼ II酵素ミックスを加え、よく混合した。
5. 25℃で4~5時間インキュベートした。
6. 1 μlの2 μg/μl プロテイナーゼKを加え、よく混合した。
7. 37℃で10分インキュベートした。
8. 5 μlの反応液を50 μlの大腸菌コンピテントセルと混和し、形質転換を行った。回復培養後、組換え体を50 μg/ml カナマイシンを含むLB寒天培地に播いた。
9. カナマイシン耐性コロニーを5~10個単離し、アルカリSDS法でプラスミドDNAを抽出した。アガロースゲル電気泳動により、目的のプラスミドと予想されるクローンを2~3個選んだ。これらの候補プラスミドを適当な制限酵素で消化したのち、アガロースゲル電気泳動を行い、目的のプラスミドであることを確認した。
10. 得られた 5’、 3’ 各エントリークローンを市販のキットで精製し、定量した。
11. LR組換え反応により、ターゲティングベクター(pHPRT-IRES-Hyg)を作製した(図2B)。各サンプルを下記のとおりに0.5 mlチューブ内で混合した。
pDEST R4-R3 または pDEST R4-R3 SA-IRES-DTA-pA 20 fmoles
5’-エントリークローン 10 fmoles
3’-エントリークローン 10 fmoles
pENTR IRES-Hyg 10 fmoles
全量 8 μl(TE溶液であわせる)
12. 上記反応液4 μl に1 μlのLRクロナーゼプラス酵素ミックスを加え、よく混合した。
13. 25℃で16時間インキュベートした。
14. 1 μlの2 μg/μl プロテイナーゼKを加え、よく混合した。
15. 37℃で10分インキュベートした。
16. 5 μlの反応液を50 μlの大腸菌コンピテントセルと混和し、形質転換を行った。回復培養後、組換え体を50 μg/ml アンピシリンを含むLB寒天培地に播いた。
17. アンピシリン耐性コロニーを5~10個単離し、アルカリSDS法でプラスミドDNAを抽出した。アガロースゲル電気泳動により、目的のプラスミドと予想されるクローンを2~3個選んだ。これらの候補プラスミドを適当な制限酵素で消化したのち、アガロースゲル電気泳動を行い、目的のプラスミド(すなわちターゲティングベクター)であることを確認した。
18. ターゲティングベクターをキットで精製し、定量した。
準備するもの
1. 制限酵素 I-SceI、10x I-SceI反応バッファー、10 mg/ml BSA(New England Biolabs)
2. 3 M 酢酸ナトリウム:酢酸ナトリウム 40.81 gを純水80 mlに溶かしたのち、酢酸でpHを5.2に合わせ、全量を100 mlとしたもの
3. TE溶液:10 mM トリス塩酸緩衝液(pH 8.0)、0.1 mM EDTA (pH 8.0)(4℃保存)
1. ターゲティングベクターを制限酵素 I-SceI で消化した。各試薬を下記のとおりに混合し、37℃で4時間以上インキュベートした。
ターゲティングベクター 50 μg
10x I-SceI バッファー 40 μl
100x BSA (10 mg/ml) 4 μl
I-SceI 15 ユニット
全量 400 μl(滅菌水であわせる)
2. 40 μlの3 M酢酸ナトリウムと0.9 mlのエタノールを加え、よく混合した。
3. 15,000 rpmで5分遠心した。
4. 0.5 mlの70%エタノールで3回洗浄した。
5. 3回目の遠心後、クリーンベンチ内で滅菌済みチップを使って上清を除去し、風乾した。
6. TE溶液を加え、DNAを溶解した(DNA濃度が2~4 μg/μlとなるようにした)。
7. 65℃で15分インキュベートした。
準備するもの
1. 増殖培地:ES培地(日水製薬)に10% 仔ウシ血清(HyClone)を加えたもの。湯浴で37℃に保温しておく。
2. 直鎖化したターゲティングベクター
3. Cell Line Nucleofector(登録商標) Kit T(Lonza)
4. 選択薬剤(100 mg/ml ハイグロマイシンB):1 gのハイグロマイシンBを純水10 mlに溶かし、ろ過滅菌したもの(4℃保存)
1. 対数増殖期にあるヒトHT1080細胞(2×106個以上)を50 ml遠心チューブに回収した。
2. 1,100 rpmで5分遠心し、上清を静かに除いた。
3. 細胞塊にSolution Tを100 μl加え、細胞数をカウントした。
4. 2×106個の細胞と直鎖化したターゲティングベクター2 μgを混合し、Solution Tで100 μlとしたのち、専用のキュベットに移した。
5. キュベットをエレクトロポレーション装置(Nucleofector II, Lonza)にセットした。
6. プログラムL-005を実行した。
7. 全量をただちに増殖培地4 mlの入った60 mmディッシュに移した。
8. 上記細胞液を1 mlずつ増殖培地9 mlの入った90 mmディッシュ (×4枚) に移した。
9. 37℃で48時間培養した。
10. トリプシン処理した後1,100 rpmで5分間遠心し、細胞を回収した。
11. 適量の増殖培地に懸濁し、細胞数をカウントした。
12. 2~4×105 cells/dishとなるように、90 mmディッシュ(増殖培地9 ml)に播種した。
13. 37℃で24時間培養した後、25~40 μlの選択薬剤(100 mg/ml ハイグロマイシン)を加えた。
14. 37℃で2週間培養し、コロニー形成を行った。
準備するもの
1. 選択培地(ハイグロマイシンB含有培地):増殖培地にハイグロマイシンBを0.25~0.4 mg/mlとなるよう加えたもの
2. 0.1%トリプシン液:0.1%トリプシン/0.02%EDTA/PBS-
3. 溶解バッファー:20 mM トリス塩酸緩衝液(pH 8.0)、250 mM 塩化ナトリウム、1% SDS
4. 10 mg/ml プロテイナーゼK:100 mgのプロテイナーゼKを純水10 mlに溶かし、ろ過滅菌したもの(-20℃保存)
5. 飽和NaCl溶液
6. ExTaqTM ポリメラーゼ
7. PCRプライマー(遺伝子ターゲティングの確認用; Iiizumi et al. Nucleic Acids Res. 2008 Nov;36(19):6333-6342.)
HPRT-F, 5'-TGAGGGCAAAGGATGTGTTACGTG-3' (配列番号5)
HPRT-R, 5'-TTGATGTAATCCAGCAGGTCAGCA-3' (配列番号6)
1. 選択培地を1.5 ml, 増殖培地を0.75 mlずつそれぞれ24ウェルプレートに分注した。
2. 0.1%トリプシン液で処理した後、軽くピペッティングしながら選択培地1.5 mlの入ったウェルに移した (ゲノム抽出用)。
3. 細胞を移した24ウェルプレートから、0.25 ml細胞液を取り増殖培地0.75 mlの入ったウェルに移した(継代用)。
4. 37℃で2~3日培養した。
5. ゲノム抽出用24ウェルプレートの各培養液を除去したのち、250 μlの溶解バッファーと1 μlの10 mg/mlプロテイナーゼKを加え、よくピペッティングしながら1.5 mlチューブへ移した。
6. 37℃で一晩(または55℃で1時間)インキュベートした。
7. 80 μlの飽和NaCl溶液を加え、よく混合した。
8. さらに250 μlの2-プロパノールを加え、よく混合した。
9. 15,000 rpm、4℃で15分遠心を行った。
10. 沈殿を除き、0.5 mlの70%エタノールで洗浄した。
11. 沈殿を30~100 μlのTE溶液に溶解させた。
12. 調製したゲノムDNAを鋳型として、プライマーHPRT-FとHPRT-Rを用いて下記の反応条件でPCRを行い、ターゲットクローンをスクリーニングした。
(結果)
ヒトHT1080細胞を用いて行った遺伝子ターゲティングの結果を下記の表にまとめる。「ネガティブ(負)選択(DT-A)あり」では、SA-IRES-DTA-pAカセットを5'アームの上流に付加したターゲティングベクターを使用した。
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。
配列番号1は、ヒトHPRT遺伝子を標的とする5’アーム増幅用のフォワードプライマーのDNA配列を示す。
5’- GGGGACAACTTTGTATAGAAAAGTTGCACATCACAGGTACCATATCAGTG -3’
(下線部はattB4配列である)
<配列番号2>
配列番号2は、ヒトHPRT遺伝子を標的とする5’アーム増幅用のリバースプライマーのDNA配列を示す。
5’- GGGGACTGCTTTTTTGTACAAACTTGCACATCTCGAGCAAGACGTTCAGT -3’
(下線部はattB1配列である)
<配列番号3>
配列番号3は、ヒトHPRT遺伝子を標的とする3’アーム増幅用のフォワードプライマーのDNA配列を示す。
5’- GGGGACAGCTTTCTTGTACAAAGTGGCCTGCAGGATCACATTGTAGCCCTCTGTGTGC -3’
(下線部はattB2配列である)
<配列番号4>
配列番号4は、ヒトHPRT遺伝子を標的とする3’アーム増幅用のリバースプライマーのDNA配列を示す。
5’- GGGGACAACTTTGTATAATAAAGTTGCTATATTACCCTGTTATCCCTAGCGTAACTCAGGGTAGAAATGCTACTTCAGGC -3’
(下線部はattB3配列である)
<配列番号5>
配列番号5は、遺伝子ターゲティングの確認用のPCRプライマー(HPRT-F)のDNA配列を示す。
HPRT-F, 5'-TGAGGGCAAAGGATGTGTTACGTG-3'
<配列番号6>
配列番号6は、遺伝子ターゲティングの確認用のPCRプライマー(HPRT-R)のDNA配列を示す。
HPRT-R, 5'-TTGATGTAATCCAGCAGGTCAGCA-3'
<配列番号7>
配列番号7は、プライマー DTA-Sal Fw (5'-GTCGACATGGATCCTGATGATGTTGTTGAT-3')のDNA配列を示す。
<配列番号8>
配列番号8、プライマーDTA-Not Rv (GCGGCCGCTTAGAGCTTTAAATCTCTGTAGGTA)のDNA配列を示す。
Claims (6)
- 標的部位の5’上流領域に相同なDNAと標的部位の3’下流領域に相同なDNAとでポジティブ選択マーカーを挟む構造を持つ遺伝子ターゲティングベクターであって、前記ポジティブ選択マーカーの5’上流にスプライスアクセプター部位とバイシストロン性発現を可能にするDNA配列とが付加されており、さらに、前記標的部位の5’上流領域に相同なDNAの5’上流にもスプライスアクセプター部位が付加されている前記ベクター。
- ポジティブ選択マーカーにポリA配列が付加されているが、プロモーターは付加されていない請求項1記載のベクター。
- 標的部位の5’上流領域に相同なDNAの5’上流に付加されたスプライスアクセプター部位の3’下流にポリA配列が付加されている請求項1又は2記載のベクター。
- 標的部位の5’上流領域に相同なDNAの5’上流に付加されたスプライスアクセプター部位と、その3’下流に付加されたポリA配列との間に、ネガティブ選択マーカーが導入されている請求項3記載のベクター。
- さらにリニア化部位が導入されている請求項1~4のいずれかに記載のベクター。
- 請求項1~5のいずれかに記載の遺伝子ターゲティングベクターを用いて、細胞に遺伝子変異を導入することを含む、遺伝子ノックアウト細胞を作製する方法。
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US14/364,922 US9303272B2 (en) | 2011-12-13 | 2012-12-12 | Gene targeting vector, and method for using same |
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Also Published As
Publication number | Publication date |
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US20140335621A1 (en) | 2014-11-13 |
JP6037290B2 (ja) | 2016-12-07 |
US9303272B2 (en) | 2016-04-05 |
EP2792744B1 (en) | 2017-07-26 |
JPWO2013089123A1 (ja) | 2015-04-27 |
EP2792744A1 (en) | 2014-10-22 |
CN104053773A (zh) | 2014-09-17 |
EP2792744A4 (en) | 2015-08-19 |
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