JP2006333742A - Cell and method for transducing dna into cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for transducing a DNA into a cell, by which the cell capable of being used for RNAi method or the like can efficiently be produced in a short time. <P>SOLUTION: This method for transducing the DNA into the cell comprises making Cre enzyme to act on a cell and a donor vector to stably transduce the insertion DNA of the donor vector into the cell. The cell is a cell in which an accept vector having lox 66 sequence represented by a base sequence having a specific sequence or lox 71 sequence represented by a base sequence having a specified sequence, and lox P sequence represented by a base sequence having a specific sequence are stably inserted into a region not containing exon and intron on the genome. The donor vector has an accept vector-free sequence among the lox 66 sequence and the lox 71 sequence, the loxP sequence, and an inserted DNA inserted between the lox 66 sequence or lox 71 sequence and the loxP sequence. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell having a predetermined base sequence stably, and a method for introducing DNA into a cell in which a desired enzyme is stably introduced by allowing a recombinant enzyme to act on the cell.

  In recent years, genome projects of various species have been elucidated by the genome project, and the types and numbers of genes responsible for life phenomena have been estimated, but there are many unknown functions. In humans, technologies (transcriptome analysis, proteome analysis, etc.) have been developed for the purpose of identifying disease-causing genes, and comprehensive analysis of gene region identification and protein expression from genes based on genomic information. ing. However, the functions of individual genes have not been elucidated, and there is a limit to the estimation of disease mechanisms from genome sequence information.

  Conventionally, as a method for analyzing individual gene functions in mammals, there is a method in which the target gene is lost (knocked out) or introduced (knocked in) in a living body (in vivo) or a cultured cell (in vitro) and the effect is examined. is there.

  The knockout method is a method of introducing a mutation into a target gene and eliminating the gene.

  In the in vivo knockout method, a mouse having a genome sequence close to that of humans and a short maturation period is frequently used, but at least one year per strain is required to produce a mouse individual knocked out by introducing a mutation into a target gene. Cost. In the in vitro system, cells collected from the knocked-out mouse body are used, and therefore the period required to obtain the knocked-out cell is about one year, as in the in vivo system.

  Since the knockout method takes time to produce individuals and cells in both in vivo and in vitro systems, a method that suppresses gene expression without losing the target gene is recently attracting attention (knockdown) Law).

  As a typical method of knockdown, there is an RNAi (RNA interference) method that specifically inhibits the function of expressed mRNA. In the RNAi method, siRNA (small interference RNA) or shRNA (short hairpin RNA) having a sequence complementary to mRNA expressed from a target gene is expressed in the genome of a living body or a cultured cell. There are a method of introducing DNA to be performed, a method of transiently introducing oligo-synthesized siRNA and shRNA into cells, and the like. siRNA and shRNA are cleaved by Dicer, which is a kind of double-stranded RNA cleaving enzyme, and then incorporated into a siRNA / protein complex (RNA-induced silencing complex (RISC)). mRNA having a sequence complementary to siRNA is recognized by the RISC complex and cleaved by the protein contained in RISC to lose its function.

  In the RNAi method, siRNA or shRNA-expressing DNA can be inserted as long as it is a region on the genome that does not inhibit the original function of the cell, so the range of insertion is wider compared to the knockout method. Moreover, what is necessary is just to insert the DNA which expresses siRNA or shRNA in any one of the double strands of DNA, and compared with the knockout method which needs to introduce | transduce a mutation into the target gene which exists in both double strands, The period for producing mouse individuals and cultured cells in which gene expression is suppressed can be dramatically shortened. In the RNAi method, the period for producing an in vivo mouse individual is about half a year, and the period for producing an in vitro cultured cell is about 3 months.

  Patent Document 1 describes a cell and a knockout mouse in which a vector having a loxP sequence and a mutant lox sequence is introduced into a predetermined gene. According to this document, an arbitrary gene can be inserted into a lox site in a vector introduced into a cell by a lox / Cre system using a recombinant enzyme (Cre enzyme).

In the cells and knockout mice described in this document, a predetermined gene is disrupted with a vector containing loxP and a mutant lox sequence, so it is necessary to extinguish that gene when examining the functions of other genes. Therefore, the preparation period of the knockout mouse itself is not shortened. Even if any DNA is inserted into the cell by a recombinant enzyme, the intrinsic function of the cell has already been lost due to the destruction of the endogenous gene, so the influence of the inserted DNA on the cell can be accurately determined. Therefore, the RNAi method cannot be applied.
JP 2002-369689 A (Claims)

  In the RNAi method, when a DNA having a sequence that expresses siRNA or shRNA is inserted into the genome of a cell, a method based on electrical stimulation or virus infection is often used. In the insertion of DNA by these methods, the insertion position and the number of inserted DNAs cannot be controlled, and the DNA is randomly inserted into the genome. As described above, the RNAi method is a method for determining the function of an endogenous gene by examining the lost function of a cell by inhibiting the function of mRNA expressed from the genome. Therefore, the DNA expressing siRNA or shRNA must be It is required to be introduced at a position that does not interfere with the original function of the cell. Therefore, when performing the RNAi method, each time DNA is inserted, it is necessary to specify the insertion position by genome analysis and select a cell introduced at a position that does not interfere with the function of the cell. I need it.

  The present inventor stably inserts an accept vector having a predetermined sequence into a region that does not damage an endogenous gene on the cell genome in advance, and the accept vector and a donor vector into which a desired DNA is inserted It was found that by recombining with a recombination enzyme, cells in which the desired DNA was stably inserted at a predetermined position in the genome could be easily obtained. Furthermore, it was confirmed that the cells obtained by appropriately selecting the DNA to be inserted can be used for knocking down the target gene using the target gene knock-in or RNAi method, and the present invention has been completed.

  That is, the present invention for solving the above problems is described below.

  [1] An accept vector having a lox66 sequence represented by SEQ ID NO: 1 or a lox71 sequence represented by SEQ ID NO: 2 and a loxP sequence represented by SEQ ID NO: 3 is stable in an exon and intron-free region on the genome. Inserted cells.

  [2] A cell in which the lox71 / 66 sequence represented by SEQ ID NO: 4, the loxP sequence represented by SEQ ID NO: 3, and the inserted DNA are stably inserted into a region not containing exons and introns on the genome. , Cells in which the inserted DNA is stably inserted between the lox71 / 66 and loxP sequences.

  [3] The cell according to [2], which has a marker gene, a promoter for expressing the marker gene, and a transcription termination signal for the marker gene between the lox71 / 66 sequence and the loxP sequence.

  [4] A cell in which an accept vector having the lox66 sequence represented by SEQ ID NO: 1 and the loxP sequence represented by SEQ ID NO: 3 is stably inserted into a region not containing exons and introns on the genome; The Cre vector is allowed to act on a donor vector having the indicated lox71 sequence, the loxP sequence, and the inserted DNA inserted between the lox71 and loxP sequences, and the inserted DNA of the donor vector is stably introduced into the cells. To introduce DNA into cells.

  [5] A cell in which an accept vector having the lox71 sequence represented by SEQ ID NO: 2 and the loxP sequence represented by SEQ ID NO: 3 is stably inserted into a region not containing exons and introns on the genome; The Cre vector is allowed to act on a donor vector having the lox66 sequence shown, the loxP sequence, and the inserted DNA inserted between the lox66 and loxP sequences, and the inserted DNA of the donor vector is stably introduced into the cells. To introduce DNA into cells.

  [6] A cell in which an accept vector having a 3′-mutated loxP sequence or a 5′-mutated loxP sequence and the loxP sequence represented by SEQ ID NO: 3 is stably inserted into a region not containing exons and introns on the genome .

  [7] A cell in which an accept vector having a 3 ′ mutant loxP sequence and the loxP sequence represented by SEQ ID NO: 3 is stably inserted into a region not containing exons and introns on the genome, and a 5 ′ mutant loxP sequence , A Cre enzyme is allowed to act on a donor vector having a loxP sequence and an inserted DNA inserted between the 5 ′ mutant loxP sequence and the loxP sequence, thereby stably introducing the inserted DNA of the donor vector into the cell. To introduce DNA into cells.

  [8] A cell in which an accept vector having a 5′-mutated loxP sequence and a loxP sequence represented by SEQ ID NO: 3 is stably inserted into a region not containing exons and introns on the genome, and a 3′-mutated loxP sequence , A Cre enzyme is allowed to act on a donor vector having a loxP sequence and an inserted DNA inserted between the 3 ′ mutant loxP sequence and the loxP sequence, thereby stably introducing the inserted DNA of the donor vector into the cell. To introduce DNA into cells.

  In the present invention, since the target DNA is introduced into an accept vector that has been previously introduced into a region that does not destroy the endogenous gene on the genome, a complicated genome analysis is performed every time a clone is prepared, and the desired DNA is obtained. There is no need to select cells introduced into the location. For this reason, it is possible to obtain cells into which the target DNA has been stably and efficiently introduced in a short period of time. Since the introduction of DNA into cells uses a combination of the loxP sequence and the lox66 and lox71 sequences in which mutations are introduced into the loxP sequence, the dropout rate after introduction is low, and a transformant in which the target DNA is efficiently introduced Can be obtained.

  The cells obtained by the DNA introduction method of the present invention can be used for knocking in a target gene, knocking down a target gene such as RNAi method, etc. by selecting the DNA to be introduced according to the purpose.

  Hereinafter, the DNA introduction method of the present invention will be described with reference to FIG.

  An accept vector 7 having a sequence 3 (lox71 sequence) and a sequence 5 (loxP sequence) is stably inserted in advance into a cell (parent strain) 1 into which DNA is introduced.

loxP is a sequence derived from E. coli P ₁ phage, as shown in FIG. 2, in order toward 'from the 3' end 5 to end, the sense side repeats, spacers, made up of three regions of the antisense side repeats It is known. In the sequence of loxP, the sequence from the 5 ′ end to the 3 ′ end and the sequence from the 3 ′ end to the 5 ′ end are complementary to each other.

  lox71 is a base sequence represented by SEQ ID NO: 3, and is a sequence in which a mutation is introduced into the sense side repetitive sequence of the loxP sequence.

  The insertion position of the accept vector 7 in the cell 1 is a region not involved in protein expression on the genome, that is, a region not containing exons and introns. Examples of regions that do not contain exons and introns on the genome include untranslated regions and intergenic DNA regions.

  In addition to lox71 and loxP, other sequences such as a marker gene 27, a promoter 25 for marker gene expression, a transcription termination signal 29 of the marker gene may be inserted into the accept vector 7. Known marker genes can be used, and examples thereof include a zeocin resistance gene and a blasticidin resistance gene.

  The cell 1 (parent strain) having the accept vector can be produced, for example, by the following method.

  First, a DNA fragment containing an accept vector having a lox71 sequence and a loxP sequence is prepared by a known method using a restriction enzyme or the like. Next, this DNA fragment is introduced into the genome of a mammalian cultured cell (HeLa cell) by electrical stimulation. When a marker gene is introduced into the accept vector, the cells are cultured in a medium to which a drug is added according to the introduced marker gene, and colonies formed by the cells into which the marker gene has been introduced into the genome are selected. . The obtained cells are subjected to genome analysis by Southern blotting, PCR, etc., and a clone in which one copy of the accept vector is introduced into a region not containing exons and introns on the genome is selected and designated as cell 1 (parent strain).

  On the other hand, the donor vector 9 has a lox66 indicated by 11 in FIG. 1 and a loxP sequence indicated by 13. lox66 is a base sequence represented by SEQ ID NO: 1, and is a sequence in which mutations are introduced into the antisense sequence of loxP. A DNA 15 having a desired sequence to be inserted into the cell 1 (hereinafter referred to as insert DNA) is introduced between lox66 and loxP of the donor vector.

  Between the lox66 sequence and the loxP sequence introduced into the donor vector 9, in addition to the inserted DNA 15, a marker gene 33, a promoter 31 for marker gene expression, a transcription termination signal 35 for the marker gene, for expression of the inserted DNA May have a promoter or the like. As the marker gene, a known gene different from the marker genes 27 and 28 introduced into the cell 1 can be used.

  For example, the donor vector 9 can be prepared as follows.

  First, a DNA fragment into which inserted DNA 15 is introduced between lox66 and loxP is prepared by a known method using a restriction enzyme or the like. Next, a known vector such as a plasmid or phage and a DNA fragment are treated with the same restriction enzyme, and the respective sticky ends are joined together with a DNA ligase.

  The insertion DNA 15 is introduced into the cell 1 by allowing the recombinant enzyme 41 to act on the cell 1 and the donor vector 9 to recombine the DNA between the accept vector 7 and the donor vector 9 introduced into the cell 1.

  As the recombinant enzyme, Cre enzyme is used. The Cre enzyme is an enzyme that recombines by cleaving the loxP spacer sequence and joining sticky ends together. The cleavage position when the Cre enzyme is allowed to act on loxP base pairs is shown in FIG.

  When the Cre enzyme is allowed to act on the accept vector 7 and the donor vector 9, a method of introducing the Cre enzyme expression vector into the cell 1 by a known method, a method of culturing the cell 1 in a medium to which the Cre enzyme is added, or the like can be employed.

  The Cre enzyme has a spacer sequence even when one or both of the two loxP strands forming a base pair are replaced with a mutant lox sequence in which a mutation is introduced on either the sense side or the antisense side. Cleavage and binding are performed (provided that the mutation lox is used for both double strands, and the mutation introduction position is a combination of the sense sides or the antisense sides). Therefore, sequence recombination is performed between loxP and lox71, between loxP and lox66, and between lox71 and lox66 as in the case of loxP.

  When the Cre enzyme is allowed to act on the accept vector 7 and the donor vector 9, recombination by the Cre enzyme occurs between the accept vector lox71 and the donor vector lox66, and between the accept vector loxP and the donor vector loxP. As a result, a cell (child strain) 17 in which the sequence 19 between lox66 and loxP of the donor vector 9 is stably integrated into the genome is obtained.

  By this recombination, the lox71 / 66 sequence indicated by 21 and the loxP sequence indicated by 23 in FIG. 1 are formed at both ends of the sequence 19 derived from the donor vector of the cell 17. The lox71 / 66 sequence is a sequence having a lox71 sense repeat sequence and a lox66 antisense repeat sequence at both ends of the spacer sequence. The base sequence of lox71 / 66 is the sequence shown in SEQ ID NO: 4.

  The Cre enzyme hardly acts on the lox71 / 66 sequence in which mutations are introduced into both the sense side repeat sequence and the antisense side repeat sequence of loxP. Therefore, the loss of the sequence 19 from the cell 17 is negligible, and the cell 17 in which the inserted DNA 15 is stably introduced onto the genome can be obtained in high yield. In the present invention, “stable” refers to a state where a predetermined base sequence is not dropped from the genome during cell division and is semipermanently retained.

  In the present invention, by appropriately selecting the sequence of the inserted DNA 15 according to the purpose, various functions can be imparted to the obtained cells, and as a result, it can be used for various applications.

  For example, when a DNA having a sequence that expresses siRNA or shRNA that inhibits mRNA expressed from the target gene in the inserted DNA is selected, the obtained cells can be used in the RNAi method. When a reporter gene or a reporter gene response element is incorporated into the inserted DNA, the resulting cells can be used for reporter assays.

  Although FIG. 1 shows the case where lox71 is used for sequence 3 of the accept vector and lox66 is used for sequence 11 of the donor vector, the insertion DNA 15 is similarly applied when lox66 is used for sequence 3 and lox71 is used for sequence 11. Can be introduced into cell 1.

  Furthermore, the accept vector sequence 3 and the donor vector sequence 11 have a single 5 ′ mutant loxP in which the loxP sense side repeat sequence is partially mutated instead of lox71 and lox66, and the loxP antisense side repeat sequence. It can be used in combination with a partially mutated 3 ′ mutant loxP. The 5'-side mutation loxP is a sequence in which 2 to 7 bases of the loxP sense-side repeat sequence is substituted, and the 3'-side mutation loxP is a sequence in which 2 to 7 bases of the loxP antisense-side repeat sequence are substituted with other bases. However, in the combination in which the base sequence from the spacer sequence of the 5′-mutant loxP toward the 5′-end and the base sequence from the spacer sequence of the 3′-mutant loxP to the 3′-end are the same sequence, the ratio at which the sequence 19 is dropped Since it becomes high, it is preferable to select a different combination.

  An example of 5 ′ side mutation loxP and 3 ′ side mutation loxP is shown below. In the following sequences, the bases shown in lower case are different bases from the loxP sequence.

[5 'side mutation loxP]
(a) 5'-AatgCaTgcTATA GCATACAT TATACGAAGTTAT-3 '(SEQ ID NO: 24)
(b) 5'-taccgggCGTATA GCATACAT TATACGAAGTTAT-3 '(SEQ ID NO: 25)
(c) 5'-tagcgTTCGTATA GCATACAT TATACGAAGTTAT-3 '(SEQ ID NO: 26)
(d) 5'-taccgggCGTATA GCATACAT TATACGAAGTTAT-3 '(SEQ ID NO: 27)
[3 'side mutation loxP]
(a) 5'-ATAACTTCGTATA GCATACAT TATAgGtAccgAg-3 '(SEQ ID NO: 28)
(b) 5'-ATAACTTCGTATA GCATACAT TATACGcccggta-3 '(SEQ ID NO: 29)
(c) 5'-ATAACTTCGTATA GCATACAT TATACGcAcggta-3 '(SEQ ID NO: 30)
(d) 5'-ATAACTTCGTATA GCATACAT TATAgGtAccgta-3 '(SEQ ID NO: 31)
(e) 5'-ATAACTTCGTATA GCATACAT TATACGtAccggg-3 '(SEQ ID NO: 32)

Production Example 1 (Production of Accept Vector)
(1) Amplification of lox71 fragment The following components were placed in a PCR tube and mixed.
・ PDNR-1r (Clontech) 1 ml
-5'Xho-lox71 (5'-TAGCTCgagAAAGATCCtaccgTTCGTATAGC-3 ': SEQ ID NO: 5) [10 mM]
1 ml
・ 3'Xho-lox71 (5'-cgcCTCgagTCACTAAATAATAG-3 ': SEQ ID NO: 6) [10 mM] 1 ml
・ DNTP [2 mM] 10 ml
・ Mg ²⁺ [10 mM] 6 ml
・ 10 × buffer (accessory) 10 ml
・ DdH ₂ O 69 ml
・ KOD plus (TOYOBO; enzyme) 2 ml
100 ml
After the PCR tube was capped, the lox71 fragment with the XhoI site added at both ends was amplified by PCR using pDNR-1r as a template. PCR conditions were 95 ° C. for 30 seconds, 53 ° C. for 30 seconds, and 68 ° C. for 30 seconds for 30 cycles.

(2) Amplification of loxP fragment The following components were placed in a PCR tube and mixed.
・ PDNR-1r (Clontech) 1 ml
・ 5'Xba-loxP (5'-GCTTCtagAGCTCCTGAAAGATCC-3 ': SEQ ID NO: 7) [10 mM] 1 ml
・ 3'Xba-loxP (5'-TcCtCTAgATAATAGTGAACGGC-3 ': SEQ ID NO: 8) [10 mM] 1 ml
・ DNTP [2 mM] 10 ml
・ Mg ²⁺ [10 mM] 6 ml
・ 10 × buffer (accessory) 10 ml
・ DdH ₂ O 69 ml
・ KOD plus (TOYOBO; enzyme) 2 ml
100 ml
After the PCR tube was capped, the loxP fragment with the XbaI site added to both ends was amplified by PCR using pDNR-1r as a template. PCR conditions were 95 ° C. for 30 seconds, 53 ° C. for 30 seconds, and 68 ° C. for 30 seconds for 30 cycles.

(3) Preparation of accept vector
The PCR product (about 110 bp) obtained in (1) was purified by PCR Purification Kit (manufactured by QIAGEN) and then digested with XhoI. The XhoI-digested PCR purified product (about 110 bp) and XhoI-digested pCMVZeo (manufactured by Invitrogen) were joined with ligase to prepare p71CMVZeo.

  Next, the PCR product (about 110 bp) obtained in (2) was purified by PCR Purification Kit (manufactured by QIAGEN) and then digested with XbaI. The XbaI-digested PCR purified product (about 110 bp) and XbaI-digested p71CMVZeo were connected with ligase to prepare p71CMVZeoP (about 3.8 Kbp), which was used as an accept vector. The block diagram of the obtained accept vector (p71CMVZeoP) is shown in FIG.

  The lox71 and loxP sequences inserted into the accept vector were confirmed to be correct in sequence and orientation by a sequencer (Beckman).

Production Example 2 (Production of cells (parent strain))
(1) Insertion of accept vector into HeLa cell 20 mg of the accept vector (p71CMVZeoP) prepared in Example 1 was digested with PvuII to make it linear. The linear acceptor vector was dissolved in 50 ml of TE buffer, and 50 ml of ice-cold PBS (−) was further added to make 100 ml.

The cultured HeLa cells were collected from the adhered dish by trypsin digestion and suspended in 0.7 ml of ice-cold PBS (−). At this time, when the number of cells was measured, it was about 1.0 × 10 ⁷ .

  Transfer cell suspension and linear acceptor vector lysate to Gene Pluser Cuvette (size: 0.4cm electrode; BioRad), set to Gene Pluser (BioRad), and apply electrical stimulation under conditions of 800V and 3mF. Gave.

  This mixture was transferred to a 10 cm dish for cell culture and cultured in DMEM medium containing 400 mg / ml zeocin for 10 to 14 days.

  The appearing colonies were picked up under a microscope and transferred to a 48-well cell culture plate (cloning). The cell culture was continued in a normal DMEM medium until the area ratio was about 12 times, and then the cells were detached with trypsin, dissolved in a cell banker, and stored at −80 ° C.

(2) Cell line selection
a) The cells obtained in (1) were grown and subjected to genome extraction (QIAGEN).

  b) The following operation was performed to confirm that the accept vector was inserted by genomic PCR.

The following components were placed in a PCR tube and mixed.
・ Genomic DNA 1 ml of each clone
・ Zeo-S2 (5'-GAGGAGCAGGACTGA-3 ': SEQ ID NO: 9) [10 mM] 0.2 ml
CMVZeo-MCS-A1 (5'-GTGCTGCAAGGCGATTAAG-3 ': SEQ ID NO: 10) [10 mM] 0.2 ml
・ DNTP [2.5 mM] 1.6 ml
・ 10 × buffer (accessory) 2 ml
・ DdH ₂ O 15ml
・ EX Taq (TAKARA; enzyme) 0.1ml
20 ml
After capping the PCR tube, the genome was amplified by PCR. PCR conditions were 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds for 30 cycles. The PCR product was subjected to agarose gel electrophoresis, and the presence or absence of a band at a position of about 300 bp was confirmed.

  c) The following operation was performed to confirm that the accept vector was inserted by genomic PCR.

The following components were placed in a PCR tube and mixed.
・ Genomic DNA 1 ml of each clone
・ Ori-S1 (5'-CTACACCGAACTGAGATAC-3 ': SEQ ID NO: 11) [10 mM] 0.2 ml
・ Lox71-zeo (5'-CCGCATAACTTCGTATAATG-3 ': SEQ ID NO: 12) [10 mM] 0.2 ml
・ DNTP [2.5 mM] 1.6 ml
・ 10 × buffer (accessory) 2 ml
・ DdH ₂ O 15ml
・ EX Taq (TAKARA; enzyme) 0.1ml
20 ml
After capping the PCR tube, the genome was amplified by PCR. PCR conditions were 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds for 30 cycles. The PCR product was subjected to agarose gel electrophoresis, and the presence or absence of a band at a position of about 400 bp was confirmed.

  d) In order to confirm that the accept vector was inserted by Southern blotting, the following operation was performed.

  Genomic DNA was digested with EcoRI and BglII and Southern blotted. Detection with an NcoI probe confirmed the presence or absence of a band at a position of about 1.5 Kb.

  e) In order to confirm that the accept vector was inserted by Southern blotting, the following operation was performed.

  Genomic DNA was digested with BglII and Southern blotted. Detection with a HinfI probe confirmed whether a single band appeared.

  f) For each clone, the genomic sequence near the accept vector insertion site was cloned according to the following procedure.

  The genomic DNA of the clone (No. 55) whose results were confirmed by the PCR analysis performed in b) and c) and the Southern blot performed in d) and e) was digested with BglII. Self-ligation with ligase and transformation into E. coli stbl4. After culturing at 30 ° C. for 1 hour, the cells were plated on a plate containing ampicillin and cultured at 30 ° C. The appearing colonies were cultured, and the sequence 5 'upstream side of the accept vector insertion site was read by a sequencer (manufactured by Beckman). As a result, the obtained genome sequence was searched on the database and confirmed to be a partial sequence on human chromosome 9.

Next, across the accept vector insertion site, 4 types of primers (5′-gtcttgttctgtagcccaagc-3 ′ (SEQ ID NO: 13), 5′-cagcttgttgagagaacttgc-3 ′ (SEQ ID NO: 14), on the 3 ′ downstream genome side, 5'-catctgtctggagcctcaactttc-3 '(SEQ ID NO: 15) and 5'-gactcctcccacagtcaccactg-3' (SEQ ID NO: 16)) were set, and PCR was performed.
・ Genomic DNA (Clone No. 55) 1 ml
・ Zeo-S2 (5'-GAGGAGCAGGACTGA-3 ': SEQ ID NO: 17) [10 mM] 0.2 ml
・ Genome side primers [10 mM] 0.2 ml
・ DNTP [2.5 mM] 1.6 ml
・ 10 × buffer (accessory) 2 ml
・ DdH ₂ O 15ml
・ Platinum Taq (Invitrogen; enzyme) 0.1ml
20 ml
The above components were mixed, put into a PCR tube and capped, and then the genome was amplified by PCR. PCR conditions were 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds for 30 cycles.

  The PCR product was subjected to agarose gel electrophoresis, the band that appeared was excised and purified using the Gel Extraction Kit (QIAGEN), and the sequence 3 'downstream of the accept vector insertion site was read using a sequencer (Beckman). It is. As a result, the obtained genome sequence was searched on the database and confirmed to be a partial sequence on human chromosome 9. An insertion map of the accept vector on the genome is shown in FIG.

Production Example 3 (Preparation of donor vector (p66CMVBsdP))
(1) Amplification of lox66 fragment The following components were placed in a PCR tube and mixed.
・ PDNR-1r (Clontech) 1 ml
・ 5'Xho-lox66 [10mM] (5'-TAGCTCgaGAAAGATCCATAACTTCGTATAGCATACATTATACGAAc
ggtaGCG-3 ': SEQ ID NO: 18) 1 ml
・ 3'Xho-lox71 (5'-cgcCTCgagTCACTAAATAATAG-3 ': SEQ ID NO: 19) [10 mM] 1 ml
・ DNTP [2 mM] 10 ml
・ Mg ²⁺ [10 mM] 6 ml
・ 10 × buffer (accessory) 10 ml
・ DdH ₂ O 69 ml
・ KOD plus (TOYOBO; enzyme) 2 ml
100 ml
After the PCR tube was capped, the lox66 fragment with the XhoI site added at both ends was amplified by PCR using pDNR-1r as a template. PCR conditions were 95 ° C. for 30 seconds, 53 ° C. for 30 seconds, and 68 ° C. for 30 seconds for 30 cycles.

(2) Introduction of lox66 fragment into vector
The PCR product (about 110 bp) obtained in (1) was purified by PCR Purification Kit (QIAGEN) and then digested with XhoI. Furthermore, pCMVBsd (Invitrogen) was also digested with XhoI. The purified PCR product (about 110 bp) digested with XhoI and pCMVBsd were joined with ligase to prepare p66CMVBsd.

(3) Amplification of loxP fragment The following components were placed in a PCR tube and mixed.
・ PDNR-1r (Clontech) 1 ml
・ 5'Nar-loxP (5'-TTAGgcgCCTTAGCTCCTGAA-3 ': SEQ ID NO: 20) [10 mM] 1 ml
・ 3'Nar-loxP (5'-CATggCgCcAAATAATAGTGAACG-3 ': SEQ ID NO: 21) [10 mM] 1 ml
・ DNTP [2 mM] 10 ml
・ Mg ²⁺ [10 mM] 6 ml
・ 10 × buffer (accessory) 10 ml
・ DdH ₂ O 69 ml
・ KOD plus (TOYOBO; enzyme) 2 ml
100 ml
After the PCR tube was capped, the loxP fragment with the NarI site added to both ends was amplified by PCR using pDNR-1r as a template. PCR conditions were 95 ° C. for 30 seconds, 53 ° C. for 30 seconds, and 68 ° C. for 30 seconds for 30 cycles.

(4) Introduction of loxP fragment into vector
The PCR product (about 110 bp) obtained in (1) was purified by PCR Purification Kit (QIAGEN) and digested with XhoI. Furthermore, pCMVBsd (Invitrogen) was also digested with XhoI. The PCR purified product (about 110 bp) digested with XhoI and pCMVBsd were joined with ligase to prepare p66CMVBsd.

  The PCR product (about 110 bp) obtained in (3) was purified by PCR Purification Kit (QIAGEN) and digested with NarI. Furthermore, p66CMVBsd obtained in (2) was digested with NarI. The XbaI-digested PCR purified product (about 110 bp) and p66CMVBsd were joined with ligase to prepare p66CMVBsdP (about 3.8 Kbp), which was used as a donor vector. A block diagram of the obtained donor vector is shown in FIG.

  The lox66 and loxP sequences inserted into the donor vector were confirmed to be correct in sequence and orientation by a sequencer (Beckman).

Production Example 4 (Preparation of donor vector (p66BsdP / Green))
The 3581 bp NarI site of p66CMVBsdP prepared in Production Example 3 was replaced with G → A by site-directed mutagenesis to obtain p66CMVBsdP mut. Furthermore, p66CMVBsdPmut was digested with NarI and blunted (blunted) with Klenow enzyme. The obtained product was subjected to agarose gel electrophoresis, and the appearing band was cut out and purified using Gel Extraction Kit (QIAGEN).

  pZsGreen1 (Clontech) was digested with BspLUll I, blunted with Klenow enzyme + dNTP, phosphorylated, and further digested with SspI. The obtained product was subjected to agarose gel electrophoresis, and the appearing band (CMV-ZsGreen-pA fragment; about 1.6 Kb) was cut out and purified using Gel Extraction Kit (QIAGEN).

  The nicked p66CMVBsdP mut and the approximately 1.6 Kb CMV-ZsGreen-pA fragment were ligated together with a ligase to create a donor vector p66BsdP / Green. The block diagram of the obtained p66BsdP / Green is shown in FIG.

Production Example 5 (Preparation of donor vector (p66Bsd / TNFR RNAi))
TNFR (Tumor Necrosis Factor Receptor; Acc No., NM 001065.2) is targeted to the sense strand (5'-caccGGAGCTTACTTGTATGATGATgtgtgctgtccGTCATTGTACAAGTAGGTTCCttttt-3 'and SEQ. Designed and an equal volume was dissolved in an annealing buffer (10 mM Tris-HCl, pH 7.5 / 1 mM EDTA / 100 mM NaCl). After reacting at 99 ° C. for 2 minutes, the mixture was cooled to 4 ° C. over 2 hours. After digestion with BfuI, pUC6 / TNFR was obtained by ligation with pUC-hU6 and ligase.

  PU6 / TNFR was digested with EcoRI-HindIII, subjected to agarose gel electrophoresis, and a pU6-TNFRshRNA-pT fragment (about 1.4 kb) was excised and purified using Gel Extraction Kit (QIAGEN).

  p66CMVBsdP was digested with EcoRI-HindIII and ligated with the pU6-TNFRshRNA-pT fragment to generate the donor vector p66BsdP / TNFR RNAi. FIG. 7 shows a structural diagram of the obtained p66BsdP / TNFR RNAi.

Example 1
500 to 1000 cells (parent strain) prepared in Production Example 2 were seeded in a cell culture dish having a diameter of 10 cm. The green fluorescent protein (GFP) expression donor vector (p66BsdP / Green, circular) prepared in Production Example 4 and the Cre enzyme expression vector (circular, provided by Prof. Izumi Saito, University of Tokyo) were introduced into the parent strain by lipofection. . After 24 hours, the medium was changed to a medium (DMEM) containing 10 μg / ml blasticidin, and cultured for 10 days while changing the medium every other day. Colonies produced in the medium were isolated under a microscope, transferred to a 48-well plate, and cultured for 10 to 20 days until filled in a 6 cm diameter dish. Then, when the obtained cells (child strains) were observed under a fluorescence microscope and checked for the presence or absence of green fluorescence, 2/6 clones (33.3%) were GFP positive cells. The cells into which the GFP-expressing donor vector was introduced were confirmed to have green fluorescence even after 4 weeks.

Example 2
500 to 1000 cells (parent strain) prepared in Production Example 2 were seeded in a cell culture dish having a diameter of 10 cm. Tumor necrosis factor receptor I (TNFRI) expression donor vector (p66Bsd / TNFR RNAi, circular) prepared in Production Example 5 and Cre enzyme expression vector (circular) were introduced into the parent strain by lipofection. After 24 hours, the medium was changed to a medium (DMEM) containing 10 μg / ml blasticidin, and cultured for 10 days while changing the medium every other day. Twenty-one clones were isolated from the colonies generated in the medium under a microscope, transferred to a 48-well plate, and cultured for 10 to 20 days until the dish was 6 cm in diameter. Total RNA was extracted from the cultured cells, and cDNA was synthesized. For 18 clones, the expression level of TNFRI mRNA was measured by quantitative RT-PCR. Among 18 clones, the clone in which the expression level of TNFRI was most suppressed was designated as a TNFRI RNAi stable cell line. When this stable cell line was cultured until the 10th generation (about 2 months), the gene expression inhibitory effect of the 10th generation TNFRI RNAi stable cell line was retained.

Test example 1
In order to examine the effect of TNF-α stimulation, which is a ligand of TNFRI, on the survival of TNFRI RNAi cells produced in Example 2, the following test was performed.
5000 cells / well of TNFRI RNAi cells were seeded in 6 wells, and after 24 hours, 0.24-1,000 ng / ml TNF-α and 1 ug / ml CHX were co-exposed. The exposure time was 24 hours, and MTT analysis (using Cell Counting Kit-8, manufactured by Dojindo) was performed after exposure.
TNFRI RNAi cells (stable cell lines that suppressed TNFRI expression) had a higher survival rate after TNF-α exposure than TNRFI-expressing strains (24 hours exposure; Hela55 (parent strain) approximately 20%, TNFRI RNAi Strain; 80%).

It is explanatory drawing which shows the DNA introduction | transduction method of this invention. It is explanatory drawing which shows the cutting | disconnection position by the Cre enzyme of loxP arrangement | sequence and loxP arrangement | sequence. 3 is a configuration diagram of an accept vector prepared in Production Example 1. FIG. 10 is an insertion map showing an insertion position of an accept vector in a cell produced in Production Example 2. It is a block diagram of the donor vector produced in Production Example 3. It is a block diagram of the donor vector produced in the manufacture example 4. 6 is a configuration diagram of a donor vector prepared in Production Example 5. FIG.

Explanation of symbols

1, 17 cells 3, 5, 11, 13, 19, 21, 23 Sequence 7 Accept vector 9 Donor vector 15 Inserted DNA
25, 31 Promoter 27, 28, 33 Marker gene 29, 35 Transcription termination signal

[About SEQ ID NOs: 1-4 and 24-32]
Artificial sequence from bacteriophage P1

Claims (8)

An accept vector having the lox66 sequence shown in SEQ ID NO: 1 or the lox71 sequence shown in SEQ ID NO: 2 and the loxP sequence shown in SEQ ID NO: 3 was stably inserted into an exon and intron-free region on the genome. cell. A cell in which the lox71 / 66 sequence represented by SEQ ID NO: 4, the loxP sequence represented by SEQ ID NO: 3, and the inserted DNA are stably inserted into a region not containing exons and introns on the genome, Are stably inserted between the lox71 / 66 and loxP sequences. The cell according to claim 2, which has a marker gene, a promoter for expression of the marker gene, and a transcription termination signal of the marker gene between the lox71 / 66 sequence and the loxP sequence. A cell in which an accept vector having a lox66 sequence represented by SEQ ID NO: 1 and a loxP sequence represented by SEQ ID NO: 3 is stably inserted into an exon and intron-free region on the genome, and lox71 represented by SEQ ID NO: 2 To a cell that stably introduces the inserted DNA of the donor vector into the cell by causing Cre enzyme to act on the donor vector having the sequence, the loxP sequence, and the inserted DNA inserted between the lox71 sequence and the loxP sequence DNA introduction method. A cell in which an accept vector having a lox71 sequence represented by SEQ ID NO: 2 and a loxP sequence represented by SEQ ID NO: 3 is stably inserted into a region not containing exons and introns on the genome, and lox66 represented by SEQ ID NO: 1 To a cell that stably introduces the inserted DNA of the donor vector into the cell by causing the Cre enzyme to act on the donor vector having the sequence, the loxP sequence, and the inserted DNA inserted between the lox66 sequence and the loxP sequence DNA introduction method. A cell in which an accept vector having a 3'-mutated loxP sequence or a 5'-mutated loxP sequence and a loxP sequence represented by SEQ ID NO: 3 is stably inserted into a region not containing exons and introns on the genome. A cell in which an accept vector having a 3 ′ mutant loxP sequence and the loxP sequence represented by SEQ ID NO: 3 is stably inserted into a region not containing exons and introns on the genome, a 5 ′ mutant loxP sequence, and loxP To a cell that stably introduces the inserted DNA of the donor vector into the cell by causing Cre enzyme to act on the donor vector having the sequence and the inserted DNA inserted between the 5 ′ mutant loxP sequence and the loxP sequence DNA introduction method. A cell in which an accept vector having a 5 ′ mutant loxP sequence and the loxP sequence represented by SEQ ID NO: 3 is stably inserted into a region not containing exons and introns on the genome, 3 ′ mutant loxP sequence, and loxP To a cell in which the inserted DNA of the donor vector is stably introduced into the cell by causing Cre enzyme to act on the donor vector having the sequence and the inserted DNA inserted between the 3 ′ mutant loxP sequence and the loxP sequence. DNA introduction method.

Images