CN106987560A - The construction method of the stable strain of the cell HB gene knockouts of RK 13 - Google Patents

The construction method of the stable strain of the cell HB gene knockouts of RK 13 Download PDF

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CN106987560A
CN106987560A CN201710200878.3A CN201710200878A CN106987560A CN 106987560 A CN106987560 A CN 106987560A CN 201710200878 A CN201710200878 A CN 201710200878A CN 106987560 A CN106987560 A CN 106987560A
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gene
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plasmid
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CN106987560B (en
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刘光清
谭永贵
刘腾
朱杰
陈宗艳
李传锋
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Shanghai Veterinary Research Institute CAAS
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • 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
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Abstract

The invention discloses a kind of construction method of the stable strain of cell HB gene knockouts of RK 13；Methods described comprises the following steps：The sgRNA of a pair of complementary targeting rabbit HB second extrons of gene of design, using pSpCas9 (BB) 2A Puro (PX459) V2.0 plasmids as carrier, builds eukaryotic expression recombinant plasmid；After sequencing identification, eukaryotic expression recombinant plasmid cotransfection is entered in the cells of RK 13, positive cell screening is carried out using puromycin, separation monoclonal cell is cultivated；Effect is knocked out with sequencing identification.The plasmid that the present invention is built carries the Cas9 endonuclease genes of micrococcus scarlatinae II type CRISPR/Cas9 systems；CRISPR/Cas9 gene editings technology success permanent knockout RK 13 cell HB genes, obtain HB and knock out stable cell line, are taken a firm foundation for the function of studying HB genes.

Description

The construction method of the stable strain of RK-13 cell HB gene knockouts
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of structure of the stable strain of RK-13 cells HB gene knockouts Construction method.
Background technology
CRISPR (clustered, regularly interspaced, short palindromic repeats) is one Plant the viral DNA invaded from bacterial degradation or the immunologic mechanism of other exogenous DNAs.Cas9 albumen first with crRNA and TracrRNA is combined into compound, then by recognizing PAM sequences (5 '-NGG-3 ') combination and invading DNA, then cutting DNA Double-strand, causes missing or the insertion of base, causes gene silencing.Scientist carries out gene using CRISPR/Cas9 systems The work of editor, is the gene orientation editing technique of a new generation.Sweden female microbiologist Emmanuelle in 2011 Charpenti exists《Nature》On publish thesis, illustrate the mechanism of action of the immune system Cas9 in bacterial body, pass through this set RNA mediated dnas cutting mechanism makes bacterium from the invasion of exogenous DNA and proposes 4 key factors Cas9, crRNA, TracrRNA, and RNaseIII.The scientist Jennifer Doudna of subsequent Univ California-Berkeley in 2012 《Science》On publish thesis, realize the external editor of genome first using this immune system, indicate CRISPR Formal birth.The scientist Zhang Feng Polymorphisms group expert George Church of Chinese origin of Blanc moral research institute in 2013 are common 《Science》On delivered landmark CRISPR achievements in research, determining for gene is realized on mammalian cell first Point editor, indicates that this technology has moved to maturity, to realize that the point of application of technology has determined important foundation.The current technology is Only need to express Cas9 endonucleases and corresponding sgRNA molecules in the cell through developing into, almost can be to arbitrary DNA Sequence is cut.The key point of this technology is exactly to design sgRNA molecules, and is linked in carrier, easy to operate.
Compared to preceding two generations gene targeting：Zinc finger endonuclease (zinc finger endonuclease, ZFN) With class activating transcription factor effector nuclease (transcription activator-like effector nuclease, The classical gene targeting such as TALEN), CRISPR/Cas9 gene editing system addresses with low cost, easy to operate, system The standby cycle is short, the advantages of efficiency high, and the rapid worldwide laboratories of CRISPR/Cas9, this technology makes gene editing technology It can be carried out in common Molecular Laboratory.Within short 1 year, bio-science field is had become most very powerful and exceedingly arrogant Research tool.
Current CRISPR/Cas9 technologies are successfully applied to the genome essence of human cell, zebra fish and mouse and bacterium True modification, modified types include gene site-directed InDe1 be mutated, it is gene site-directed knock in, two site simultaneous mutations and small fragment lack Lose.Because its mutation efficiency is high, make the characteristics of simple and cost is low, it is considered to be a kind of gene with broad prospect of application Group fixed point transformation molecular tool.
Hemoglobin β-chain thought the protein simply with oxygen carrying capacity in the past always, during bacterium infection Blood transportation can be accelerated, and then promote the removing to bacterium.There is document report classical swine fever virus C protein can be with HB Interact and propagation of the CSFV on cell can be suppressed, interference HB expression virus titers rise, and we can profit Downstream experiment is carried out with the cell line of knockout,.Rabbit hemorrhagic disease virus belongs to Caliciviridae Lagovirus, very long one section in the past Time is because without suitable cell culture system, cause the viral research relatively backward with respect to other positive chain RNA virus, nothing Method carries out appropriate cell experiment, the nucleotide sequence that our laboratories pass through the capsid protein surface amino groups acid molecule to RHDV Rite-directed mutagenesis is carried out, improved virus can be bred on RK-13 cells, achieve breakthrough development, this causes us RHDV can be allowed to rise in value on cell and carry out downstream experiment.
The content of the invention
It is an object of the invention to provide a kind of construction method of the stable strain of RK-13 cells HB gene knockouts；It is specifically sharp HB genes in RK-13 cells are knocked out with the CRISPR/Cas9 gene editings system of improvement, RK-13 cell HB gene knockouts are built Stable strain.
The purpose of the present invention is achieved through the following technical solutions：
The present invention relates to a kind of construction method of the stable strain of RK-13 cells HB gene knockouts, methods described includes following step Suddenly：
S1, a pair of complementary targeting rabbit HB second extrons of gene of design sgRNA, with pSpCas9 (BB) -2A- Puro (PX459) V2.0 plasmids are carrier, construct an eukaryotic expression recombinant plasmid；
S2, after sequencing identification, the eukaryotic expression recombinant plasmid is transfected into RK-13 cells, using fast jointly Purine mycin carries out positive cell screening, and separation monoclonal cell is cultivated；Effect is knocked out with sequencing identification, you can obtain RK- The stable strain of 13 cell HB gene knockouts.
As a result：Sg RNA are properly inserted into PX459 plasmid vectors, are transfected and are screened WB in the cell after monoclonal and verify HB protein expressions are lacked.
Conclusion：Successfully construct the stable strain of RK-13 cell HB gene knockouts.
It is preferred that, in step S1, the sequence of the sgRNA includes the forward primer HB- as shown in SEQ ID NO.1 SgRNAF sequences, and the reverse primer HB-sgRNAR sequences as shown in SEQ ID NO.2.
It is preferred that, in step S1, the structure specifically includes following steps：
S1-1, using Bbs I restriction enzymes pSpCas9 (BB) -2A-Puro (PX459) V2.0 plasmids are entered into line Property, and be incubated using alkaline phosphatase, remove plasmid end sequence phosphorylation after digestion；Then agarose gel electrophoresis is pure Change, reclaim DNA fragmentation；
S1-2, the single-stranded annealing of two sgRNA oligonucleotides formed into double-strand, and through T4 tyrosine phosphorylations；Annealing Product and the DNA fragmentation of the recovery are attached using T4 DNA ligases, and are converted into Stb13 competent cells, are chosen Take monoclonal bacterium colony；
S1-3, it is sequenced as primer with U6 promoters, extracts sequencing correct plasmid.
It is preferred that, in step S1-2, the program of T4 tyrosine phosphorylations is 37 DEG C of 30min, 95 DEG C of 5min, and 90 DEG C to 25 DEG C are every Minute declines 5 DEG C, 4 DEG C of preservations.
The invention further relates to a kind of eukaryotic expression recombinant plasmid of targeting knock out RK-13 cells HB genes, the restructuring is true Nuclear expression plasmid is the sgRNA by designing a pair of complementary targeting rabbit HB second extrons of gene, with pSpCas9 (BB) -2A-Puro (PX459) V2.0 plasmids are eukaryotic expression recombinant plasmid obtained by vector construction.
It is preferred that, the sequence of the sgRNA includes the forward primer HB-sgRNAF sequences as shown in SEQ ID NO.1, and Reverse primer HB-sgRNAR sequences as shown in SEQ ID NO.2.
The present invention has the advantages that：
The present invention is carried suppurative using pSpCas9 (BB) -2A-Puro (PX459) V2.0 (PX459-HB) built The Cas9 endonuclease genes of streptococcus II type CRISPR/Cas9 systems；The success of CRISPR/Cas9 gene editings technology is permanent Property knocked out RK-13 cell HB genes, obtain HB knock out stable cell line, be in the future research HB genes function lay heavily fortified point Real basis.
Brief description of the drawings
By reading the detailed description made with reference to the following drawings to non-limiting example, further feature of the invention, Objects and advantages will become more apparent upon：
Fig. 1 is two sgRNA design synthesis principle schematic diagrames；
Fig. 2 is RK-13 cells and knocks out strain HB gene order-checking result AB1 files comparison schematic diagram；Wherein, upper figure is RK-13 cellular genome sequencing result AB1 files, figure below is knockout strain HB gene order-checking result AB1 files；
Fig. 3 is RK-13 cells and knocks out strain HB gene order-checking result SEQ files comparison schematic diagram；Wherein, upper figure is RK-13 cellular genome sequencing result SEQ files, figure below is knockout strain HB gene order-checking result SEQ files；
Fig. 4 is WB detection RK-13 cells and knocks out strain HB gene knockouts effect and β-actin controls；Wherein, left figure is RK-13 knocks out cell line effect, and right figure compares for β-actin.
Embodiment
With reference to embodiment, the present invention is described in detail.Following examples will be helpful to those skilled in the art The present invention is further understood, but the invention is not limited in any way.It should be pointed out that to one of ordinary skill in the art For, without departing from the inventive concept of the premise, it can also make certain adjustments and improvements.These belong to the guarantor of the present invention Protect scope.
Embodiment
1：Materials and methods
1.1 experiment material：1.1.1 plasmid, bacterial strain and cell CRISPR/Cas9n plasmids pSpCas9 (BB) -2A-Puro (PX459) V2.0 is preserved purchased from addgene and by laboratory, and RK-13 cells are purchased from Wuhan University's cyropreservation center.Competence Cell Stb13 is purchased from Beijing Quan Shi King Companies.Plasmid map is found in http：//www.addgene.org websites.
1.1.2 main agents：Bbs I restriction enzymes, T4 ligase, 10xT4Ligation Buffer, 10mmol/L ATP are purchased from NEB companies；AxyPrep DNA gels QIAquick Gel Extraction Kit is limited purchased from love biotechnology (Hangzhou) of pursuing progress Company；The a large amount of extracts kits of plasmid are purchased from QIAGEN companies；Lipo2000 transfection reagents are purchased from Invitrogen companies； Opti-MEM culture mediums are purchased from Gbico companies；MEM culture mediums are purchased from Hyclone companies, and mouse source HB polyclonal antibodies are by this experiment Room prepares and preserved, and mouse source β-actin antibody, the anti-mouse IgG of horseradish peroxidase mark are century purchased from health.Gene sequencing and SgRNA synthesis is completed by Shanghai Hua Jin bio tech ltd.
1.2 method：
1.2.1 the design of sgRNA oligo sequences
The design application http of sgRNA oligo sequences：1 pair of //crispr.mit.edu/ website designs are directed to HB cds The sgRNA sequences of the extron in area.Design method is following (principle such as Fig. 1)：(1) the HB genome cds areas issued according to NCBI Sequence, the sequence that about 200bp is rich in NGG is found behind initiation codon, the website is committed to, and sgRNA sequences can only be On same extron, it is impossible to across two extrons because extron in genome across intron sequences.(2) basis The result that website is provided, higher several to sequence of selection fraction, restriction enzyme site is added at its two ends.In every sgRNA sequence F chain 5 ' end addition CACC, 5 ' end addition AAAC of R chains, the cohesive terminus,cohesive termini formed with pX4592.0 plasmids after Bbs I digestions is mutual Mend.5 ' first base in end such as F chains are not G, then add a G behind 5 ' end CACC, and correspondingly 3 ' ends of R chains are further added by One C.2 pX4592.0 recombinant plasmids are referred to as pX459- Δs HB below.
The sgRNA oligonucleotide sequences of table 1
Title Sequence (5-3)
HB-sgRNA F CACCGCAAAGGACTCGAAGAACCTC(SEQ ID NO.1)
HB-sgRNA R AAACGAGGTTCTTCGAGTCCTTTGC(SEQ ID NO.2)
1.2.2 the structure of recombinant plasmid
Enzyme pSpCas9 (BB) -2A-Puro (PX459) V2.0 plasmids are carried out using Bbs I restriction enzymes linear Change, and 30min is incubated at 37 DEG C using alkaline phosphatase (CIP), remove plasmid end sequence phosphorylation after digestion；Then 1% Agarose gel electrophoresis is purified, and wall scroll DNA fragmentation is remained as after pSpCas9 (BB) -2A-Puro (PX459) V2.0 plasmid enzyme restrictions, DNA fragmentation is reclaimed with glue reclaim kit, and linearisation 50ng/ul is diluted to, moved back two sgRNA oligonucleotides are single-stranded Fire forms double-strand, and through T4 tyrosine phosphorylations, and program is 37 DEG C of 30min, 95 DEG C of 5min, and 90 DEG C to 25 DEG C per minute to decline 5 DEG C, 4 DEG C of preservations；Annealed product is diluted with ultra-pure water 1: 200, is then attached using T4 DNA ligases, and is converted extremely In Stb13 competent cells, picking monoclonal bacterium colony is sequenced with U6 promoters as primer.Will sequencing correct plasmid profit Extraction reagent kit in endotoxin is gone to extract plasmid with Qiagene.Positive cell is screened：Cell transfecting：RK-13 cells, which are used, contains 10% tire The MEM cultures of cow's serum (FBS) are based on 5%CO2, and 37 DEG C incubated.24h before transfection, phase cell of taking the logarithm is with 5 × 105/ hole 6 orifice plate cultures are inoculated into, and are named, and set up negative control group.Step will knock out plasmid with lipo3000 and turn to specifications Contaminate into RK-13 cells, changed after 6h after liquid, 48h plus puromycin screens vitellophag after positive cell, then 48h, and progressively added Big puromycin concentration, will grow the cell dissociation after stabilization, collects cell and simultaneously counts, infinite dilution is micro- into 96 hollow plates Microscopic observation individual cells are simultaneously marked, and carry out Colony Culture, when individual cells are agglomerating, then expand culture step by step, are collected Part cell extraction genomic DNA, target gene after being expanded through PCR be connected and be sequenced with carrier T, is entered with wild type gene group Row is compared, checking HB genes whether successful knockout.The cell expansion culture of base will be successfully lacked, extract total protein of cell, WB Checking knocks out efficiency.
Gene order-checking result as shown in Figure 2,3, from Fig. 2,3, knocks out strain HB genes and 10 alkali is lacked at sgRNA Base, causes frameshift mutation.
WB detects HB gene knockouts result as shown in figure 4, as shown in Figure 4, successful knockout HB genes.
The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow Ring the substantive content of the present invention.
SEQUENCE LISTING
<110>China Agriculture Academe Shanghai Veterinary Institute
<120>The construction method of the stable strain of RK-13 cell HB gene knockouts
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<170> PatentIn version 3.5
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caccgcaaag gactcgaaga acctc 25
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aaacgaggtt cttcgagtcc tttgc 25

Claims (6)

1. the construction method of the stable strain of a kind of RK-13 cells HB gene knockouts, it is characterised in that methods described includes following step Suddenly：
S1, a pair of complementary targeting rabbit HB second extrons of gene of design sgRNA, with pSpCas9 (BB) -2A-Puro (PX459) V2.0 plasmids are carrier, construct an eukaryotic expression recombinant plasmid；
S2, after sequencing identification, the eukaryotic expression recombinant plasmid is transfected into RK-13 cells jointly, it is mould using purine Element carries out positive cell screening, and separation monoclonal cell is cultivated；Effect is knocked out with sequencing identification, you can obtain RK-13 thin The stable strain of born of the same parents HB gene knockouts.
2. the construction method of the stable strain of RK-13 cells HB gene knockouts according to claim 1, it is characterised in that step In S1, the sequence of the sgRNA includes the forward primer HB-sgRNA F sequences as shown in SEQ ID NO.1, and such as SEQ ID Reverse primer HB-sgRNA R sequences shown in NO.2.
3. the construction method of the stable strain of RK-13 cells HB gene knockouts according to claim 1, it is characterised in that step In S1, the structure specifically includes following steps：
S1-1, using Bbs I restriction enzymes pSpCas9 (BB) -2A-Puro (PX459) V2.0 plasmids are linearized, And be incubated using alkaline phosphatase, remove plasmid end sequence phosphorylation after digestion；Then agarose gel electrophoresis is purified, and is reclaimed DNA fragmentation；
S1-2, the single-stranded annealing of two sgRNA oligonucleotides formed into double-strand, and through T4 tyrosine phosphorylations；Annealed product It is attached, and is converted into Stbl3 competent cells, picking list using T4 DNA ligases with the DNA fragmentation of the recovery Colonies；
S1-3, it is sequenced as primer with U6 promoters, extracts sequencing correct plasmid.
4. the construction method of the stable strain of RK-13 cells HB gene knockouts according to claim 3, it is characterised in that step In S1-2, the program of T4 tyrosine phosphorylations is 37 DEG C of 30min, 95 DEG C of 5min, 90 DEG C to 25 DEG C 5 DEG C of declines per minute, 4 DEG C of guarantors Deposit.
5. a kind of eukaryotic expression recombinant plasmid of targeting knock out RK-13 cells HB genes, it is characterised in that the restructuring eucaryon table It is the sgRNA by designing a pair of complementary targeting rabbit HB second extrons of gene up to plasmid, with pSpCas9 (BB) -2A- Puro (PX459) V2.0 plasmids are eukaryotic expression recombinant plasmid obtained by vector construction.
6. eukaryotic expression recombinant plasmid according to claim 5, it is characterised in that the sequence of the sgRNA includes such as SEQ Forward primer HB-sgRNA F sequences shown in ID NO.1, and the reverse primer HB-sgRNA R sequences as shown in SEQ ID NO.2 Row.
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CN109456995A (en) * 2018-11-08 2019-03-12 杜以军 Gene knockout plasmid, cell line and preparation method and application
WO2019237396A1 (en) * 2018-06-16 2019-12-19 深圳市博奥康生物科技有限公司 Method for targeted knockout of human tnfsf18 gene by applying crispr/cas9 system
WO2020000456A1 (en) * 2018-06-29 2020-01-02 深圳市博奥康生物科技有限公司 Method for targeted knockout of human cnih2 gene by applying crispr/cas9 system
CN112111528A (en) * 2019-06-21 2020-12-22 华东师范大学 Method for repairing abnormal splicing of intron

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108085298A (en) * 2017-11-30 2018-05-29 中山珐玛斯医药科技有限公司 The drug absorption screening system and its construction method of a kind of MDCK-MDR1
WO2019237396A1 (en) * 2018-06-16 2019-12-19 深圳市博奥康生物科技有限公司 Method for targeted knockout of human tnfsf18 gene by applying crispr/cas9 system
WO2020000456A1 (en) * 2018-06-29 2020-01-02 深圳市博奥康生物科技有限公司 Method for targeted knockout of human cnih2 gene by applying crispr/cas9 system
CN109456995A (en) * 2018-11-08 2019-03-12 杜以军 Gene knockout plasmid, cell line and preparation method and application
CN112111528A (en) * 2019-06-21 2020-12-22 华东师范大学 Method for repairing abnormal splicing of intron
CN112111528B (en) * 2019-06-21 2024-04-02 华东师范大学 Repair method for abnormal splicing of introns

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