CN105671045B - A kind of method of sheep embryo fibroblast homologous recombination repair frequency after raising gene editing - Google Patents
A kind of method of sheep embryo fibroblast homologous recombination repair frequency after raising gene editing Download PDFInfo
- Publication number
- CN105671045B CN105671045B CN201610104796.4A CN201610104796A CN105671045B CN 105671045 B CN105671045 B CN 105671045B CN 201610104796 A CN201610104796 A CN 201610104796A CN 105671045 B CN105671045 B CN 105671045B
- Authority
- CN
- China
- Prior art keywords
- lig4
- sheep embryo
- gene
- embryo fibroblast
- sirna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- 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
- C12N15/1137—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 against enzymes
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of methods of sheep embryo fibroblast homologous recombination repair frequency after raising gene editing.The application in promoting the in vitro sheep embryo fibroblast homologous recombination repair product containing gene editing DNA fragmentation is being prepared the present invention provides the substance of Lig4 gene expressions in the in vitro sheep embryo fibroblast containing gene editing DNA fragmentation is inhibited.The experiment proves that, the present invention screens the siRNA for inhibiting sheep Lig4 gene expressions, inhibit sheep Lig4 gene expressions by siRNA, the fibroblastic NHEJ of sheep embryo is inhibited to repair approach to stimulate intracellular HR to repair approach, the frequency that cell uses HR to repair is improved, basis is provided to improve sheep embryo fibroblast gene targeting efficiency and research ovine genome accurate edits.
Description
Technical field
The present invention relates to sheep embryo fibroblast after biotechnology more particularly to a kind of raising gene editing is same
The method of source recombinantal repair frequency.
Background technology
Using artificial endonucleases (Engineered endonuclease, EEN), such as zinc finger enzyme (Zinc finger
Nucleases, ZFNs), class activating transcription factor effector nuclease (Transcription activator-like
Effector nucleases, TALENs) and CRISPR/Cas9 (Clustered regularly interspaced short
Palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)), fixed point cutting genome generates special
Anchor point genome double-strand break (Double-strand brakes, DSBs) lacks gram (Nicks), promotes accurate edits lactation
The research of Animal genome.
Cell is mainly repaired by non-homologous end joining approach (Non-homologous end joining, NHEJ)
DSBs generates gene knockout phenotype.The donor template of common transfection EEN and single stranded oligonucleotide or homologous sequence, pass through dependence
Homologous templates repair approach, and such as micro- homology arm mediates connection (Microhomology-mediated end joining, MMEJ)
With homologous recombination (Homologous recombination, HR), external source target gene is accurately knocked in into cellular genome, it is real
Existing gene editing.Even if generating the DSBs of genome using efficient CRISPR/Cas9 systems, cell selects NHEJ repair for event
Probability of happening be more than 90%, using homologous templates repairability probability be less than 10%.Illustrate in most cells, NHEJ considerably beyond
Rely on the probability of happening of homologous templates repair for event.
Inhibit NHEJ approach, promotes cell to repair approach using HR, HR maintenance frequencies can be improved.With EEN selective cleavages
Drosophila cell genome, RNA interfere (RNA inhibite, RNAi) instantaneously to inhibit Lig4 genes (DNA Ligase 4, Lig4)
Translation, the short left and right homology arm of PCR amplification (80 and 60nt) donor template, in the cell of drug screening, homologous recombination efficiency
Up to 50%.EEN handles drosophila embryos, the arabidopsis of Lig4-/- mutant, and compared with wild type, homologous recombination efficiency significantly carries
It is high.Regulation and control inhibit the Lig4 protein factors in nematode (Caenorhabditis elegans) body cell to make to inhibit NHEJ
Organism repairs DSBs approach and turns to MMEJ.Bmku70 albumen is the necessary factor of NHEJ, knocks out the silkworm embryo of Bmku70 genes
Tire, microinjection CRISPR/Cas9 systems plasmid and donor template, compared with wild type silkworm embryos, HR is more efficient.SCR7
(5,6-bis (benzylideneamino) -2-mercapto-pyrimidin-4-ol) specificity is the same as mankind's Lig4 albumen
DNA binding domain (DNA binding domain, DBD) combines, and Lig4 has been blocked to be combined with DNA, inhibits NHEJ to repair approach, carries
High 4-5 times of HR maintenance frequencies.
The drosophila that Lig4 is knocked out can normal existence, but mouse Lig4 gene knockouts show as embryonic death, so far to feeding
The research of newborn animal Lig4 gene knockouts is less.Mammalian cell repairs 3 main paths of DSBs：NHEJ, MMEJ and HR,
Complementary competitive relation is presented, inhibits NHEJ that will stimulate cell that HR is selected to repair approach.
The quantitative study of the laboratories Gorbunova NHEJ and HR repair approach, cell aging state difference are obtained, to gene
It is different that group DSBs repairs result；Human somatic cell repairs DSBs, mainly uses NHEJ approach, and in cell cycle in each stage
NHEJ can be used；HR reparations occur mainly in the S phases.
Gene targeting is a kind of experiment hand of directed change biological living hereditary information based on homologous recombination
Section, promotes biological study.Normal mammalian cell generation homologous recombination probability is extremely low, causes gene targeting efficiency extremely low
(10-6)。
Invention content
It is an object of the present invention to provide the purposes for inhibiting Lig4 gene expressions and/or active substance.
The in vitro sheep embryo of promotion is being prepared into fibre the present invention provides Lig4 gene expressions and/or active substance is inhibited
Dimension cell carries out the application in homologous recombination repair product in gene editing.
In above application, the gene editing is that will import in vitro sheep by exogenous DNA molecule after specific site is sheared
In embryo fibroblast, exogenous DNA molecule carries out homologous recombination repair in cell after making the shearing；
Or the gene editing is by exogenous DNA molecule homologous recombination to the in vitro sheep embryo after specific site is sheared
On tire fibroblast genome.
The specific site shearing is realized by I-SceI digestions.
It is described to inhibit the substance of Lig4 gene expressions for for interfering in vitro sheep embryo fibroblast in above application
The siRNA of middle Lig4 gene expressions.
In above application, the nucleotides sequence of the siRNA is classified as sequence 3 or sequence 4 in sequence table.
It is homologous heavy that another object of the present invention is that one kind makes in vitro sheep embryo fibroblast be carried out in gene editing
The method that group maintenance frequency improves.
Method provided by the invention, includes the following steps：Inhibit the in vitro sheep embryo for carrying out gene editing at fiber finer
Lig4 gene expressions in born of the same parents realize that in vitro sheep embryo fibroblast carries out homologous recombination repair frequency in gene editing and carries
It is high.
In the above method, Lig4 gene expressions in the in vitro sheep embryo fibroblast of the inhibition progress gene editing
Will to be used to inhibit the substance of Lig4 gene expressions in vitro sheep embryo fibroblast to import the progress gene editing
In in vitro sheep embryo fibroblast.
In the above method, the gene editing is that will import in vitro sheep by exogenous DNA molecule after specific site is sheared
In embryo fibroblast, exogenous DNA molecule carries out homologous recombination repair in cell after making the shearing；
Or the gene editing is by exogenous DNA molecule homologous recombination to the in vitro sheep embryo after specific site is sheared
On tire fibroblast genome.
It is described to inhibit the substance of Lig4 gene expressions in vitro sheep embryo fibroblast for for doing in the above method
Disturb the siRNA of Lig4 gene expressions in vitro sheep embryo fibroblast；
The nucleotide sequence of the siRNA is specially sequence 3 or sequence 4 in sequence table.
It is described that in vitro sheep embryo fibroblast is made to carry out homologous recombination repair frequency in gene editing in the above method
Rate rises to the cell-isogenic recombination of Lig4 gene expressions in the in vitro sheep embryo fibroblast for inhibiting to carry out gene editing
Maintenance frequency is higher than the fibroblastic homologous recombination repair frequency of in vitro sheep embryo for carrying out gene editing.
Above-mentioned exogenous DNA molecule is HR plasmids.
The experiment proves that the present invention screens the siRNA for inhibiting sheep Lig4 gene expressions, pressed down by siRNA
Sheep Lig4 gene expressions processed inhibit the fibroblastic NHEJ of sheep embryo to repair approach to stimulate intracellular HR to repair way
Diameter improves the frequency that cell uses HR to repair, to improve sheep embryo fibroblast gene targeting efficiency and research sheep
Genome accurate edits provide basis.
Description of the drawings
Fig. 1 is the sites the siRNA schematic diagram designed for sheep Lig4 genes.
Fig. 2 is the reporter gene construct (a) and DNA breakage cohesive end (b) figure analyzed HR and repair approach.
Fig. 3 is sheep Lig4 gene C DS sequence PCR amplification results.
Fig. 4 is the PCR detections of pMD18T-ShLig4 thalline.
Fig. 5 is the real-time fluorescence quantitative PCR result that siRNA influences sheep Lig4 gene expressions.
Fig. 6 is the Western blot figures that siRNA interferes sheep Lig4 gene expressions.
Fig. 7 is HR repair vector reconnection fluorescence imaging results.
Fig. 8 is HR repair vector reconnection flow cytomery results.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The nucleotides sequence of Lig4 genes is classified as sequence 1, and the amino acid sequence for encoding albumen is sequence 2.
Some materials are as follows：pcDNA3.1+Carrier (Invitrogen), pMD18-T (TaKaRa), HR plasmids
The laboratories Gorbunova give, pDsRed2-N1 (Clontech), pEGFP-N1 (Clontech), stbl3 strains
(Invitrogen), electroporation (Amax Nucleofector II), flow cytometer (BDFACSAria III), sheep embryo at
Fibrocyte is preserved by this laboratory original cuiture.
Portion of reagent is as follows：RealBand 10KB(0.25-10kb)DNA Ladder(Cat NO.B600031-0500)
Give birth to work in Shanghai；DL 2000DNA Marker (Cat NO.3427A, TaKaRa).Taq DNA Polymerase(Cat
NO.ET101-02), mini-scale plasmid extracts kit (Cat NO.DP103-02, Tiangeng biochemical technology Co., Ltd).
PrimerSTAR DNA Polymerase (Cat NO.R045, Takara), plastic recovery kit (Cat NO.D2500-02,
OMEGA)；Restriction enzyme (Thermo Fisher)；Lig4 monoclonal antibodies ((Cat NO.GTX100100, GeneTex)；
EasyScript All-in-one First–Strand cDNA Synthesis SuperMix for qPCR(Cat
The Beijing Quanshijin Biotechnology Co., Ltd NO.AT314)；Real-time fluorescence quantitative PCR kit (Cat NO.B639271, Shanghai
Raw work).
Trizol (Cat NO.15596-026, Invitrogen)；Mammalian proteins extraction agent (Cat
NO.CW0889), BCA protein quantifications kit (Cat NO.CW0014), protease inhibitor cocktail (Cat
NO.CW2200), PAGE gel reagent preparation box (Cat NO.CW0022), speed swimming SDS-PAGE electrophoretic buffers (10 ×)
(Cat NO.CW2566), albumen tracer sample-loading buffer (5 ×) (Cat NO.CW0027A), goat anti-rabbit igg HRP (Cat
NO.CW0103), anti-GAPDH rabbit polyclonal antibodies (Cat NO.CW0101), Western Blot confining liquids I (BSA) (Cat
NO.CW0054), Western antibody diluents (Cat NO.CW2340), highly sensitive chemical luminescence detection kit (Cat
NO.CW0049B), western blot membrane regeneration liquor (Cat NO.CW0056A) is century purchased from health；The double-colored pre-dyed eggs of 15-150kDa
White Marker (Cat NO.C610011) gives birth to work purchased from Shanghai；Consumptive material gives birth to work purchased from Shanghai.
The water of use for laboratory DEPC processing prepares electricity and turns liquid.SolutionⅠ(10ml)：2g ATP-Na2, 1.2g MgCl2*
6H2O；SolutionⅡ(500ml)：6g KH2PO4, 0.6g NaHCO3, 0.2g C6H12O6.Solution I is filtered using 0.2 μm
Device filtration sterilization, -20 DEG C of preservations；The NaOH of Solution II adjust 7.2,0.2 μm of filter filtration sterilization of pH value, 4 DEG C of preservations；With
When by solution I and solution II according to 1:50 volume ratios mix.
Tissue Culture Dish, centrifuge tube (Nunc)；DMEM high sugar culture solutions (Gibco)；Fetal calf serum (BI,
LOT1407738)；Pancreatin substitute TrypLETMExpress (1X) (12604-021, Gibco).
The screening of the siRNA of Lig4 gene expressions in embodiment 1, interference sheep embryo fibroblast
One, the structure of eukaryon expression plasmid pcDNA3.1-Shlig4
1, design of primers
According to the sheep Lig4 gene orders (XM_004012236) and GAPDH gene orders (NM_ predicted in Genbank
001289746.1) design primer, by Shanghai, Sheng Gong Bioisystech Co., Ltd synthesizes.Primer sequence is shown in Table 1.
Table 1 is Primer and sequence
Underscore is restriction enzyme site BamHI and XbaI, and black matrix is protectiveness base.
2, the structure of pcDNA3.1-Shlig4 recombinant plasmids
Be free of introne according to the cDNA sequence of mammal Lig4 genes, with primer Shlig4F/Shlig4R, directly with
Ovine genome DNA is template, carries out PCR amplification, obtains the PCR product that 2736bp is consistent with expected size, as sheep
CDNA (Fig. 3, wherein 1 of Lig4 genes：Lig4；2：Negative control；M.Maker).
By PCR product in 1% Ago-Gel electrophoresis.According to OMEGA DNA glue recovery purifying kit specifications into
The operation of row glue recycling.The PCR product of recovery purifying is connect for 16 DEG C overnight with pMD18-T carriers, and connection product converts Stbl3 senses
By state cell and the LB tablets of Amp resistances are inoculated in, PCR detections are carried out to clone with primer ShLig4RTF3/ShLig4RTR3,
Obtain positive colony (Fig. 4, wherein 1-7 of the purpose band of 250bp：Thalline PCR；8：Negative control；M：DL2000).Picking
Positive colony extraction plasmid is named as pMD18T-shlig4 and serves the raw work sequencing in sea.
Using pMD18T-Shlig4 as template, Lig4 genes are expanded with primer PC3.1lig4F/PC3.1lig4RPCR.PCR
Product glue recovery purifying.The PCR product and pcDNA3.1 of purifying+Through BamHI and XbaI double digestions, gel extraction purifies purpose piece
Section, 16 DEG C of T4 ligases connection overnight, is inoculated with the LB tablets of Amp resistances, obtains eukaryon expression plasmid pcDNA3.1- after conversion
Work sequencing is given birth in Shlig4, Shanghai.
By sequencing, pcDNA3.1-Shlig4 is that DNA fragmentation shown in sequence in sequence table 1 is inserted into pcDNA3.1+It carries
The carrier obtained between the BamHI and XbaI enzyme cutting site of body (Invitrogen).
Two, the siRNA designs of interference sheep Lig4 gene expressions
According to sheep Lig4 gene sequencing as a result, synthesizing the interference sequence in 4 sites by the design of Shanghai Ji Ma companies.Synthesis
Sequence is shown in Table 2, and action site is shown in Fig. 1.
The siRNA sequence of 2 sheep Lig4 genes of table designs
Three, sheep embryo Fibroblast cell-culture
The pregnant 1 monthly age tire sheep of Sheep Fibroblast original cuiture selection, takes musculature to cut into fritter, by 0.5cm
Uniformly plantation waits for that tissue fritter is adherent to spacing in culture dish, and culture solution (+15% serum+1% of DMEM stostes is dual anti-) is added, sets
In 37 DEG C, 5%CO2Culture in incubator.3-4d changes liquid 1 time, when waiting for that primary cell converges the 80-90% for accounting for culture dish in flakes,
Carry out secondary culture；4-6h changes liquid, after cell grow to 90% or more converge after digestion process, appropriate complete culture solution is added and suspends
1 after cell:3 expand culture.Cell dissociation uses Gibco pancreatin substitute TrypLE, obtains in vitro sheep embryo into fiber finer
Born of the same parents.
Four, the screening of best Lig4 gene siRNAs
1, electrotransfection
Respectively by 4 kinds of siRNA, plasmid pcDNA3.1-Shlig4 and plasmid pDSRed2-N1 electrotransfections in vitro sheep embryo
It is specific as follows in tire fibroblast：
In vitro sheep embryo fibroblastic growth to culture dish 80% when, digestion collects 1.8 × 106A cell, 100
μ L electricity turns liquid suspension (S1：S2=1：50) corresponding 4 kinds of siRNA, the plasmid pcDNA3.1- of above-mentioned two synthesis, are separately added into
Shlig4 and plasmid pDSRed2-N1 (Clontech).Electric revolving cup is positioned in electric turn trough, CZ-167 electricity carryover sequences, room are run
Temperature stands 10min, is laid in 6cm Tissue Culture Dish after mixing, 37 DEG C, 5%CO2It is cultivated in incubator, 9h cells are completely adherent
Afterwards, 1 cell culture fluid is replaced, 72h collects cell, obtains transfection negative control siRNA cells, transfection Lig4-siRNA-49
Cell, transfection Lig4-siRNA-1132 cells, transfection Lig4-siRNA-1827 cells and transfection Lig4-siRNA-2629 are thin
Born of the same parents, transfection pcDNA3.1-Shlig4 cells and transfection pDSRed2-N1 cells.
2, sheep Lig4 expression conditions quantitative PCR detection
Extract transfection negative control siRNA cells, transfection Lig4-siRNA-49 cells, transfection respectively with Trizol methods
Lig4-siRNA-1132 cells, transfection Lig4-siRNA-1827 cells, transfection Lig4-siRNA-2629 cells, transfection
PcDNA3.1-Shlig4 cells, transfection pDSRed2-N1 cells, the in vitro sheep embryo fibroblast electricity of blank turn and do not appoint
The normal in vitro fibroblastic total serum IgE of sheep embryo of where reason, reverse transcription obtain cDNA.
The preparation of above-mentioned cDNA is carried out using following reaction systems：1 μ g, 5 × Trans Script All-in- of total serum IgE
5 μ L, gRNA Remover of one SuperMix for qPCR, 1 μ L, are added with RNase-free Water to 20 μ L.Simultaneously
A pipe negative control is done with 5 × Trans Script All-in-one No-RT control SuperMix5 μ L.Gently mix
It is even, 42 DEG C of 15min, 85 DEG C of 5s, after reaction cDNA be stored in -20 DEG C.
Selection GAPDH is reference gene, ShLig4RTF3/ShLig4RTR3 and GAPDHF/GAPDHR primers are respectively adopted
The cDNA of the cell of siRNAs different to above-mentioned 4 kinds transfections and the cell of transfection pcDNA3.1-Shlig4 carries out PCR amplification, according to
Real-time fluorescence quantitative PCR kit (work is given birth in Cat NO.B639271, Shanghai) illustrates to operate.Real-time PCR reaction systems
Preparation (20 μ L)：2 × qPCR Mix, 10 μ L, each 0.4 μ L of upstream and downstream primer, template cDNA1 μ L, 8.2 μ L of deionized water.
Real-time PCR response procedures：95℃5min；95 DEG C of 5s, 60 DEG C of 20s, 72 DEG C of 20s,；Fluorescence letter is collected at 72 DEG C of 20s
Number, the mode of collecting signal is single, totally 45 cycles；95 DEG C of 5s, 65 DEG C of 15s, 95 DEG C of 0s；Fluorescence is collected in 95 DEG C of 0s
The mode of signal, collecting signal is continuous；Last 40 DEG C of 30s.
Quantitative fluorescent PCR data analysis is according to Ct methods (Qr=2-ΔΔCt), it determines after different siRNA electrotransfections to sheep embryo
The influence of tire fibroblast Lig4 gene mRNA expression amounts.
Amplification curve and solubility curve are shown in Fig. 5, A：Amplification curve；B：Solubility curve；C：SiRNA influences sheep Lig4 genes
The real-time fluorescence quantitative PCR comparative analysis block diagram of expression；Lig4：Transfect pcDNA3.1-Shlig4 cells；Red：Transfection
PDSRed2-N1 cells；sc：Transfect negative control siRNA cells；Si-4：Transfect Lig4-siRNA-2629 cells；Si-3：Turn
Contaminate Lig4-siRNA-1827 cells；Si-2：Transfect Lig4-siRNA-1132 cells；Si-1：It is thin to transfect Lig4-siRNA-49
Born of the same parents；KC：The in vitro sheep embryo fibroblast electricity of blank turns；cell：Normal in vitro sheep embryo without any processing is at fibre
Tie up cell；As a result show that amplification curve is good, each only there are one unimodal no non-specific amplifications for solubility curve.Not appoint
The same batch sheep embryo fibroblast Lig4 gene expression doses of where reason are that 1, Real-time PCR results show blank
In vitro sheep embryo fibroblast electricity turns in (KC) and transfection pDSRed2-N1 cells, transfection negative control siRNA cells
The all slightly above normal in vitro sheep embryo fibroblast of the expression quantity of Lig4 genes；It transfects Lig4-siRNA-2629 cells, turn
Contaminate the Lig4 bases in Lig4-siRNA-1827 cells, transfection Lig4-siRNA-1132 cells, transfection Lig4-siRNA-49 cells
Because the Lig4 gene mRNAs after SiRNA is interfered expression quantity be significantly lower than normal cell, wherein Lig4-siRNA-1132 and
Lig4-siRNA-2629 interference effects are more satisfactory.Illustrate that Lig4-siRNA-1132 and Lig4-siRNA-2629 can effectively press down
Make the expression of Lig4 gene mRNAs in vitro sheep embryo fibroblast.
3, Western blot are tested
To transfection pcDNA3.1-Shlig4 cells, transfection pDSRed2-N1 cells, transfection negative control siRNA cells, turn
Lig4-siRNA-2629 cells, transfection Lig4-siRNA-1132 cells, the in vitro sheep embryo fibroblast electricity of blank is contaminated to turn
Use health for century mammalian proteins extraction agent with normal in vitro sheep embryo fibroblast without any processing
(Cat NO.CW0889) extracts albumen, and BCA protein quantifications kit (Cat NO.CW0014) measures albumen concentration, and albumen is dense
Degree adjusts consistent rear denaturation, and pvdf membrane, 5% 4 DEG C of BSA solution closing overnight, after TBST washes film are transferred to after SDS-PAGE electrophoresis
It is added 1:1000 diluted Lig4 monoclonal antibodies (GeneTex) are incubated at room temperature 4h；TBST washes film each 10min three times, is added
1:5000 diluted goat antirabbit secondary antibodies are incubated at room temperature 1.5h；TBST washes film each 15min three times, and a certain amount of chemistry is added
Luminous agent is incubated at room temperature 3min, then exposure fixing.
Film after exposure is taken out, suitable regenerated liquid is added.It is incubated at room temperature 1h, TBST cleans 5min × 3 time.BSA mistakes
The closing overnight of 4 DEG C of night, then carries out the incubation of internal reference antibody again.Step is identical as Lig4, anti-GAPDH rabbit polyclonal antibodies (Cat
NO.CW0101 1) is pressed:2500 dilutions, goat antirabbit secondary antibody 1:5000 dilutions.
The results are shown in Figure 6, Lig4：Transfect pcDNA3.1-Shlig4 cells；Red：Transfect pDSRed2-N1 cells；sc：
Transfect negative control siRNA cells；Si-4：Lig4-siRNA-2629；Si-2：Lig4-siRNA-1132；KC：Blanc cell electricity
Turn；cell：Cell without any processing；For siRNA (Lig4-siRNA-2629 and the Lig4- of the design of sheep Lig4 genes
SiRNA-1132), after electrotransfection Sheep Fibroblast, Lig4 expressing quantities are significantly lower than the cell sample of other experiment process
Product.Illustrate that Lig4-siRNA-2629 and Lig4-siRNA-1132 can effectively inhibit sheep Lig4 gene expressions.Electric Pignus pignoris grain
PDSRed2-N1, negative control siRNA, blank electricity turn cell Lig4 protein expressions and are all slightly above normal cell, with Real-time
PCR results are consistent.Overexpression Lig4 gene mRNA expressions amount is very high and expressing quantity is only slightly higher than normal cell, Ke Nengyuan
Because of the translation result of cell selective.
Therefore best Lig4 gene siRNAs are Lig4-siRNA-2629 and Lig4-siRNA-1132.
Embodiment 2 improves sheep embryo fibroblast genomic DNA homologous recombination repair frequency
Lig4 gene siRNAs are improving HR remediation efficiencies in vitro sheep embryo fibroblast, specific as follows：
Since sheep primary fibroblast passage capacity is limited, it is substantially infeasible to establish stable clone.In order to quantitative
DNA end joint efficiencies and fidelity are measured, using plasmid reconnection method of testing (plasmid rejoining assay).
The HR plasmids that the laboratories Gorbunova give, using GFP (Green Fluorescent Protein) as weight
Structure reporter gene.This report system contains the GFP-Pem1 copies of 2 mutation.In first 1 genetic fragment of GFP-Pem,
Deletion containing 22bp bases longs in first part's exon of GFP, and the I-SceI enzymes for being inserted into two reverse symmetries are known
Other site.Second GFP-Pem1 genetic fragment lack promoter, ATG initiation codon and second part GFP exon.When
After the fracture of the sites I-SceI, not complementary cohesive end DSBs is generated.In the cell, the HR reporting systems of fracture, homologous heavy
Under group repair, homologous recombination occurs in broken site using the connected homologous GFP masterplates of donor, restores GFP expression (figures
2), specific as follows：
1, gene editing
HR plasmids (are documented in：Seluanov A,Mao Z,Gorbunova V.Analysis of DNADouble-
strand Break(DSB)Repair in Mammalian Cells.J Vis Exp,2010,8:(43) and Mao Z,
Bozzella M,Seluanov A,Gorbunova V.DNArepair by nonhomologous end joining and
homologous recombination during cell cycle in human cells.Cell Cycle.2008,7
(18):2902-2906) I-SceI enzymes is used to linearize, gel extraction, the linearisation HR for obtaining the fracture of specific site DNA double chain is carried
Body.
2, HR plasmids reconnection reparation
Turn scheme by following 4 groups of electricity and realizes HR plasmid reconnection reparations：
DNA and siRNA to be transfected are adjusted to debita spissitudo first：Linearisation HR carriers are adjusted to 1 μ g/ μ L,
PDSRed2-N1 plasmids are adjusted to 0.1 μ g/ μ L, siRNA and are diluted to 20uM concentration.
HR+Red：100 μ L electricity turn 1.6 ╳ 10 of liquid suspension6A sheep embryo fibroblast, in this liquid, addition 2.0
μ L linearisation HR carriers (in the dense 2.0 μ g/100 μ L of reaction system), 1 μ L pDSRed2-N1 plasmids are (in the dense of reaction system
0.1 μ g/100 μ L) and 1 μ L water, gently mixing.
Lig4-siRNA-1132+HR+Red：100 μ L electricity turn 1.6 ╳ 10 of liquid suspension6A sheep embryo fibroblast,
In this liquid, 2.0 μ L linearisation HR carriers (2.0 μ g/100 μ L) of addition, 1 μ L pDSRed2-N1 plasmids (0.1 μ g/100 μ L) and
The 20uM Lig4-siRNA-1132 (final concentration 200nM) of 1 μ, gently mixing.
Lig4-siRNA-2629+HR+Red：100 μ L electricity turn 1.6 ╳ 10 of liquid suspension6A sheep embryo fibroblast,
In this liquid, 2.0 μ L linearisation HR carriers (2.0 μ g/100 μ L) of addition, 1 μ L pDSRed2-N1 plasmids (0.1 μ g/100 μ L) and
The 20uM Lig4-siRNA-2629 (final concentration 200nM) of 1 μ, gently mixing.
Negative control SiRNA+HR+Red：100 μ L electricity turn 1.6 ╳ 10 of liquid suspension6A sheep embryo fibroblast, at this
In liquid, 2.0 μ L linearisation HR carriers (2.0 μ g/100 μ L) of addition, 1 μ L pDSRed2-N1 plasmids (0.1 μ g/100 μ L) and 1 μ
20uM negative control SiRNA (final concentration 200nM), gently mixing.
Above-mentioned pDSRed2-N1 expression red fluorescent protein RFP (Red Fluorescent Protein), cotransfection
PDSRed2-N1 is to determine that electric rotaring carrier DNA enters the transfection efficiency of cell；The HR carrier electricity of linearisation is rotated into thin
Born of the same parents, after being repaired by homologous recombination approach, by fluoresced green (GFP)；The HR carriers and plasmid pDSRed2-N1 of linearisation turn
Enter cell, in the same cell inner expression GFP and RFP, pink colour is presented under green fluorescence light source.
Above-mentioned each group is used into CZ-167 program electrotransfections, 3 Duplicate Samples of each experiment process.It is supervised using fluorescence microscope
Survey fibroblast GFP and RFP expression.
Sheep embryo fibroblast electricity turns 36h after the HR plasmids linearized and pDSRed2-N1 plasmids, you can observes
Red fluorescent cell.After electrotransfection cell, cultivate to 72h, two kinds of fluorescence can observe expression.
Lig4-siRNA-1132+HR+Red groups electrotransfection cell culture 72h, fluorescence microscope result such as Fig. 7, A natural lights
Source；B green fluorescence light sources, pink colour cell are the colors that GFP and RFP coexpression cells are presented；C red fluorescence light sources；It observes
The cell (see Fig. 7 B) of fluoresced green illustrates the HR carriers of linearisation, can be repaired by HR in Sheep Fibroblast
Approach realizes reconnection.
Each group electricity turns liquid electrotransfection cell culture 72h, collects cell, be resuspended in 0.5ml PBS (NaCl 137mmol/L,
KCl 2.7mmol/L, Na2HPO410mmol/L, KH2PO42mmol/L, pH 7.2~7.4) in, flow cytometry analysis
(BDFACSAria III, FACSDiva Version 6.1.3), the expression of FITC Air conduct measurement green fluorescences, the inspection of the channels PE
Survey red fluorescence.Every part of sample cell counts 10000, repeats 3 parts of Duplicate Samples.
HR maintenance frequencies (homologous recombination efficiency)=(green cells percentage+red green fluorescence cell percentages)/(red
Color fluorecyte percentage+red green fluorescence cell percentages).
The results are shown in Figure 8 for flow cytomery, A：Blanc cell (in vitro sheep embryo fibroblast)；B：GFP
Single positive (the in vitro sheep embryo fibroblast of transfection pEGFP-N1 plasmids (Clontech))；C：Mono- positive (the transfections of RFP
The in vitro sheep embryo fibroblast of pDSRed2-N1 plasmids)；D：(Lig4-siRNA-1132+HR+Red is thin by GFP+RFP
Born of the same parents)；E：Each group electricity turns liquid electrotransfection cell HR repair vector reconnection flow cytomery block diagrams, KC：HR+Red；Si-2：
Lig4-siRNA-1132+HR+Red；Si-4：Lig4-siRNA-2629+HR+Red；SC：Negative control SiRNA+HR+Red；It can
To find out, Lig4-siRNA-1132+HR+Red groups and Lig4-siRNA-2629+HR+Red groups and negative control SiRNA+HR+
Significant difference between Red control groups, compared with KC control groups, Lig4-siRNA-2629 and Lig4-siRNA-1132 instantaneous interferences
Sheep Lig4 genes, HR maintenance frequencies improve 3-4 times.
Claims (6)
1. Lig4 gene expressions and/or active substance is inhibited to prepare the in vitro sheep embryo fibroblast of promotion in gene volume
The application in homologous recombination repair product is carried out when collecting；
The inhibition Lig4 gene expressions and/or active substance are for interfering Lig4 in vitro sheep embryo fibroblast
The siRNA of gene expression；The nucleotides sequence of the siRNA is classified as sequence 3 or sequence 4 in sequence table.
2. application according to claim 1, it is characterised in that：
The gene editing is that will import in vitro sheep embryo fibroblast by exogenous DNA molecule after specific site is sheared
In, exogenous DNA molecule carries out homologous recombination repair in cell after making the shearing；
Or the gene editing be by exogenous DNA molecule homologous recombination to the in vitro sheep embryo after specific site is sheared at
On fibrocyte genome.
3. a kind of method for making in vitro sheep embryo fibroblast carry out homologous recombination repair frequency raising in gene editing,
Include the following steps：The substance of Lig4 gene expressions in vitro sheep embryo fibroblast is inhibited to inhibit to carry out gene editing
Lig4 gene expressions in vitro sheep embryo fibroblast, realize in vitro sheep embryo fibroblast in gene editing into
Row homologous recombination repair frequency improves；
It is described to inhibit the substance of Lig4 gene expressions in vitro sheep embryo fibroblast for for interfering in vitro sheep embryo
The siRNA of Lig4 gene expressions in fibroblast；
The nucleotides sequence of the siRNA is classified as sequence 3 or sequence 4 in sequence table.
4. according to the method described in claim 3, it is characterized in that：The inhibition carry out the in vitro sheep embryo of gene editing at
Lig4 gene expressions are that will be used to inhibit the substance of Lig4 gene expressions in vitro sheep embryo fibroblast in fibrocyte
It imports in the in vitro sheep embryo fibroblast for carrying out gene editing.
5. according to the method described in claim 4, it is characterized in that：
The gene editing is that will import in vitro sheep embryo fibroblast by exogenous DNA molecule after specific site is sheared
In, exogenous DNA molecule carries out homologous recombination repair in cell after making the shearing；
Or the gene editing be by exogenous DNA molecule homologous recombination to the in vitro sheep embryo after specific site is sheared at
On fibrocyte genome.
6. according to any method in claim 3-5, it is characterised in that：
It is described make in vitro sheep embryo fibroblast carried out in gene editing homologous recombination repair frequency rise to inhibit into
In the in vitro sheep embryo fibroblast of row gene editing the cell-isogenic recombinantal repair frequency of Lig4 gene expressions be higher than into
The fibroblastic homologous recombination repair frequency of in vitro sheep embryo of row gene editing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610104796.4A CN105671045B (en) | 2016-02-25 | 2016-02-25 | A kind of method of sheep embryo fibroblast homologous recombination repair frequency after raising gene editing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610104796.4A CN105671045B (en) | 2016-02-25 | 2016-02-25 | A kind of method of sheep embryo fibroblast homologous recombination repair frequency after raising gene editing |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105671045A CN105671045A (en) | 2016-06-15 |
| CN105671045B true CN105671045B (en) | 2018-11-13 |
Family
ID=56305395
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610104796.4A Expired - Fee Related CN105671045B (en) | 2016-02-25 | 2016-02-25 | A kind of method of sheep embryo fibroblast homologous recombination repair frequency after raising gene editing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105671045B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755049B (en) * | 2016-11-14 | 2020-04-21 | 西北农林科技大学 | Visual cell repair efficiency report system based on artificial nuclease and establishment method thereof |
| CN108866100A (en) * | 2017-05-16 | 2018-11-23 | 中国科学院上海生命科学研究院 | A kind of efficient gene editing method |
| CN111088290B (en) * | 2019-12-30 | 2021-10-08 | 同济大学 | Application of farrerol in gene editing |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1217074A1 (en) * | 2000-12-22 | 2002-06-26 | Universiteit Leiden | Nucleic acid integration in eukaryotes |
| US8906607B2 (en) * | 2010-04-30 | 2014-12-09 | Cellectis | Method for modulating double-strand break-induced homologous recombination |
| EP2718446A2 (en) * | 2011-06-07 | 2014-04-16 | Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) | Improved recombination efficiency by inhibition of nhej dna repair |
| CN104894115B (en) * | 2015-05-26 | 2018-11-13 | 中国科学院青岛生物能源与过程研究所 | A kind of Aspergillus terreus and its construction method and application with efficient homologous recombination ability |
-
2016
- 2016-02-25 CN CN201610104796.4A patent/CN105671045B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN105671045A (en) | 2016-06-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104726494B (en) | The method that CRISPR-Cas9 technologies build chromosome translocation stem cell and animal model | |
| CN106191113B (en) | Preparation method of MC3R gene knockout pig | |
| CN104968784B (en) | Composition and application thereof comprising guide RNA and CAS protein-encoding nucleic acid or CAS protein | |
| CN108342480A (en) | A kind of genetic mutation detection Quality Control object and preparation method thereof | |
| CN106893739A (en) | For the new method and system of target gene operation | |
| CN107058320A (en) | The preparation and its application of IL7R gene delection zebra fish mutant | |
| CN108642055A (en) | The sgRNA of pig miR-17-92 gene clusters can effectively be edited | |
| CN109266680B (en) | Method for preparing CKO/KI animal model by using Cas9 technology | |
| CN107043782A (en) | A kind of gene knockout method and its sgRNA fragments and application | |
| CN105671045B (en) | A kind of method of sheep embryo fibroblast homologous recombination repair frequency after raising gene editing | |
| CN110804628A (en) | High-specificity non-off-target single-base gene editing tool | |
| CN113278618B (en) | gRNA for specifically recognizing porcine COL1A1 gene, and biological material, kit and application thereof | |
| CN107365803A (en) | Exempt from the method that drug screening quickly obtains mouse Rosa26 Gene targeting foreign genes | |
| JP2017184639A (en) | Method for introducing cas9 protein into fertilized egg of mammal | |
| CN106754949B (en) | Pig flesh chalone gene editing site 864-883 and its application | |
| CN109055434A (en) | A method of pig KIT gene structural mutation is corrected using CRISPRCas9 technology | |
| CN109735541B (en) | ACADSB gene knockout dairy cow mammary gland epithelial cell line and construction method thereof | |
| CN107034234B (en) | A kind of kit for being used to knock out two kinds of genes of FUT8 and DHFR in Chinese hamster ovary celI | |
| CN108138165A (en) | The ovum of birds, the production method of birds and birds | |
| CN104894255B (en) | A kind of method and its application detecting inefficient genome editor based on polyacrylamide gel electrophoresis | |
| CN106399370A (en) | Method for establishing stable transgenic flounder embryo cell strain | |
| CN104232643B (en) | RNAi interference fragments, interference carrier, preparation method and applications | |
| CN109929876A (en) | The construction method of Vps28 knock out mice animal model and application | |
| CN110499316B (en) | Milk cow mammary gland epithelial cell line with CD44 gene knocked out and construction method thereof | |
| CN107201365B (en) | A kind of sgRNA sequences of specific knockdown dihydrofolate reductase gene and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20181113 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |