CN106434752A - Process of knocking out Wnt3a gene and verification method thereof - Google Patents
Process of knocking out Wnt3a gene and verification method thereof Download PDFInfo
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Abstract
The invention discloses a process of knocking out Wnt3a gene and a verification method thereof. The knockout and verification of Wnt3a gene are finished through the following steps: establishment of a Cas9 lentiviral vector for Wnt3a gene, culture and passage of HepG2 cell, lentivirus infection and screening of target cell, verification of gene knockout efficiency through a mispairing enzyme method, cell protein analysis and cell proliferation detection by a CCK-8 method. The invention has the following advantages: the Wnt3a gene is knocked out by establishing a Cas9 double-vector lentivirus system for the first time; Crispr/Cas9 is a technology for accurately editing specific site of the genome of any species, and the cell-level single gene or multiple genes can be knocked out by the technology; compared with other gene editing technologies, the method has the advantages that the targeting accuracy is higher; only if the RNA target sequence is completely matched with the genome sequence, can the Cas9 cut the DNA and realize simultaneous knockout of multiple sites of the target gene; and moreover, the experimental period of vector establishment is short, the time and the cost are remarkably saved, and species limit is avoided.
Description
Technical field
The invention belongs to medical domain is and in particular to a kind of process of knockout Wnt3a gene and its verification method.
Background technology
Hepatocellular carcinoma（Hepatocellular Carcinoma, HCC）It is one of modal malignant tumor, rank
The 3rd of the tumor correlation cause of the death.There is the complicated mistake that development is that multi-pathogenesis, polygenes, multi-step and multiple-factor are worked in coordination with HCC
Journey, is related to oncogene activation, multi signal path and the gene such as antioncogene inactivation and some oncogenes of period of embryo are brought back to life again
Regulation and control.Hepatocellular carcinoma based on operative treatment, auxiliary chemotherapy, local treatment etc..Early hepatocarcinoma may be selected excision and
Liver transplantation, but lost operative treatment chance during most of patient assessment.Therefore, early diagnosiss and effectively treatment are to closing weight
Will.
Wnt signal path is considered to develop closely related, especially classical Wnt/β-catenin signal with the generation of HCC
The abnormal activation of approach, is the key link of HCC pathogenesis.Can make as the Wnt3a protein overexpression in classical pathway
For hepatocarcinoma early diagnosis mark, but also as the related novel targets of prognosis in hcc, there is exploitation and application prospect.
Content of the invention
Present invention aims to the deficiencies in the prior art, now provide a kind of knockout with exploitation and application prospect
The process of Wnt3a gene and its verification method.
For solving above-mentioned technical problem, the technical solution used in the present invention is：Knock out process and its checking of Wnt3a gene
Method, its innovative point is：Through building the Cas9 slow virus carrier for Wnt3a gene, the culture of HepG2 cell and biography
Generation, aim cell slow virus infection and screening, mispairing enzyme process checking gene knockout efficiency, cell protein analysis, the detection of CCK-8 method
The step of cell proliferation completes knockout and checking to Wnt3a gene；
Described structure concretely comprising the following steps for the Cas9 slow virus carrier of Wnt3a gene：Build Cas9 complex carries slow viruss, bag
Containing Lenti-sgRNA-EGFP virus 2, respectively expression target gene sgRNA sequence and comparison sgRNA sequence, titre 1E+8 with
On；1, Lenti-CAS9-puro virus, expresses CAS9 albumen, carries puro resistance, titre is in more than 1E+8；
The culture of described HepG2 cell and the concrete steps passing on include cell recovery and passage.
Further, the culture of described HepG2 cell and concretely comprising the following steps of the cell recovery passing in step：
（1）Take out cell cryopreservation tube from liquid nitrogen container, put into rapidly in 37 DEG C of water-baths, and shake makes it thaw as early as possible frequently；
（2）After thawing completely, under 1000rpm environment, it is centrifuged 2min；
（3）Then, after adopting 70% alcohol wipe cryopreservation tube sterilization, move to super-clean bench；
（4）Suck frozen stock solution supernatant, add the fresh complete medium re-suspended cell of 1ml, by cell suspension inoculation to containing 3ml
In the Tissue Culture Flask of complete medium, gently shake even after be placed in 37 DEG C, 5%CO2Cultivate in incubator；
（5）Next day is cultivated for after changing a culture fluid.
Further, the culture of described HepG2 cell and concretely comprising the following steps of the passage passing in step：
1）The cell that growth 90% is converged is passed on, and discards old culture fluid, adds the D-Hanks solution of 2ml sterilizing, washing
Cell growth face；
2）Then discard this solution, add 1ml pancreatin Digestive system, digest 1-2 min under 37 DEG C of environment, until cell disappears completely
Change is got off；
3）Add complete medium 2ml, with measuring pipette piping and druming, the cell on wall is rinsed；
4）Divide to two new Tissue Culture Flasks after mixing cell, supply complete medium to 4ml, continue culture.
Further, described aim cell slow virus infection and concretely comprising the following steps of screening：
A. slow viruss ghost to be screened will be uninfected by be inoculated in 6 orifice plates to 70-80% degrees of fusion；
B., after cell attachment, puromycin medicine is added to carry out the screening of ghost lethal least concentration, 1 μ g/ml, 2.5 μ g/
Ml, 5 μ g/ml and 10 μ g/ml are screened, and draw the lowest concentration of drug of complete cell death after drug treating 48h；
C. cultivate and carry out pancreatin digestion to the aim cell of exponential phase, make cell suspension；
D. by cell number be 5 × 104The cell suspension inoculation of/ml in 6 orifice plates, 5%CO under 37 DEG C of environment2Incubator is cultivated
Treat that cell fusion degree reaches 30%；
E. according to cell MOI value, virus, observation of cell state after 12h are added：Without obvious cytotoxic effect, continue
Culture medium is changed after continuous culture 24h；If there are obvious cytotoxic effect, change culture medium immediately；
F.48 after hour, add puromycin drug screening 48h, obtain the mixing clone of stable expression CAS9；
G. the cell immediate observation cell state after screening is it is ensured that cell state is good；
H. bed board again, infects the slow viruss of expression sgRNA according to slow virus infected cell step；
I. dye 3 days after observe slow viruss on reporter gene GFP expression, fluorescence efficiency 80% about cell start into
Row subsequent experimental.
Further, described mispairing enzyme process verifies concretely comprising the following steps of gene knockout efficiency：
（One）DNA extracts：Collect the cell of successfully infection, extract genomic DNA using genome DNA extraction test kit；
（Two）PCR expands：Around sgRNA binding site, design one pair of genes group PCR primer, enter performing PCR according to following system anti-
Should：2 × Taq Plus Master Mix 10 μ L, primers F 0.5 μ L, primer R 0.5 μ L, genomic DNA 30ng, plus distilled water
To 20 μ L；
PCR response procedures are arranged：94 DEG C of denaturations 90s, 94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 60s/kb, repeat
Degeneration, annealing, extend step 35 circulation, 72-98 DEG C melts, and naturally cools to less than 40 DEG C；
（Three）Enzyme action screening positive clone：It is formulated as follows reaction system in sterilizing PCR pipe：PCR primer 2 μ L, Detecase
Buffer 2 μ L, Detecase 2 μ L, plus distilled water is to 10 μ L, 45 DEG C of reaction 20min, immediately after to above-mentioned 10 μ L systems
Interior addition 2 μ L stop buffers, the agarose gel electrophoresiies subsequently carrying out 2% detect, electrophoresis 105V, 45min, after electrophoresis terminates,
Gel image analyser is taken pictures.
Further, what described cell protein was analyzed concretely comprises the following steps：
I. Protein Extraction and concentration mensuration：Rinse attached cell with PBS, scraped with scraper plate, each sample takes 2 × 106Individual cell
Crack on ice, extract total protein by Protein Extraction Reagent box operating procedure, concentration is measured with BCA method, is diluted to optimal loading
Measure 2 μ g/ μ L, the albumen collected immediately using or -80 DEG C of environment under preserve stand-by；
II. western blot test：Preparation 10%SDS polyacrylamide gel separation gel and 5% concentration glue, take 20 μ g testing protein samples
Product are put in EP pipe and Jia 5 × SDS sample-loading buffer, and boiling 5min makes albuminous degeneration, add enough running buffer into electrophoresis tank
Liquid, the formula of described electrophoretic buffer is：Tris3.02g, glycine 18.8g, 10%SDS10mL, ddH2O1000mL, loading,
Electrophoresis constant voltage 60 V × 40 min, rear 110V × 60 min, enter in the transfer groove that sample is placed with after terminating transferring film buffer
Row transferring film, the formula of described transferring film buffer is：Tris 3.02g, glycine 14.4g, 100% methanol 200 mL, ddH2O
800mL, constant current 300mA × 110min；Take out after protein delivery to pvdf membrane, rinse 5min with lavation buffer solution, rinse altogether
4 times, the formula of described lavation buffer solution is：TBS-T, Tris 2.42g, NaCl 8.0g, Tween-20 0.5mL, ddH2O
It is 7.5 that 1000mL, enriching HCl 120mL make its pH value, 5% skim milk confining liquid decolorization swinging table closing 1h under room temperature；TBS-
T rinses 5 min, rinses 4 times altogether, adds the anti-human Wnt3a of Mus and the employing diluting 1000 times using Dako antibody diluent
Dako antibody diluent dilutes 2000 times of Mus anti-human β-actin antibody as internal reference, and, TBS-T rinses under 4 DEG C of environment overnight
5min, rinses 4 times, dilutes the sheep anti-mouse igg two of 1000 times of horseradish peroxidase HP labelling using Dako antibody diluent
Anti- 37 DEG C of incubation 1 h, after washing, ECL develops the color and takes pictures.
Further, described CCK-8 method detects concretely comprising the following steps of cell proliferation：If blank group, negative control group, experiment
Group, trophophase cell of taking the logarithm, prepare cell suspension with trypsinization, be inoculated in 96 orifice plates, every hole 100 μ L respectively, incite somebody to action
Culture plate is placed on 37 DEG C, 5% CO2Incubator in preculture, take out respectively at given time point and change liquid, 10 μ are added in every hole
LCCK-8 reagent, continues to be incubated 1-4 h in incubator, detects A using microplate reader450Value.
Beneficial effects of the present invention are as follows：The present invention passes through to build Cas9 complex carries slow viruss system knockout Wnt3a first
Gene, Crispr/Cas9 is a kind of technology that the specific site of any species gene group can be carried out with accurate edits, uses
This technology can carry out cellular level single-gene or polygenes knock out, and the method is than other gene editing technology targeting accuracies more
Height, RNA target must mate completely to sequence and genome sequence, and Cas9 just can shear to DNA, and can achieve to target gene
Multiple sites knock out simultaneously, and vector construction is short for experimental period, save plenty of time and cost, and no species limit.
Brief description
Fig. 1 is Wnt3a ImmunohistochemistryResults Results comparison diagram in liver cancer tissue and cancer beside organism；
Fig. 2 is the first subinfection virus of the Cas9 with puromycin labelling and its selection result；
Fig. 3 is the second subinfection band fluorescently-labeled sgRNA virus；
Fig. 4 detects gene knockout efficiency agarose gel electrophoresiies result figure for mispairing enzyme process；
Fig. 5 is Western interpretation of result figure；
Fig. 6 is CCK8 interpretation of result figure.
Specific embodiment
Hereinafter embodiments of the present invention are illustrated by particular specific embodiment, those skilled in the art can be by this explanation
Content disclosed by book understands other advantages and effect of the present invention easily.
Embodiment 1
The following example is the process of knockout Wnt3a gene and the verification step of the present invention.
Knock out process and its verification method of Wnt3a gene, carry through building the Cas9 slow viruss for Wnt3a gene
Body, the culture of HepG2 cell and pass on, aim cell slow virus infection and screening, mispairing enzyme process checking gene knockout efficiency, thin
Born of the same parents' analysis of protein, CCK-8 method detect that the step of cell proliferation completes the knockout to Wnt3a gene and checking；
Build concretely comprising the following steps of the Cas9 slow virus carrier for Wnt3a gene：Build Cas9 complex carries slow viruss, comprise
Lenti-sgRNA-EGFP virus 2, respectively expression target gene sgRNA sequence and comparison sgRNA sequence, titre 1E+8 with
On, target gene sgRNA sequence is specifically as shown in table 1；1, Lenti-CAS9-puro virus, expresses CAS9 albumen, and band puro resists
Property, titre is in more than 1E+8；
Table 1 is SgRNA template sequence
Table 1
The culture of HepG2 cell and the concrete steps passing on include cell recovery and passage；The culture of HepG2 cell and biography
Ride instead of walk rapid in the concretely comprising the following steps of cell recovery：
（1）Take out cell cryopreservation tube from liquid nitrogen container, put into rapidly in 37 DEG C of water-baths, and shake makes it thaw as early as possible frequently；
（2）After thawing completely, under 1000rpm environment, it is centrifuged 2min；
（3）Then, after adopting 70% alcohol wipe cryopreservation tube sterilization, move to super-clean bench；
（4）Suck frozen stock solution supernatant, add the fresh complete medium re-suspended cell of 1ml, by cell suspension inoculation to containing 3ml
In the Tissue Culture Flask of complete medium, gently shake even after be placed in 37 DEG C, 5%CO2Cultivate in incubator；
（5）Next day is cultivated for after changing a culture fluid.
The culture of HepG2 cell and concretely comprising the following steps of the passage passing in step：
1）The cell that growth 90% is converged is passed on, and discards old culture fluid, adds the D-Hanks solution of 2ml sterilizing, washing
Cell growth face；
2）Then discard this solution, add 1ml pancreatin Digestive system, digest 1-2 min under 37 DEG C of environment, until cell disappears completely
Change is got off；
3）Add complete medium 2ml, with measuring pipette piping and druming, the cell on wall is rinsed；
4）Divide to two new 6-cm dish after mixing cell, supply complete medium to 4ml, continue culture.
Aim cell slow virus infection is concretely comprised the following steps with screening：
A. slow viruss ghost to be screened will be uninfected by be inoculated in 6 orifice plates to 70-80% degrees of fusion；
B., after cell attachment, puromycin medicine is added to carry out the screening of ghost lethal least concentration, 1 μ g/ml, 2.5 μ g/
Ml, 5 μ g/ml and 10 μ g/ml are screened, and draw the lowest concentration of drug of complete cell death after drug treating 48h；
C. cultivate and carry out pancreatin digestion to the aim cell of exponential phase, make cell suspension；
D. by cell number be 5 × 104Cell suspension inoculation in 6 orifice plates, 5%CO under 37 DEG C of environment2Incubator culture is treated thin
Born of the same parents' degrees of fusion reaches 30%；
E. according to cell MOI value, virus, observation of cell state after 12h are added：Without obvious cytotoxic effect, continue
Culture medium is changed after continuous culture 24h；If there are obvious cytotoxic effect, change culture medium immediately；
F.48 after hour, add puromycin drug screening 48h, obtain the mixing clone of stable expression CAS9；
G. the cell immediate observation cell state after screening is it is ensured that cell state is good；
H. bed board again, infects the slow viruss of expression sgRNA according to slow virus infected cell step；
I. the expression of reporter gene GFP on slow viruss is observed in infection after 3 days, and fluorescence efficiency starts in 80% about cell
Carry out subsequent experimental.
Table 2 is target spot corresponding primer information
Table 2
It is illustrated in figure 2 the first subinfection virus of the Cas9 with puromycin labelling and its selection result, A group is infected group, B group
For matched group, add puromycin screening postoperative infection group survival, matched group is dead；It is illustrated in figure 3 the second subinfection band fluorescence
The sgRNA virus of labelling, A1-3 is sgRNA（+）Infection group and viral infection group, A4 is sgRNA（-）Infection group and viral infection group, B1-4 fluorescence efficiency
Reach more than 80% representative to infect successfully.
Mispairing enzyme process verifies concretely comprising the following steps of gene knockout efficiency：
（One）DNA extracts：Collect the cell of successfully infection, extract genomic DNA using genome DNA extraction test kit；
（Two）PCR expands：Around sgRNA binding site, design one pair of genes group PCR primer, enter performing PCR according to following system anti-
Should：2 × Taq Plus Master Mix 10 μ L, primers F 0.5 μ L, primer R 0.5 μ L, genomic DNA 30ng, plus distilled water
To 20 μ L；
PCR response procedures are arranged：94 DEG C of denaturations 90s, 94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 60s/kb, repeat
Degeneration, annealing, extend step 35 circulation, 72-98 DEG C melts, and naturally cools to less than 40 DEG C.
（Three）Enzyme action screening positive clone：It is formulated as follows reaction system in sterilizing PCR pipe：PCR primer 2 μ L,
Detecase Buffer2 μ L, Detecase2 μ L, plus distilled water is to 10 μ L, 45 DEG C of reaction 20min, immediately after to above-mentioned 10 μ
Add 2 μ L stop buffers in L system, subsequently carry out 2% agarose gel electrophoresiies detection, electrophoresis 105V, 45min, electrophoresis is tied
Shu Hou, gel image analyser is taken pictures.It is illustrated in figure 4 mispairing enzyme process detection gene knockout efficiency agarose gel electrophoresiies knot
Really.
What cell protein was analyzed concretely comprises the following steps：
I. Protein Extraction and concentration mensuration：Rinse attached cell with PBS, scraped with scraper plate, each sample takes 2 × 106Individual cell
Crack on ice, extract total protein by Protein Extraction Reagent box operating procedure, concentration is measured with BCA method, is diluted to optimal loading
Measure 2 μ g/ μ L, collected albumen immediately using or -80 DEG C of environment under preserve stand-by；
II. western blot test：Preparation 10%SDS polyacrylamide gel separation gel and 5% concentration glue, take 20 μ g testing protein samples
Product are put in EP pipe and Jia 5 × SDS sample-loading buffer, and boiling 5min makes albuminous degeneration, add enough running buffer into electrophoresis tank
Liquid, the formula of described electrophoretic buffer is：Tris3.02g, glycine 18.8g, 10%SDS10mL, ddH2O1000mL, loading,
Sample is placed with after terminating in the transfer groove of transferring film buffer by electrophoresis constant voltage 60 V × 40 min, 110 V × 60 min afterwards
Carry out transferring film, the formula of described transferring film buffer is Tris 3.02g, glycine 14.4g, 100% methanol 200 mL, ddH2O
800mL, constant current 300mA × 110min；Take out after protein delivery to pvdf membrane, rinse 5min with lavation buffer solution, rinse altogether
4 times, the formula of described lavation buffer solution is：TBS-T, Tris2.42 g, NaCl8.0g, Tween-20 0.5mL, ddH2O
It is 7.5 that 1000mL, enriching HCl120mL make its pH value, and under room temperature, 5% skim milk confining liquid decolorization swinging table closes 1 h；TBS-
T rinses 5 min, rinses 4 times altogether, adds the anti-human Wnt3a of Mus and the employing diluting 1000 times using Dako antibody diluent
Dako antibody diluent dilutes 2000 times of Mus anti-human β-actin antibody as internal reference, and, TBS-T rinses under 4 DEG C of environment overnight
5min, rinses 4 times, dilutes the sheep anti-mouse igg two of 1000 times of horseradish peroxidase HP labelling using Dako antibody diluent
Anti- 37 DEG C of incubation 1 h, after washing, ECL develops the color and takes pictures.It is illustrated in figure 5 the structural analyses of Western, as seen from the figure,
The expression of sgRNA2 target spot corresponding hepatoma carcinoma cell Wnt3a is substantially lowered.
CCK-8 method detects concretely comprising the following steps of cell proliferation：If blank group, negative control group, experimental group, growth of taking the logarithm
Phase cell, prepares cell suspension with trypsinization, is inoculated in 96 orifice plates, every hole 100 μ L respectively, culture plate is placed on 37
DEG C, 5% CO2Incubator in preculture, take out respectively at given time point and change liquid, 10 μ LCCK-8 reagent are added in every hole, continue
It is incubated 1-4 h in incubator, A is detected using microplate reader450Value.It is illustrated in figure 6 CCK-8 method testing result, as seen from the figure,
Hepatoma cell proliferation ability after successful knockout Wnt3a gene is substantially suppressed.
The present invention passes through to build Cas9 complex carries slow viruss system knockout Wnt3a gene first, and Crispr/Cas9 is one
Plant the technology that the specific site of any species gene group can be carried out with accurate edits, cellular level can be carried out using this technology
Single-gene or polygenes knock out, and principle is that Cobra venom endonuclease Cas9 albumen identifies specific gene group site simultaneously by guidance quality RNA
Double-stranded DNA is cut, the method is higher than other gene editing technology targeting accuracies, and RNA target is to sequence and gene
Group sequence must be mated completely, and Cas9 just can shear to DNA, and can achieve that sites multiple to target gene knock out simultaneously, carries
Body structure is short for experimental period, saves plenty of time and cost, and no species limit.
Above-described embodiment is presently preferred embodiments of the present invention, is not the restriction to technical solution of the present invention, as long as
The technical scheme that can realize on the basis of above-described embodiment without creative work, is regarded as falling into patent of the present invention
Rights protection scope in.
Claims (7)
1. knock out the process of Wnt3a gene and its verification method it is characterised in that：Through building the Cas9 for Wnt3a gene
Slow virus carrier, the culture of HepG2 cell and pass on, aim cell slow virus infection with screening, mispairing enzyme process checking clpp gene
Except efficiency, cell protein analysis, CCK-8 method detect that the step of cell proliferation completes the knockout to Wnt3a gene and checking；
Described structure concretely comprising the following steps for the Cas9 slow virus carrier of Wnt3a gene：Build Cas9 complex carries slow viruss, bag
Containing Lenti-sgRNA-EGFP virus 2, respectively expression target gene sgRNA sequence and comparison sgRNA sequence, titre 1E+8 with
On；1, Lenti-CAS9-puro virus, expresses CAS9 albumen, carries puro resistance, titre is in more than 1E+8；
The culture of described HepG2 cell and the concrete steps passing on include cell recovery and passage.
2. the process of knockout Wnt3a gene according to claim 1 and its verification method it is characterised in that：Described HepG2
The culture of cell and concretely comprising the following steps of the cell recovery passing in step：
Take out cell cryopreservation tube from liquid nitrogen container, put into rapidly in 37 DEG C of water-baths, and shake makes it thaw as early as possible frequently；
After thawing completely, under 1000rpm environment, it is centrifuged 2min；
Then, after adopting 70% alcohol wipe cryopreservation tube sterilization, move to super-clean bench；
Suck frozen stock solution supernatant, add the fresh complete medium re-suspended cell of 1ml, cell suspension inoculation is extremely complete containing 3ml
In the Tissue Culture Flask of full culture medium, gently shake even after be placed in 37 DEG C, 5%CO2Cultivate in incubator；
Next day is cultivated for after changing a culture fluid.
3. the process of knockout Wnt3a gene according to claim 1 and its verification method it is characterised in that：Described HepG2
The culture of cell and concretely comprising the following steps of the passage passing in step：
The cell that growth 90% is converged is passed on, and discards old culture fluid, adds the D-Hanks solution of 2ml sterilizing, and washing is thin
Intracellular growth face；
Then discard this solution, add 1ml pancreatin Digestive system, digest 1-2 min under 37 DEG C of environment, until cell catapepsises
Get off；
Add complete medium 2ml, with measuring pipette piping and druming, the cell on wall is rinsed；
Divide to two new Tissue Culture Flasks after mixing cell, supply complete medium to 4ml, continue culture.
4. the process of knockout Wnt3a gene according to claim 1 and its verification method it is characterised in that：Described purpose
Cell slow virus infection is concretely comprised the following steps with screening：
Slow viruss ghost to be screened will be uninfected by be inoculated in 6 orifice plates to 70-80% degrees of fusion；
After cell attachment, add puromycin medicine carry out ghost lethal least concentration screening, 1 μ g/ml, 2.5 μ g/ml,
5 μ g/ml and 10 μ g/ml are screened, and draw the lowest concentration of drug of complete cell death after drug treating 48h；
Cultivate and carry out pancreatin digestion to the aim cell of exponential phase, make cell suspension；
Cell number is 5 × 104The cell suspension inoculation of/ml in 6 orifice plates, 5%CO under 37 DEG C of environment2Incubator culture is treated thin
Born of the same parents' degrees of fusion reaches 30%；
According to cell MOI value, add virus, observation of cell state after 12h：Without obvious cytotoxic effect, continue
Culture medium is changed after culture 24h；If there are obvious cytotoxic effect, change culture medium immediately；
After 48 hours, add puromycin drug screening 48h, obtain the mixing clone of stable expression CAS9；
Cell immediate observation cell state after screening is it is ensured that cell state is good；
Bed board again, infects the slow viruss of expression sgRNA according to slow virus infected cell step；
Infection 3 days after observe slow viruss on reporter gene GFP expression, fluorescence efficiency 80% about cell start into
Row subsequent experimental.
5. the process of knockout Wnt3a gene according to claim 1 and its verification method it is characterised in that：Described mispairing
Enzyme process verifies concretely comprising the following steps of gene knockout efficiency：
（One）DNA extracts：Collect the cell of successfully infection, extract genomic DNA using genome DNA extraction test kit；
（Two）PCR expands：Around sgRNA binding site, design one pair of genes group PCR primer, enter performing PCR according to following system anti-
Should：2 × Taq Plus Master Mix 10 μ L, primers F 0.5 μ L, primer R 0.5 μ L, genomic DNA 30ng, plus distilled water
To 20 μ L；
PCR response procedures are arranged：94 DEG C of denaturations 90s, 94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 60s/kb, repeat
Degeneration, annealing, extend step 35 circulation, 72-98 DEG C melts, and naturally cools to less than 40 DEG C；
（Three）Enzyme action screening positive clone：It is formulated as follows reaction system in sterilizing PCR pipe：PCR primer 2 μ L, Detecase
Buffer 2 μ L, Detecase 2 μ L, plus distilled water is to 10 μ L, 45 DEG C of reaction 20min, immediately after to above-mentioned 10 μ L systems
Interior addition 2 μ L stop buffers, the agarose gel electrophoresiies subsequently carrying out 2% detect, electrophoresis 105V, 45min, after electrophoresis terminates,
Gel image analyser is taken pictures.
6. the process of knockout Wnt3a gene according to claim 1 and its verification method it is characterised in that：Described cell
The concretely comprising the following steps of analysis of protein：
I. Protein Extraction and concentration mensuration：Rinse attached cell with PBS, scraped with scraper plate, each sample takes 2 × 106Individual cell in
Crack on ice, extract total protein by Protein Extraction Reagent box operating procedure, concentration is measured with BCA method, is diluted to optimal applied sample amount 2
μ g/ μ L, the albumen collected immediately using or -80 DEG C of environment under preserve stand-by；
II. western blot test：Preparation 10%SDS polyacrylamide gel separation gel and 5% concentration glue, take 20 μ g testing protein samples
Product are put in EP pipe and Jia 5 × SDS sample-loading buffer, and boiling 5min makes albuminous degeneration, add enough running buffer into electrophoresis tank
Liquid, the formula of described electrophoretic buffer is：Tris3.02g, glycine 18.8g, 10%SDS10mL, ddH2O1000mL, loading,
Electrophoresis constant voltage 60 V × 40 min, rear 110V × 60 min, enter in the transfer groove that sample is placed with after terminating transferring film buffer
Row transferring film, the formula of described transferring film buffer is：Tris 3.02g, glycine 14.4g, 100% methanol 200 mL, ddH2O
800mL, constant current 300mA × 110min；Take out after protein delivery to pvdf membrane, rinse 5min with lavation buffer solution, rinse altogether
4 times, the formula of described lavation buffer solution is：TBS-T, Tris 2.42g, NaCl 8.0g, Tween-20 0.5mL, ddH2O
It is 7.5 that 1000mL, enriching HCl 120mL make its pH value, 5% skim milk confining liquid decolorization swinging table closing 1h under room temperature；TBS-
T rinses 5 min, rinses 4 times altogether, adds the anti-human Wnt3a of Mus and the employing diluting 1000 times using Dako antibody diluent
Dako antibody diluent dilutes 2000 times of Mus anti-human β-actin antibody as internal reference, and, TBS-T rinses under 4 DEG C of environment overnight
5min, rinses 4 times, dilutes the sheep anti-mouse igg two of 1000 times of horseradish peroxidase HP labelling using Dako antibody diluent
Anti- 37 DEG C of incubation 1 h, after washing, ECL develops the color and takes pictures.
7. the process of knockout Wnt3a gene according to claim 1 and its verification method it is characterised in that：Described CCK-8
Method detects concretely comprising the following steps of cell proliferation：If blank group, negative control group, experimental group, trophophase cell of taking the logarithm, use pancreas egg
Cell suspension is prepared in white enzymic digestion, is inoculated in 96 orifice plates, every hole 100 μ L respectively, culture plate is placed on 37 DEG C, 5% CO2Culture
Preculture in case, takes out respectively at given time point and changes liquid, 10 μ LCCK-8 reagent are added in every hole, continues to be incubated in incubator
1-4 h, detects A using microplate reader450Value.
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