CN110295147A - The rapid identification method and its application of proliferation phenotype after functional gene knocks out in a kind of esophageal carcinoma cell line - Google Patents
The rapid identification method and its application of proliferation phenotype after functional gene knocks out in a kind of esophageal carcinoma cell line Download PDFInfo
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Abstract
The rapid identification method and its application of proliferation phenotype after being knocked out the invention belongs to functional gene in technical field of molecular biology more particularly to a kind of esophageal carcinoma cell line.Then the present invention is utilized selected by flow cytometry apoptosis cell, is seeded in six orifice plates by the zero load of plasmid and CRISPR/Cas9 after transfection target gene sgRNA respectively and CRISPR/Cas9 carrier homologous recombination.Remaining cell cracking obtains DNA, makees Ago-Gel identification verifying after PCR.After the cell of sorting cultivates 2 weeks in cell incubator, culture is terminated, after being fixed with 4% paraformaldehyde, then crystallized purple dyeing.The clone formation number of every group of cell can be visually judged at this time, and cloning efficiency can accurately reflect the proliferative capacity and group's dependence of cell.Proliferation phenotype after can quickly judging cancer of the esophagus functional gene knockout according to Clone formation result, the present invention have the advantages such as high efficiency, high precision and high throughput.
Description
Technical field
After being knocked out the invention belongs to functional gene in technical field of molecular biology more particularly to a kind of esophageal carcinoma cell line
The rapid identification method and its application of proliferation phenotype.
Background technique
The cancer of the esophagus is one of most common malignant tumour in China, since its early symptom is unobvious, most patient with esophageal carcinoma
It has been advanced stage, poor prognosis when making a definite diagnosis.Patient with esophageal carcinoma 5 years survival rates in China's only have 20% or so at present.Although the situation is tense,
However in tumor research field, the basic research of the cancer of the esophagus but relatively lags behind with drug development, and especially the cancer of the esophagus is in world's model
Enclosing interior Area distribution has significant otherness, and China is most important district occurred frequently, and wherein the overwhelming majority belongs to squamous carcinoma.Therefore it opens
Cancer of the esophagus high throughput research platform is sent out, it is very necessary.
CRISPR/Cas9-gene editing magic scissors hand, as a kind of newest genome edit tool emerged in large numbers, energy
DNA identification and the editor for enough completing RNA guiding, are compared using CRISPR/Cas9 system development cancer of the esophagus high throughput research platform
It is convenient.The gene targeting system of CRISPR is taken Cas9 nuclease to using the RNA of target spot specificity specific on genome
Target spot leads to gene knockout etc. to cut to specific gene site.Compared with traditional gene editing method,
The targeting system of CRISPR is simple, and vector construction is simple, and target practice efficiency is very high, is extensive in cell line gene knockout modelling
Using and one of the means approved.
Traditional cell line knocks out, even if still needing to carry out after cell transfecting using CRISPR/Cas gene editing system
More wheel resistance drug screenings and Method of Limited Dilution isolate monoclonal cell and can carry out phenotypic analysis after expanding culture no matter
It is limiting dilution method or resistant screening methods, is required to expand individual cells culture to centainly after screening positive cell
Phenotypic analysis can be carried out after degree, take considerable time cost and human cost；And screening efficiency is low, and analysis phenotype is taken
When, largely delay the research progress and range of cancer of the esophagus functional gene.
Publication No. is that cancer of the esophagus function base in a kind of cell line is disclosed in CN109402179A Chinese invention patent application
Because of the rapid identification method of proliferation phenotype after knocking out, it is pointed out that RND2 gene also can be by collecting different time after cell sorting
The DNA of point carries out fragment analysis, but the technology path needs to extract the process of DNA, and further fragments analysis experiment twice
Cumbersome, required sequenator is expensive to be not easy to promote.
Summary of the invention
In view of the problems of the existing technology, the object of the present invention is to provide functional genes in a kind of esophageal carcinoma cell line to strike
Except the rapid identification method and its application of rear proliferation phenotype, this method can quickly judge the increasing after cancer of the esophagus functional gene knockout
Phenotype is grown, the molecule screening of cancer of the esophagus high throughput is realized, there is very strong directive function to esophageal cancer related gene functional study.
The invention is realized in this way in a kind of esophageal carcinoma cell line functional gene knock out after proliferation phenotype Rapid identification
Method, comprising the following steps:
Step 1: utilizing CRISPR/Cas9 gene editing technology by esophageal cancer cell functional gene PARK2 gene knockout；
Step 2: cell and zero load cellular control unit that functional gene knocks out are seeded to orifice plate training by cell sorting respectively
It supports；
Step 3: after the cell of orifice plate culture is fixed, crystallized purple dyeing is estimated the cloning efficiency of two groups of cells, is sentenced
Proliferation phenotype after disconnected cancer of the esophagus functional gene knockout.
Further, in step 2 after cell sorting, cell cracking is separately taken to extract DNA, carries out PCR, electroresis appraisal, verify function
Can gene whether successful knockout.
Further, the primer sequence of PCR detection is shown in SEQ ID NO.10 and SEQ ID NO.11.
Further, the cell that cancer of the esophagus functional gene knocks out is compareed with zero load using flow cell sorter in step 2
The cell of group is sorted, and is seeded in six orifice plates and is cultivated.
Fluidic cell sorting technology is to irradiate band under flow at high speed state using flow cell sorter with high energy laser
The cell of fluorescence labels simultaneously measures its intensity for emitting fluorescence.Fluidic cell sorting can be sorted according to unused Marker, be had
The features such as precision is high, and speed is fast.Clone forming Test is one of the effective ways for measuring ability of cell proliferation.Individual cells are in body
It is more than generation to continue 6 outside, cell mass composed by offspring is known as cloning.At this moment each to clone the cell containing 50 or more, greatly
It is small, by counting cloning efficiency, quantitative analysis to be done to the multiplication potentiality of individual cells between 0.3-1.0mm, understand thin
The proliferative capacity and independent survival capacity of born of the same parents.
Further, the fixed cell of paraformaldehyde is used in step 3.
Further, utilize CRISPR/Cas9 gene editing technology by esophageal cancer cell functional gene PARK2 base in step 1
Because the step of knocking out, includes:
S1: sgRNA target sequence is designed for functional gene, and is directed to target sequence design primer；
S2: primer annealing, pairing obtain the double chain DNA fragment for having cohesive end；
S3: the double chain DNA fragment with cohesive end is connected with the Cas9 carrier with identical cohesive end, is weighed
Group expression vector；
S4: esophageal cancer cell is transfected with recombinant expression carrier, culture is obtained esophageal cancer cell functional gene PARK2 base
Because of the cell of knockout.
Further, sgRNA target sequence is designed as two or three in step S1.
It selects three target sequences while practicing shooting, can be improved the efficiency of gene editing.
Further, the sgRNA target sequence is shown in SEQ ID NO.1 to SEQ ID NO.3.
Further, in step S3, the Cas9 carrier is pX460 carrier.
Proliferation phenotype is quick after functional gene knocks out in a kind of esophageal carcinoma cell line as described in claim 1-9 is any
Application of the identification method in the proliferation phenotype judgement after cancer of the esophagus functional gene knockout.
Further, the step of extracting DNA is as follows: by distribution to the remaining cell after 6 orifice plates, centrifugation is abandoned supernatant, is added
9-11 μ L lysate and 3-5 μ L Proteinase K, it is another that 9-10 μ L deionized water is added, then in 55-57 DEG C of 10-20min, 94-96 DEG C
3-7min is cracked.
This kind of DNA extraction method is convenient and efficient, and required cell concentration is few, and extracts obtained sample P CR amplification positive rate
It is high.
In conclusion advantages of the present invention and good effect are as follows:
Compared with prior art, the technology of the present invention route has process simple, only needs once-through operation, and result is more
Intuitively to stablize.Furthermore present invention candidate target gene PARK2 belong to inventor in cancer of the esophagus research newfound one it is new
Molecular marked compound, at present not yet it has been reported that innovative value with higher.
The rapid identification method of proliferation phenotype after cancer of the esophagus functional gene knocks out in cell line of the invention, in above
Hold, CRISPR/Cas9 technology can quickly be reflected with fluidic cell sorting technology and fragment analysis technology organic combination to together
Determine the Relevant phenotype after cancer of the esophagus functional gene knocks out.From CRISPR/Cas9 gene knockout to acquisition table in method of the invention
Type conclusion, whole flow process amounted to for two weeks, as a result precisely stablized, compared with the monoclonal after traditional resistance screening, Method of Limited Dilution
Cell strain screening, then to the proliferation phenotype analysis after monoclonal cell amplification, save a large amount of manpowers and time cost.The present invention
Using the combination of the above technology, it can be achieved that the molecule of cancer of the esophagus high throughput screens, provided point for the clinical research of the cancer of the esophagus
Sub- therapy target.
Detailed description of the invention
Fig. 1 is PCR identification PARK2 gene C RISPR-Cas9 target practice effect picture；
Fig. 2 is violet staining result；
Fig. 3 is cell clone number comparison result.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments to the present invention
It is further elaborated, equipment used in each embodiment and test example and reagent unless otherwise specified, can be from business
Approach obtains.Described herein specific examples are only used to explain the present invention, is not intended to limit the present invention.
The present invention discloses in a kind of esophageal carcinoma cell line functional gene knock out after proliferation phenotype rapid identification method and
It is applied, inventive concept are as follows: plasmid after transfecting target gene sgRNA and CRISPR/Cas9 carrier homologous recombination respectively and not
The CRISPR/Cas9 of the sgRNA containing target gene is unloaded.Then flow cell sorter is utilized, every group sorting 5000 have
The positive cell group of CRISPR/Cas9 carrier fluorescence label.After sorting, every group of cell takes out 500 cell inoculations respectively
Into three culture holes of six orifice plates, i.e. 500 cells of target gene knockout group and unloaded group are contained in the every hole of six orifice plates, and
It is each that there are three multiple holes.Remaining cell cracking obtains DNA, makees Ago-Gel identification verifying after PCR.The cell of sorting is in cell
After cultivating 2 weeks in incubator, culture is terminated, after being fixed with 4% paraformaldehyde, then crystallized purple dyeing.Can visually it judge at this time
The clone formation number of every group of cell.Proliferation after can quickly judging cancer of the esophagus functional gene knockout according to Clone formation result
Phenotype.By knocking out PARK2 gene in esophageal carcinoma cell line KYSE150, it can be observed that the gene is in esophageal carcinoma cell line
In effect.Steps are as follows for specific experiment:
1, it is directed to candidate gene PARK2, determines target practice site
According to sgRNA design principle, with the transcript (Transcript:PRKN- of human gene PARK2
206ENST00000366898.6) Exon9 therein as template, using Photographing On-line software (http: //
Crispor.tefor.net), three specific positions are obtained as sgRNA target sequence, it can also be only in certain other embodiments
Two target sequences are designed, the target sequence in the present embodiment is respectively as follows:
SgRNA1:5 '-GTCCTCCCCTATCAACCCCG-3 ' is shown in SEQ ID NO.1；
SgRNA2:5 '-TACTGCTGGTACCGGTTGTA-3 ' is shown in SEQ ID NO.2；
SgRNA3:5 '-TGGAGCGGTGTGTTCCTGTC-3 ' is shown in SEQ ID NO.3.
Then by being transcribed in vitro, synthesis obtains sgRNA.
2, design primer
According to the target sequence designed above, designs three pairs of primers and is synthesized by hundred Li Ge Bioisystech Co., Ltd of Shanghai,
Adding BbsI restriction enzyme sites at 5 ' ends of primer sequence, (base sequence for wherein adding underscore is expressed as the digestion position of addition
Point), primer sequence is respectively as follows:
H-PARK2-IVT-1:5 '-CACCGTCCTCCCCTATCAACCCCG-3 ' is shown in SEQ ID NO.4；
H-PARK2-IVT-2:5 '-AAACCGGGGTTGATAGGGGAGGAC-3 ' is shown in SEQ ID NO.5；
H-PARK2-IVT-3:5 '-CACCG TACAACCGGTACCAGCAGTA-3 ' is shown in SEQ ID NO.6；
H-PARK2-IVT-4:5 '-AAACTACTGCTGGTACCGGTTGTA C-3 ' is shown in SEQ ID NO.7；
H-PARK2-IVT-5:5 '-CACCGACAGGAACACACCGCTCCA-3 ' is shown in SEQ ID NO.8；
H-PARK2-IVT-6:5 '-AAACTGGAGCGGTGTGTTCCTGTC-3 ' is shown in SEQ ID NO.9.
It is worth noting that the U6 promoter that the pX461 carrier of expression sgRNA is, if transcription initiation site is base
If G, gene expression amount can be significantly raised, therefore is considered as this factor when redesign primer, additionally adds a bases G, this
In the case of kind, corresponding 3 ' end of downstream primer needs to add a base C, to guarantee that gene maintains higher expression quantity.
3, primer annealing, pairing obtain the double chain DNA fragment for having cohesive end
By synthesized in step 26 primers with H-PARK2-IVT-1+H-PARK2-IVT-2 and H-PARK2-IVT-3+H-
PARK2-IVT-4 is obtained there are also the combination annealed pairs of H-PARK2-IVT-5+H-PARK2-IVT-6 and is had cohesive end
Double chain DNA fragment.
Specific procedure is as follows: two pairs of primers of synthesis being distinguished phosphorylation first, phosphorylation reaction system is primer H-
There are also H-PARK2-IVT-5+H- by PARK2-IVT-1+H-PARK2-IVT-2 and H-PARK2-IVT-3+H-PARK2-IVT-4
1 μ L (100 μM) are respectively added in PARK2-IVT-6, add 1 μ 10 × T4Ligation of L Buffer (NEB), 0.5 μ is then added
L T4Polynucleotide Kinase (NEB M0201S) is eventually adding 6.5 μ L ddH2O is prepared to 10 μ L of total volume
It after reaction system, mixes well, is placed in 37 DEG C of incubation 30min；The above reaction product is taken out, is transferred in PCR instrument and is become
Property and annealing, response procedures are as follows: 95 DEG C, 5min；95–25℃at-5℃/min.
4, the vector DNA fragment of linearisation is obtained
Carrier (pX460) DNA is linearized using restriction enzyme BbsI, then purification and recovery, obtaining has viscosity
The vector DNA fragment of end, specific system are as follows: 1 μ g pX461 carrier DNA is sequentially added into 1.5ml centrifuge tube；3μL 10
×NEB Buffer 2.1；The last moisturizing of 1 μ L BbsI (NEB) is placed in 37 DEG C of incubators and is incubated overnight to 30 μ L of total volume.Enzyme
Digestion products are purified using QIAquick PCR Purification Kit after cutting and are recycled to 20 μ L ddH2In O.
5, complete sgRNA and Cas9 expression vector is obtained by connection reaction, conversion, recombinant screen
It is added obtained in the double chain DNA fragment obtained in 0.5 μ L step (3) with cohesive end and 2 μ L steps (4)
Carrier DNA with identical cohesive end adds 0.5 μ L T4DNA ligase (NEB M0202S) and 1 10 × T4 of μ L
Ligation Buffer (NEB) converts bacillus coli DH 5 alpha and applies amicillin resistance plate, is placed in 37 after reaction 1 hour
DEG C incubator is incubated overnight, and picking individual colonies in afternoon carry out sequence verification within second day, and sgRNA and Cas9 weight finally can be obtained
Group expression vector pX460-PARK2-1 and pX460-PARK2-2 and pX460-PARK2-3.
6, transfection esophageal carcinoma cell line is obtained
By mass mixings such as the expression vectors built, it is thin that the cancer of the esophagus is then transfected by liposome-mediated rotaring transfecting mode
Born of the same parents system KYSE150.
Cell culture and transfection detailed step are as follows:
The culture and passage of KYSE150 cell: cell is purchased from Chinese Academy of Sciences's cell bank, and cell is taken back laboratory and is placed in CO2
It is cultivated in incubator, after cell is completely adherent, changes liquid with DPBS to rinse out dead cell and cell metabolite, add fresh
Culture solution RPMI1640+10%FBS+1% dual anti-(penicillin+streptomysin)) continue to cultivate, seen under inverted microscope daily
Cellular morphology and upgrowth situation are examined, culture bottle bottom 90% or so is covered with to cell and starts to pass on.Observe cell growth in culture bottle
Situation absorbs the old culture solution in bottle, washs 1-2 with the DPBS of preheating when cell Proliferation is to be paved with bottom of bottle 80~90%
It is secondary, then tryptic digestive juice (0.25% pancreatin+0.02%EDTA) 1mL is added into culture bottle, it will after 37 DEG C of digestion 2min
Culture bottle is placed in microscopically observation.After cellular morphology is rounded, space between cells increases, discards trypsase and 3mL is added
RPMI1640 softly blows and beats cell to terminate digestion reaction, with pipettor repeatedly, forms cell suspension after cell detachment bottle wall,
It draws appropriate suspension to be transferred in new culture bottle, addition fresh culture, which is uniformly mixed, is placed on CO2It is trained in constant incubator
It supports.After cell up to after 90% convergence degree, will be cultivated for 24 hours in F2 cell inoculation to 24 orifice plates by above-mentioned propagating method.
The transfection of cell: pX460-PARK2-1 and pX460-PARK2-2 and pX460-PARK2-3 carrier DNA are respectively taken
1 μ g, it is experimental group that two kinds of plasmids, which amount to 3 μ g, and taking 3ug pX460 zero load is control group, is added separately to 150 μ L and subtracts serum free culture system
It is diluted in base (Opti-MEM)；It takes 0.75 μ L liposome Lipofectamine 3000 to be diluted in 150 μ L and subtracts serum free culture system
In base (Opti-MEM), then liposome dilution is added into DNA dilution, is mixed well, is placed in and incubates under room temperature
Educate 20min.Culture medium in above-mentioned steps in 24 orifice plates of inoculated and cultured cell is discarded, then divides compound in order
Be not added in corresponding cell, every hole is added 300 μ L, it is another additionally plus three holes are not to be added as parallel control experiment group and transfection
The cell of mixture is as negative control group.All cells are in 37 DEG C of 5% concentration C O2After being incubated for 4h in incubator, culture is discarded
Base replaces fresh culture medium and continues to cultivate.
7, cell mass is sorted
After transfecting 48h, culture medium is discarded, cell is resuspended in containing 500 μ L fresh cultures using trypsin digestion
In centrifuge tube, after 1500rpm is centrifuged 5min, most of culture medium supernatant is abandoned, stays about 200 μ L culture based on tube bottom, uses liquid relief
Device gently blows and beats mixing, then plus 400 μ L fresh cultures mix after be transferred in streaming pipe.It is by flow cytometer that CFP is positive
Property cell mass sort into the 15ml centrifuge tube added with 10mL fresh culture, each centrifuge tube sorts 5000 CFP sun
Property cell.After sorting, every group of cell takes out 500 cell inoculations into three culture holes of six orifice plates respectively, i.e. six holes
500 cells of target gene knockout group and unloaded group are contained in the every hole of plate, and respectively there are three multiple holes.Remaining cell cracking obtains
DNA is taken, makees Ago-Gel identification verifying after PCR.The cell of sorting cultivates 2 weeks, every other day, replacement in cell incubator
Culture solution is primary.
8, Ago-Gel verifying knocks out efficiency
Remaining cell cracking obtains DNA after six orifice plate cells will be distributed, and Ago-Gel identification verifying is run after PCR,
Wherein cracking and PCR authentication step are mainly as follows:
1. lytic cell extracts DNA
Supernatant is abandoned in cell centrifugation, adds 10 μ L lysates and 4 μ L Proteinase Ks, another to be added 9.6ul deionized water, in PCR instrument into
Row cracking, reaction condition are 56 DEG C of 15min, 95 DEG C of 5min.
2. PCR is identified:
According to PARK2 gene information, in the segment comprising target sequence, design primer:
PARK2-PCR-F:5 '-CACACTTATATGTCTTGCCAGTTGAA-3 ' is shown in SEQ ID NO.10；
PARK2-PCR-R:5 '-TCCTTCACTCCTGTCTACTTAAAGG-3 ' is shown in SEQ ID NO.11.
Include target practice site target fragment (theoretical length 750bp) by PCR amplification, carries out PCR with this primer.
PCR system are as follows: (Vazyme, P111) 2*Taq Master PCR MIX:5 μ L, PARK2-PCR-F (5 μM) and
Each 0.5 μ L of PARK2-PCR-R (5 μM)；Genomic DNA: 20ng；Supplement ddH2O to 10 μ L of total volume；Program are as follows: 95 DEG C,
5min；95 DEG C, 30s, 60 DEG C, 30s, 72 DEG C, 50s, 30 circulations；72 DEG C, 10min.
PCR product is detected with 2% agarose gel electrophoresis, as a result as shown in Figure 1, PARK2 has succeeded quilt as seen from the figure
CRISPR-Cas9 practices shooting successfully.
9, violet staining determines plate clone result
After the cell of sorting cultivates 2 weeks in cell incubator, culture is terminated, after fixing with 4% paraformaldehyde, then through tying
Crystalviolet dyeing.The clone formation number of every group of cell can be visually judged at this time.It can quickly judge to eat according to Clone formation result
Proliferation phenotype after pipe cancer functional gene knockout.As a result see Fig. 2, Fig. 3.After can determine whether that PARK2 is knocked out according to plate clone result
The growth rate of esophageal carcinoma cell line is accelerated.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Xinxiang College of Medical Science
<120>rapid identification method and its application of proliferation phenotype after functional gene knocks out in a kind of esophageal carcinoma cell line
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Claims (10)
- The rapid identification method of proliferation phenotype after 1. functional gene knocks out in a kind of esophageal carcinoma cell line, which is characterized in that including Following steps:Step 1: utilizing CRISPR/Cas9 gene editing technology by esophageal cancer cell functional gene PARK2 gene knockout；Step 2: cell and zero load cellular control unit that functional gene knocks out are seeded to orifice plate culture by cell sorting respectively；Step 3: after the cell of orifice plate culture is fixed, crystallized purple dyeing estimates the cloning efficiency of two groups of cells, judges to eat Proliferation phenotype after pipe cancer functional gene knockout.
- The Rapid identification side of proliferation phenotype after 2. functional gene knocks out in a kind of esophageal carcinoma cell line according to claim 1 Method, it is characterised in that: in step 2 after cell sorting, separately take cell cracking and extract DNA, carry out PCR, electroresis appraisal, verify function Can gene whether successful knockout.
- The Rapid identification side of proliferation phenotype after 3. functional gene knocks out in a kind of esophageal carcinoma cell line according to claim 2 Method, it is characterised in that: the primer sequence of PCR detection is shown in SEQ ID NO.10 and SEQ ID NO.11.
- The Rapid identification side of proliferation phenotype after 4. functional gene knocks out in a kind of esophageal carcinoma cell line according to claim 1 Method, it is characterised in that: the cell and unloaded control group that cancer of the esophagus functional gene is knocked out using flow cell sorter in step 2 Cell sorted, and be seeded in six orifice plates and cultivate.
- The Rapid identification side of proliferation phenotype after 5. functional gene knocks out in a kind of esophageal carcinoma cell line according to claim 1 Method, it is characterised in that: the fixed cell of paraformaldehyde is used in step 3.
- The Rapid identification side of proliferation phenotype after 6. functional gene knocks out in a kind of esophageal carcinoma cell line according to claim 1 Method, which is characterized in that utilize CRISPR/Cas9 gene editing technology by esophageal cancer cell functional gene PARK2 gene in step 1 The step of knockout includes:S1: sgRNA target sequence is designed for functional gene, and is directed to target sequence design primer；S2: primer annealing, pairing obtain the double chain DNA fragment for having cohesive end；S3: the double chain DNA fragment with cohesive end is connected with the Cas9 carrier with identical cohesive end, obtains recombination table Up to carrier；S4: esophageal cancer cell is transfected with recombinant expression carrier, culture is obtained esophageal cancer cell functional gene PARK2 clpp gene The cell removed.
- The Rapid identification side of proliferation phenotype after 7. functional gene knocks out in a kind of esophageal carcinoma cell line according to claim 6 Method, it is characterised in that: sgRNA target sequence is designed as two or three in step S1.
- The Rapid identification side of proliferation phenotype after 8. functional gene knocks out in a kind of esophageal carcinoma cell line according to claim 7 Method, it is characterised in that: the sgRNA target sequence is shown in SEQ ID NO.1 to SEQ ID NO.3.
- The Rapid identification side of proliferation phenotype after 9. functional gene knocks out in a kind of esophageal carcinoma cell line according to claim 6 Method, it is characterised in that: in step S3, the Cas9 carrier is pX460 carrier.
- 10. proliferation phenotype is quick after functional gene knocks out in a kind of esophageal carcinoma cell line as described in claim 1-9 is any Application of the identification method in the proliferation phenotype judgement after cancer of the esophagus functional gene knockout.
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| CN111593124A (en) * | 2020-06-08 | 2020-08-28 | 郑州大学第一附属医院 | UGT3A2 gene and application of protein coded by same in auxiliary diagnosis of upper gastrointestinal tumor |
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