CN108504657B - The method for knocking out HEK293T cell KDM2A gene using CRISPR-CAS9 technology - Google Patents
The method for knocking out HEK293T cell KDM2A gene using CRISPR-CAS9 technology Download PDFInfo
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Abstract
The present invention provides a kind of methods that CRISPR-CAS9 technology knocks out HEK293T cell KDM2A gene, and steps are as follows: (1), cell transfecting；(2), cellular genome is extracted；(3), PCR is identified；(4), gene level is verified；(5), protein level is verified: after gene level verifies mutant clone, it is KDM2A gene defection type that K3, K7-1 and K7-2 are further verified with protein immunoblotting (Western blotting).The present invention goes back while providing the application using the HEK293T cell line of the KDM2A gene knockout of above method preparation and using it as cell model in cell Proliferation and apoptotic signal access involved in research KDM2A methylase.
Description
Technical field
The present invention provides a kind of methods for knocking out HEK293T cell KDM2A gene using CRISPR-CAS9 technology, belong to
In gene engineering technology field.
Background technique
HEK cell is derived from source of people embryonic kidney cell, and HEK293T cell is the mutation of HEK cell, is by its transfection
What SV40 large T antigen obtained can be with the cell line of in-vitro multiplication, and this cell line proliferation is fast, and transfection efficiency is high, is usually used in albumen
Expression.Lysine dependence demethylation enzyme family, cell proliferation and apoptosis have particularly important influence, this feature is determined
Having determined it can be used as the potential application of oncotherapy.One of notable feature of cell carcinogenesis is exactly methylase and demethylation
The expression of enzyme is lacked of proper care and the epigenetic regulation dysfunction of initiation.In the demethylation enzyme family, KDM2A is so far
Study a still not very clear member.The major function of KDM2A is No. 36 lysine (H3K36) methyl for identifying histone H 3
Change site, makes its demethylation, make heterochromatin stable structure and promote the gene expression in the region, to play the original of the gene
Oncogene function.Existing research shows that express up-regulation with it related for the generation of kinds cancer, such as intestinal cancer, lung cancer and breast cancer
Deng.Existing research shows that KDM2A can promote cell to grow and migrate, but it is still unclear how to influence Apoptosis for it
Chu.Therefore, the knockout cell line of KDM2A is constructed, and is particularly important, structure with this research for apoptosis pathway
Building successful cell line can be used for protein expression, transcriptome analysis and other kinds of experiments purposes.Although there is portion at present
Divide the relevant report interfered about KDM2A gene silencing and RNA, but compared with gene knockout, the effect of gene knockout is more
Be it is thorough, be more advantageous to research KDM2A cell proliferation and apoptotic effect influence.
Summary of the invention
The present invention solves deficiency in the prior art, provides a kind of CRISPR-CAS9 technology knockout HEK293T cell
The method of KDM2A gene.The present invention goes back while providing thin using the HEK293T of the KDM2A gene knockout of above method preparation
Born of the same parents system and the answering in cell Proliferation and apoptotic signal access involved in research KDM2A methylase using it as cell model
With.
Realize technical solution used by above-mentioned purpose of the present invention are as follows:
A kind of sgRNA of the specific knockdown source of people KDM2A gene based on CRISPR/CAS9 system, the sgRNA exist
Target sequence on KDM2A gene is located on No. 14 of KDM2A gene (NM_012308.2) and 18 exons, wherein 14 extras
Target sequence on aobvious son are as follows: 5 '-ACATCGCACTCGTCTCCGTC-3 ', the target sequence on 18 exons are as follows: 5 '-
TTTGAAGCGCTCATCGCGGC-3’。
Invention also provides the expression vector pX458-KDM2A of above-mentioned sgRNA.The expression vector pX458-
The construction method of KDM2A is as follows, and 5 ' ends of the target sequence on 14 exons and on 18 exons add the enzyme of BbsI
Enzyme site caccg successively constitutes sequence SEQ ID NO:1 and SEQ ID NO:2, and artificial synthesized its complementary series SEQ ID
NO:3 and SEQ ID NO:4, by complementary series SEQ ID NO.1 and SEQ ID NO:3 and SEQ ID NO.2 and SEQ ID
NO:4 is formed by double-stranded DNA, with processBbsIThe pX458 carrier of digestion connects, so that expression vector pX458- be made
KDM2A。
Further, the present invention provides a kind of HEK293T cell line of KDM2A gene knockout and the KDM2A
The construction method of the HEK293T cell line of gene knockout, comprising the following steps: using expression vector pX458-KDM2A as KDM2A
The targeting vector of gene, transfects HEK293T cell, and the monoclonal cell strain of the KDM2A gene knockout of acquisition is abbreviated as respectively
K3, K7-1 and K7-2.
Specific construction method is as follows:
(1), cell transfecting: prepare transfection when culture HEK293T cell to 70~90% convergence degree, expression vector will be contained
The mixed liquor of pX458-KDM2A is added in HEK293T cell and is transfected, and then cultivates；
(2), cellular genome is extracted: the HEK293T cell after transfection is carried out genome extraction；
(3), PCR is identified: according to KDM2A gene order, design primer KDM2A-3F and KDM2A-3R, KDM2A-7F and
KDM2A-7R, the sequence of above-mentioned primer respectively such as: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:
Shown in 8；PCR reaction is carried out using the extracted genome of above-mentioned primer pair；
(4), gene level is verified: PCR product being sequenced, sequencing result and wild type PCR sequence alignment determine K3, K7-
1 and K7-2 is mutant；
(5), protein level is verified: after gene level verifies mutant clone, with protein immunoblotting (Western
Blotting) further verifying K3, K7-1 and K7-2 is KDM2A gene defection type.
Compared with prior art, the invention has the following advantages that (1) present invention using CRISPR/Cas9 system from
KDM2A gene is knocked out in HEK293T cell, easy to operate, effect is complete and thorough, constitutes together with HEK293T cell characteristics
Ideal cell model, for studying the change of intracellular epigenetics.(2) CRISPR/Cas9 system is based in the present invention
System knock out KDM2A gene method, with silencing, strike it is low, interference the methods of compared with, knock out effect it is more thorough, be more advantageous to and grind
Study carefully the influence of KDM2A cell proliferation and apoptotic effect.(3) present invention is confirmed from the detection of gene and protein level, KDM2A
By successful knockout, illustrate that the albumen is thoroughly changed, may cause the thorough forfeiture of KDM2A function, be suitable for KDM2A into
Row more in-depth study.
Detailed description of the invention
Fig. 1 is the mutant cell genotype identification figure that sgRNA detected and be transfected CRISPR targeting vector in embodiment；
Fig. 2 is the KDM2A protein expression content difference figure of knockout type cell strain and wild-type cell strain；
Fig. 3, Fig. 4 and Fig. 5 be a), b), c) three groups of flow cytometry wild types and each cell Proliferation knocked out between system
With apoptosis disparity map.
Specific embodiment
Detailed specific description is done to the present invention combined with specific embodiments below, following embodiment is merely to illustrate this hair
It is bright, but be not used in and limit the scope of the invention.If not special indicate, embodiment is carried out according to conventional laboratory conditions, such as J. Sa
Nurse Brooker et al. is written " Molecular Cloning:A Laboratory guide ", or the condition according to reagent manufacturers instruction suggestion.
Embodiment 1: the method for knocking out HEK293T cell KDM2A gene using CRISPR-CAS9 technology.
1, experimental material
Carrier for expression of eukaryon pSpCas9 (BB) -2A-GFP (pX458), source of people HEK293T cell.
2, the building of CRISPR/Cas9 targeting vector
(1) determination of target sequence
Source of people KDM2A gene (NM_ is obtained according to website http://asia.ensembl.org/index.html
012308.2) exon information selects No. 14 and the 18th exon as target sequence.SgRNA sequence is designed later,
Its 5 ' end adds BbsI restriction enzyme site sequence (as shown in SEQ ID NO:1,2), and synthesizes sequence (such as SEQ ID being complementary
NO:3, shown in 4).
(2) target sequence is connect with pX458 plasmid
1. pX458 empty carrier digestion, 20 μ L reaction systems are shown in Table 1,37 °C of digestions and stay overnight.
1 pX458 empty plasmid digestion system of table
2. the recovery purifying of pX458 digestion products
It is carried out according to Axygen plastic recovery kit specification.
3. the sgRNA sequence and complementary series that synthesize form double-stranded DNA, 10 μ L reaction systems are shown in Table 2, response procedures: 37 °
C, 30min；95 °C of 5min, are down to room temperature naturally.
Table 2 synthesizes segment and forms double-strand system
4. connecting after the target fragment for forming double-strand is diluted 250 times with carrier pX458, recombinant vector pX458- is obtained
KDM2A.10 μ L reaction systems are shown in Table 3, response procedures: room temperature connects 3h.
3 linked system of table
(3) bacillus coli DH 5 alpha, picking single colonie PCR identification are converted
5 μ L of connection product is added into 50 μ L DH5 α competent cells, ice bath 30 minutes after mixing, then at 42 °C
Heat-shock transformed 90 seconds, subsequent ice bath 2 minutes, 37 °C of sterile LB mediums incubated are added, and (1% tryptone, 0.5% yeast mentioned
Taking object, 1% sodium chloride) 950 μ L take 100 μ L bacterium solutions to apply shake culture 2 hours under conditions of 37 °C and revolving speed are 180 revs/min
Cloth is chosen after 37 °C of inversion cultures overnight on amicillin resistance solid LB media (ampicillin containing 100 μ g/mL)
Single colonie is taken, is cultivated in LB liquid medium of the 1mL containing 100 μ g/mL ampicillins, bacterium solution PCR identification is carried out, filters out
Plasmid with correct electrophoretic band carries out next step experiment.Sequencing primer is the forward primer sequence of U6 promoter:
5’-GAGGGCCTATTTCCCATGATTCC-3’(SEQ ID NO: 9)。
3, the preparation and identification of plasmid
Previous step is filtered out and meets expected clone, with health be century the big extraction reagent kit of high purity plasmid largely prepare nothing
Endotoxic plasmid pX458-KDM2A, measures its purity and concentration.Operating procedure by specification carries out.
4, the building of KDM2A Knockout cells system
(1) prepare transfection when being inoculated with HEK293T cell to 70~90% convergence degree, before transfection, steps are as follows for cell transfecting:
1. diluting 3000 reagent of Lipofectamine with Opti-MEM culture medium, and mix well；
2. the plasmid pX458-KDM2A mentioned greatly with the dilution of Opti-MEM culture medium, prepares DNA premixed liquid, then adds
P3000 reagent, and mix well；
3. both the above mixed liquor is mixed according to the ratio of 1:1, then it is incubated at room temperature 5 minutes, forms DNA- rouge
Liposome complex, and be added in cell, liquid is changed after cultivating 4 hours in 37 °C of lower constant incubators；
4. after culture 24 hours, filtering out the monoclonal cell with EGFP label 96 using sorting type flow cytometer
It is cultivated in orifice plate；
5. you can well imagine to cell quantity enough time-divisions takes genomic DNA to be identified.
(2) cellular genome is extracted: attached cell being handled and is abandoned on to the greatest extent for cell suspension with 2000rpm centrifugation 5 minutes
It clearly, the use of health is century universal pillar genome extraction kit, according to kit specification operating procedure by cytogene
Group extracts.
(3) PCR is identified: on KDM2A gene, design primer KDM2A-3F and KDM2A-3R, KDM2A-7F and KDM2A-
7R sequence is as follows.PCR reaction condition: 95 °C initial denaturation 5 minutes；35 circulations: 95 °C are denaturalized 30 seconds, and 58 °C are annealed 30 seconds, 72 °
C extends 1 minute；Last 72 °C extend 7 minutes eventually.PCR system is shown in Table 4.
KDM2A-3F:5 '-TGTAATCTTCTACCCTATCCA-3 ' (SEQ ID NO:5)
KDM2A-3R:5 '-AAGAGGAGGACTCAACCA-3 ' (SEQ ID NO:6)
KDM2A-7F:5 '-TGTCGGTTCAGAGGGTTTG-3 ' (SEQ ID NO:7)
KDM2A-7R:5 '-CCACAGGCTTTCAGATGC-3 ' (SEQ ID NO:8)
4 PCR amplification system of table
Wherein amplified fragments size it will meet expected PCR product recovery purifying and be sequenced.
(4) gene level is verified: PCR product being sequenced, sequencing result and wild type PCR sequence alignment determine K3, K7-1
It is mutant, sequence analysis e-g on the right side of Fig. 1 with K7-2) shown in.
(5) protein level is verified: after gene level verifies mutant clone, with protein immunoblotting (Western
Blotting) further verifying K3, K7-1 and K7-2 is KDM2A deficiency, as a result sees Fig. 2, KDM2A albumen is not detected.
Pass through the verifying of the above DNA level and protein level, it was demonstrated that the HEK293T cell construction of KDM2A gene knockout
Then success is that K7-1 and K7-2 indicate that 18 exons are prominent using 2 plants of knockouts that K3 indicates that the mutation of 14 exons generates
Sell of one's property raw 2 plants of knockouts system.
Embodiment 2: the shadow of proliferation and apoptosis that flow cytomery KDM2A is to wild-type cell system and knockout respectively
It rings
1, how specifically to be influenced to detect the knockout of KDM2A gene and can be generated on cell, we are to wild type
HEK293T cell and knockout system have carried out the detection of EdU cell Proliferation and the detection of Annexin V-FITC/PI Apoptosis respectively.
Operating process is as follows:
(1) hole every in six orifice plates is inoculated with 1 × 105~3 × 106A cell, culture to normal phase；
(2) by specification prepares EdU culture medium, and the blank control of EdU culture medium is not added in setting one, to carry out streaming
The dyestuff context analyzer of detection data.After cell culture medium is changed to EdU culture medium and control medium, 1mL is added in every hole,
It is incubated for 2 hours；
(3) cell is transferred in 1.5mL EP pipe, collects cell and abandon supernatant, then cell is resuspended with PBS, is centrifuged on abandoning
Clearly；
(4) illustrate to be dyed by EdU cell Proliferation detection explanation and the detection of Annexin V-FITC/PI apoptosis, later
Flow cytometer showed and fluorescent microscopic imaging are carried out under corresponding wavelength.The above experiment has independently carried out 3 repetitions, as a result see Fig. 3,
Fig. 4 and Fig. 5.
2, the difference by flow cytometry wild-type cell and knockout type cell in cell Proliferation and apoptosis, discovery
KDM2A itself can promote cell Proliferation and have both the function of reducing Apoptosis.
Although being made that detailed description to the present invention with a general description of the specific embodiments herein,
Those skilled in the art can be modified or improve to it on the basis of the present invention.Therefore without departing from spirit of that invention
On the basis of these modifications or improvements, fall within the scope of the claimed invention.
Sequence table
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Claims (1)
1. a kind of method for knocking out source of people KDM2A gene in HEK293T cell using CRISPR-CAS9 technology, it is characterised in that
Specific method is as follows:
(1), cell transfecting: prepare transfection when culture HEK293T cell to 70~90% convergence degree, expression vector will be contained
The mixed liquor of pX458-KDM2A is added in HEK293T cell and is transfected, and then cultivates；
The construction method of the expression vector pX458-KDM2A are as follows: the target sequence on 14 exons and 18 exons
5 ' ends add the restriction enzyme site caccg of BbsI, successively constitute sequence SEQ ID NO:1 and SEQ ID NO:2, and it is artificial synthesized
Its complementary series SEQ ID NO:3 and SEQ ID NO:4, by sequence SEQ ID NO.1 and SEQ ID NO:3 and SEQ ID
NO.2 and SEQ ID NO:4 is formed by double-stranded DNA, with processBbsIThe pX458 carrier of digestion connects, and carries so that expression be made
Body pX458-KDM2A；Target sequence on 14 exon are as follows: 5 '-ACATCGCACTCGTCTCCGTC-3 ', 18 extras are aobvious
Target sequence on son are as follows: 5 '-TTTGAAGCGCTCATCGCGGC-3 '；
(2), cellular genome is extracted: the HEK293T cell after transfection is carried out genome extraction；
(3), PCR is identified: according to KDM2A gene order, design primer KDM2A-3F and KDM2A-3R, KDM2A-7F and KDM2A-
7R, the sequence of above-mentioned primer is respectively as shown in: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8；
PCR reaction is carried out using the extracted genome of above-mentioned primer pair；
(4), gene level is verified: PCR product being sequenced, sequencing result and wild type PCR sequence alignment, is determined and obtain mutation carefully
Born of the same parents' strain；
(5), protein level is verified: after gene level verifies mutant clone, further being verified with protein immunoblotting
Stating mutant clone is KDM2A gene defection type.
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| US20150044192A1 (en) | 2013-08-09 | 2015-02-12 | President And Fellows Of Harvard College | Methods for identifying a target site of a cas9 nuclease |
| US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US9388430B2 (en) | 2013-09-06 | 2016-07-12 | President And Fellows Of Harvard College | Cas9-recombinase fusion proteins and uses thereof |
| US9340799B2 (en) | 2013-09-06 | 2016-05-17 | President And Fellows Of Harvard College | MRNA-sensing switchable gRNAs |
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
| EP3177718B1 (en) | 2014-07-30 | 2022-03-16 | President and Fellows of Harvard College | Cas9 proteins including ligand-dependent inteins |
| EP3365356B1 (en) | 2015-10-23 | 2023-06-28 | President and Fellows of Harvard College | Nucleobase editors and uses thereof |
| GB2568182A (en) | 2016-08-03 | 2019-05-08 | Harvard College | Adenosine nucleobase editors and uses thereof |
| AU2017308889B2 (en) | 2016-08-09 | 2023-11-09 | President And Fellows Of Harvard College | Programmable Cas9-recombinase fusion proteins and uses thereof |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| KR102622411B1 (en) | 2016-10-14 | 2024-01-10 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | AAV delivery of nucleobase editor |
| WO2018119359A1 (en) | 2016-12-23 | 2018-06-28 | President And Fellows Of Harvard College | Editing of ccr5 receptor gene to protect against hiv infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| WO2018165629A1 (en) | 2017-03-10 | 2018-09-13 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| EP3601562A1 (en) | 2017-03-23 | 2020-02-05 | President and Fellows of Harvard College | Nucleobase editors comprising nucleic acid programmable dna binding proteins |
| WO2018209320A1 (en) | 2017-05-12 | 2018-11-15 | President And Fellows Of Harvard College | Aptazyme-embedded guide rnas for use with crispr-cas9 in genome editing and transcriptional activation |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| EP3676376A2 (en) | 2017-08-30 | 2020-07-08 | President and Fellows of Harvard College | High efficiency base editors comprising gam |
| KR20200121782A (en) | 2017-10-16 | 2020-10-26 | 더 브로드 인스티튜트, 인코퍼레이티드 | Uses of adenosine base editor |
| CN109266680B (en) | 2018-10-17 | 2020-09-25 | 江苏集萃药康生物科技有限公司 | Method for preparing CKO/KI animal model by using Cas9 technology |
| BR112021018606A2 (en) | 2019-03-19 | 2021-11-23 | Harvard College | Methods and compositions for editing nucleotide sequences |
| DE112021002672T5 (en) | 2020-05-08 | 2023-04-13 | President And Fellows Of Harvard College | METHODS AND COMPOSITIONS FOR EDIT BOTH STRANDS SIMULTANEOUSLY OF A DOUBLE STRANDED NUCLEOTIDE TARGET SEQUENCE |
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