CN108504657A - The method for knocking out HEK293T cell KDM2A genes using CRISPR-CAS9 technologies - Google Patents

The method for knocking out HEK293T cell KDM2A genes using CRISPR-CAS9 technologies Download PDF

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CN108504657A
CN108504657A CN201810325470.3A CN201810325470A CN108504657A CN 108504657 A CN108504657 A CN 108504657A CN 201810325470 A CN201810325470 A CN 201810325470A CN 108504657 A CN108504657 A CN 108504657A
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kdm2a
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彭勇波
许文豪
梁大焱
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South Central Minzu University
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Abstract

The present invention provides a kind of methods that CRISPR CAS9 technologies knock out HEK293T cell KDM2A genes, 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 types that K3, K7 1 and K7 2 is further verified with protein immunoblotting (Western blotting).The present invention goes back while providing the HEK293T cell lines of the KDM2A gene knockouts prepared using the above method and the application using it as cell model in studying the cell Proliferation and apoptotic signal access involved by KDM2A methylases.

Description

The method for knocking out HEK293T cell KDM2A genes using CRISPR-CAS9 technologies
Technical field
The present invention provides a kind of methods knocking out HEK293T cell KDM2A genes using CRISPR-CAS9 technologies, belong to In gene engineering technology field.
Background technology
HEK cells are derived from people source embryonic kidney cell, and HEK293T cells are the mutation of HEK cells, are by its transfection What SV40 large T antigens 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.There is particularly important influence, this feature to determine for lysine dependence demethylation enzyme family, cell proliferation and apoptosis Having determined it can be 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 The still not very clear member of research.The major function of KDM2A is to identify No. 36 lysine (H3K36) methyl of 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.It is existing studies have shown that the generation of kinds cancer is related with its up-regulated expression, such as intestinal cancer, lung cancer and breast cancer Deng.It is existing studies have shown that KDM2A can promote cell growth and migration, but it is still unclear how to influence Apoptosis for it Chu.Therefore, the knockout cell line of KDM2A is built, and is particularly important, structure with this research for apoptosis pathway It builds successful cell line and 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 silencings and RNA, but compared with gene knockout, the effect of gene knockout is more It is thorough, is more advantageous to the influence of research KDM2A cell proliferations and apoptotic effect.
Invention content
The present invention solves deficiency in the prior art, provides a kind of CRISPR-CAS9 technologies knockout HEK293T cells The method of KDM2A genes.The present invention also and meanwhile provide the KDM2A gene knockouts prepared using the above method HEK293T it is thin Born of the same parents system and the answering in studying the cell Proliferation and apoptotic signal access involved by KDM2A methylases using it as cell model With.
Technical solution is used by realizing above-mentioned purpose of the present invention：
A kind of sgRNA of the specific knockdown people source KDM2A genes based on CRISPR/CAS9 systems, the sgRNA exist Target sequence on KDM2A genes is located on No. 14 of KDM2A genes (NM_012308.2) and 18 exons, wherein 14 extras Target sequence on aobvious son is：5 '-ACATCGCACTCGTCTCCGTC-3 ', the target sequence on 18 exons are：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 constitutes sequence SEQ ID NO successively：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 are formed by double-stranded DNA, are connect with the pX458 carriers Jing Guo BbsI digestions, to which expression vector pX458- be made KDM2A。
Further, the present invention provides a kind of HEK293T cell lines of KDM2A gene knockouts and the KDM2A The construction method of the HEK293T cell lines of gene knockout, includes the following steps：Using expression vector pX458-KDM2A as KDM2A The targeting vector of gene, transfects HEK293T cells, and the monoclonal cell strain of the KDM2A gene knockouts of acquisition is abbreviated as respectively K3, K7-1 and K7-2.
Specific construction method is as follows：
(1), cell transfecting：Prepare transfection when cultivating HEK293T cells to 70~90% degree of converging, expression vector will be contained The mixed liquor of pX458-KDM2A is added in HEK293T cells and is transfected, and then cultivates；
(2), cellular genome is extracted：HEK293T cells after transfection are subjected to genome extraction；
(3), PCR is identified：According to KDM2A gene orders, design primer KDM2A-3F and KDM2A-3R, KDM2A-7F and KDM2A-7R, the sequence of above-mentioned primer is respectively such as：SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7 and SEQ ID NO: Shown in 8；PCR reactions are carried out using the genome that above-mentioned primer pair is extracted；
(4), gene level is verified：PCR product is sequenced, sequencing result and wild type PCR sequence alignments 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 verification K3, K7-1 and K7-2 is KDM2A gene defection types.
Compared with prior art, the present invention has the following advantages：(1) present invention using CRISPR/Cas9 systems from KDM2A genes are knocked out in HEK293T cells, easy to operate, effect is complete and thorough, is constituted together with HEK293T cell characteristics Ideal cell model, change for studying intracellular epigenetics.(2) CRISPR/Cas9 systems are based in the present invention System knock out KDM2A genes method, with silence, 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 proliferations 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, the thorough forfeiture of KDM2A functions may be caused, be suitable for KDM2A into Row more in-depth study.
Description of the drawings
Fig. 1 is the mutant cell genotype identification figure that sgRNA detected and be transfected CRISPR targeting vectors in embodiment；
Fig. 2 is the KDM2A protein expression content difference figures 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 implementation mode
Detailed specific description is done to the present invention with reference to specific embodiment, 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. Sas Nurse Brooker et al. is written《Molecular Cloning:A Laboratory guide》, or according to the condition of reagent manufacturers instruction suggestion.
Embodiment 1：The method for knocking out HEK293T cell KDM2A genes using CRISPR-CAS9 technologies.
1, experiment material
Carrier for expression of eukaryon pSpCas9 (BB) -2A-GFP (pX458), people source HEK293T cells.
2, the structure of CRISPR/Cas9 targeting vectors
(1) determination of target sequence
According to website http://asia.ensembl.org/index.html obtains people source KDM2A genes (NM_ 012308.2) exon information selects No. 14 and the 18th exon as target sequence.SgRNA sequences are designed later, Its 5 ' end adds BbsI restriction enzyme sites sequence (such as SEQ ID NO：1, shown in 2), and synthesize sequence (such as SEQ ID being complementary to NO：3, shown in 4).
(2) target sequence is connect with pX458 plasmids
1. pX458 empty carrier digestions, 20 μ L reaction systems are shown in Table 1,37 DEG C of digestions and stay overnight.
1 pX458 empty plasmid digestion systems of table
Ingredient Dosage
pX458 1μg(x μL)
BbsI enzymes 1μL
NEBuffer 2.1(10×) 2μL
ddH2O 17-x μL
2. the recovery purifying of pX458 digestion products
It is carried out according to Axygen plastic recovery kit specifications.
3. the sgRNA sequences and complementary series that synthesize form double-stranded DNA, 10 μ L reaction systems are shown in Table 2, response procedures：37 DEG C, 30min；95 DEG C of 5min, are down to room temperature naturally.
Table 2 synthesizes segment and forms double-strand system
Ingredient Dosage
SgRNA sequences (100 μM) 1μL
Complementary series (100 μM) 1μL
T4 Polynucleotide Kinase reaction buffers 1μL
T4 PNK(NEB) 0.5μL
ddH2O 6.5μL
4. being connect with carrier pX458 after the target fragment for forming double-strand is diluted 250 times, 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
Ingredient Dosage
Target sequence 1μL(500ng)
Glue recycles pX458 plasmids 5μL
10 × T4 DNA ligase buffer solutions 1μL
T4 Ligase 1μL
ddH2O 2μL
(3) bacillus coli DH 5 alpha, the PCR identifications of picking single bacterium colony are converted
5 μ L of connection product are added into 50 μ L DH5 α competent cells, ice bath 30 minutes after mixing, then at 42 DEG C Heat-shock transformed 90 seconds, 37 DEG C of sterile LB medium (1% tryptone, 0.5% yeast incubated were added in subsequent ice bath 2 minutes Extract, 1% sodium chloride) 950 μ L, shake culture 2 hours, takes 100 μ L bacterium under conditions of 37 DEG C and rotating speed are 180 revs/min Liquid is coated on amicillin resistance solid LB media (ampicillin for containing 100 μ g/mL), and 37 DEG C are inverted culture overnight Picking single bacterium colony afterwards is cultivated in LB liquid mediums of the 1mL containing 100 μ g/mL ampicillins, carries out bacterium solution PCR identifications, sieve It selects the plasmid with correct electrophoretic band and carries out next step experiment.Sequencing primer is the forward primer sequence of U6 promoters：
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 structure of KDM2A Knockout cells system
(1) prepare transfection when being inoculated with HEK293T cells to 70~90% degree of converging, before transfection, steps are as follows for cell transfecting：
1. diluting 3000 reagents of Lipofectamine with Opti-MEM culture mediums, and mix well；
2. the plasmid pX458-KDM2A carried greatly with the dilution of Opti-MEM culture mediums, prepares DNA premixed liquids, then adds P3000 reagents, and mix well；
3. then both the above mixed liquor is incubated at room temperature 5 minutes according to 1: 1 ratio mixing, DNA- fat is formed Liposome complex, and be added in cell, change liquid after being cultivated 4 hours in constant incubator at 37 DEG C；
4. after culture 24 hours, the monoclonal cell marked with EGFP is filtered out 96 using sorting type flow cytometer It is cultivated in orifice plate；
Genomic DNA is taken to be identified 5. waiting for that cell quantity enough time-divisions you can well imagine.
(2) cellular genome is extracted：It is cell suspension by attached cell processing, is centrifuged 5 minutes with 2000rpm, is abandoned on to the greatest extent Clearly, the use of health is century universal pillar genome extracts kit, according to kit specification operating procedure by cytogene Group extracts.
(3) PCR is identified：On KDM2A genes, design primer KDM2A-3F and KDM2A-3R, KDM2A-7F and KDM2A- 7R sequences are as follows.PCR reaction conditions：95 DEG C of pre-degenerations 5 minutes；35 cycles：95 DEG C are denaturalized 30 seconds, and 58 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute；Last 72 DEG C extend 7 minutes eventually.PCR system is shown in Table 5.
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)
Table 5PCR amplification systems
Ingredient Dosage (μ L)
ddH2O 10.5
KDM2A-F 0.5
KDM2A-R 0.5
Genomic DNA 1
2×Pfu Mix 12.5
Wherein amplified fragments size it will meet expected PCR product recovery purifying and be sequenced.
(4) gene level is verified：PCR product is sequenced, sequencing result and wild type PCR sequence alignments determine K3, K7-1 It is mutant, sequence analysis such as Fig. 1 right side d-f with K7-2) shown in.
(5) protein level is verified：After gene level verifies mutant clone, with protein immunoblotting (Western Blotting) further verification K3, K7-1 and K7-2 is KDM2A deficiencies, as a result sees Fig. 2, KDM2A albumen is not detected.
Pass through the verification of the above DNA level and protein level, it was demonstrated that the HEK293T cell constructions of KDM2A gene knockouts Then success uses K3 to indicate that 2 plants that the mutation of 14 exons generates knock out system, K7-1 and K7-2 indicate the mutation of 18 exons 2 plants generated knock out system.
Embodiment 2：Flow cytomery KDM2A is respectively to the shadow of wild-type cell system and the proliferation and apoptosis that knock out system It rings
1, how specifically to be influenced to detect the knockout of KDM2A genes and can be generated on cell, we are to wild type HEK293T cells and knockout system have carried out the detection of EdU cell Proliferations and the detection of Annexin V-FITC/PI Apoptosis respectively. Operating process is as follows：
(1) 1 × 10 will be inoculated in six orifice plates per hole5~3 × 106A cell, culture to normal phase；
(2) by specification prepares EdU culture mediums, a blank control for being not added with EdU culture mediums is arranged, to carry out streaming The dyestuff context analyzer of detection data.After cell culture medium is changed to EdU culture mediums and control medium, 1mL is added per hole, It is incubated 2 hours；
(3) cell is transferred in 1.5mL EP pipes, collects cell and abandon supernatant, then cell is resuspended with PBS, centrifugation is abandoned Clearly；
(4) illustrate to be dyed by EdU cell Proliferations 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 is found 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, belong to the scope of protection of present invention.
Sequence table
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Claims (8)

1. a kind of sgRNA of the specific knockdown people source KDM2A genes based on CRISPR/CAS9 systems, it is characterised in that：It is described Target sequences of the sgRNA on KDM2A genes is located on No. 14 of KDM2A genes (NM_012308.2) and 18 exons, wherein Target sequence on 14 exons is：5 '-ACATCGCACTCGTCTCCGTC-3 ', the target sequence on 18 exons are：5’- TTTGAAGCGCTCATCGCGGC-3’。
2. the expression vector pX458-KDM2A of sgRNA described in claim 1.
3. the construction method of expression vector pX458-KDM2A described in claim 2, it is characterised in that：In 14 exons and 5 ' ends of the target sequence on 18 exons constitute sequence SEQ ID NO successively plus the restriction enzyme site caccg of BbsI：1 He 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 are formed by double-stranded DNA, with process BbsI digestions The connection of pX458 carriers, to which expression vector pX458-KDM2A be made.
4. the expression vector pX458-KDM2A described in claim 2 is in the HEK293T cell lines for preparing KDM2A gene knockouts Application.
5. a kind of HEK293T cell lines of KDM2A gene knockouts.
6. the construction method of the HEK293T cell lines of the KDM2A gene knockouts described in claim 5, it is characterised in that including with Lower step：Using expression vector pX458-KDM2A as the targeting vector of KDM2A genes, HEK293T cells are transfected, obtain KDM2A The monoclonal cell strain of gene knockout, is abbreviated as K3, K7-1 and K7-2 respectively.
7. the construction method of the HEK293T cell lines of KDM2A gene knockouts according to claim 6, it is characterised in that tool The construction method of body is as follows：
(1), cell transfecting：Prepare transfection when cultivating HEK293T cells to 70~90% degree of converging, expression vector will be contained The mixed liquor of pX458-KDM2A is added in HEK293T cells and is transfected, and then cultivates；
(2), cellular genome is extracted：HEK293T cells after transfection are subjected to genome extraction；
(3), PCR is identified：According to KDM2A gene orders, design primer KDM2A-3F and KDM2A-3R, KDM2A-7F and KDM2A- 7R, the sequence of above-mentioned primer is respectively such as：SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7 and SEQ ID NO:Shown in 8； PCR reactions are carried out using the genome that above-mentioned primer pair is extracted；
(4), gene level is verified：PCR product is sequenced, sequencing result and wild type PCR sequence alignments, determine K3, K7-1 and K7-2 is mutant；
(5), protein level is verified：Gene level verify mutant clone after, further verified with protein immunoblotting K3, K7-1 and K7-2 is KDM2A gene defection types.
8. the HEK293T cell lines of the KDM2A gene knockouts described in claim 5 are in the proliferation and apoptosis of research KDM2A albumen And the application in potential the Molecular Biology Mechanism research.
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