CN108342416A - A kind of conditionity induction knocks out the construction method for the liver cancer cell lines for being overexpressed Chd1l genes - Google Patents
A kind of conditionity induction knocks out the construction method for the liver cancer cell lines for being overexpressed Chd1l genes Download PDFInfo
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Abstract
It is induced the invention discloses a kind of conditionity and knocks out the liver cancer cell lines and its construction method that are overexpressed Chd1l genes.First, structure is overexpressed the liver cancer cell lines of Chd1l and green fluorescent protein；Secondly, the carrier of conditionity induced expression Cas9 is transferred in above-mentioned liver cancer cell lines；Finally, structure carries red fluorescence and with the targeting cutting active pLVX mCherry hU6 Chd1l SgRNA carrier of Chd1l, is gone to above-mentioned cell line, finally obtains conditionity induction and knocks out the liver cancer cell lines for being overexpressed Chd1l.This cell line feature is the liver cancer cell lines for overexpression Chd1l before adding fortimicin (Dox) induction, is the liver cancer cell lines of knockout Chd1l expression after induction.The cell line controls of the present invention are good, cultivated while can reducing multiple cell lines, simplify research work, are the ideal tools of gene functional research.
Description
Technical field
The invention belongs to tumor research fields, and the Chd1l- for knocking out Chd1l genes is induced the present invention relates to a kind of conditionity
EGFP liver cancer cell lines and its construction method.
Background technology
Chd1l (chromodomain helicase/ATPase DNA binding protein 1-like gene) base
Because being located at the areas chromosome 1q21, containing conservative SNF2-N functional domains, unwindase functional domain and Macro functional domains, belong to SWI2/
SNF2 correlation ATP enzyme superfamilies, it is related with the processes such as chromatin unwinding and DNA reparations.The gene is in addition to specific in liver cancer
Height expression is outer, there is abnormal amplification and high expression in a variety of solid tumors, and with the close phase of generation, development and prognosis of kinds of tumors
It closes.In hepatocellular carcinoma (HCC), huge control is shown in its treatment after so that Chd1l expression is reduced with siRNA perturbation techniques
Potential is treated, especially shows the sensibility to 5-FU chemotherapy, becomes a potential treatment target spot for the treatment of liver cancer.
CRISPR-Cas9 gene editing technologies have become the powerful of functional genomics research.By CRISPR-Cas9
System is combined to form Tet-on-CRISPR-Cas9 systems with tetracycline inducible expression (Tet-on systems), may be implemented
The spatio-temporal ediating of gene is carried out to animal and cell model, and then preferably studies gene function.The system, which is not only avoided that, strikes
Except after gene cell line due to occur apoptosis and can not follow-up study, and induce and knock out front and back two groups of cells (control group and realities
Test group) it is good compared to data accuracy.
Tet-on systems have obtained wide because having extremely strict ON/OFF adjusting function in terms of making transgenic animals
General application.The system can be carried out on the time to the foreign gene being transferred in vitro and the regulating and expressing of tissue specificity.
The embryonic period, embryonic phase of animal, when not needing destination gene expression, addition Dox can bring a negative impact the growth and development of fetus.
Tet-on systems are closed in the case where not adding derivant, avoid shadow of the addition derivant to development of fetus
It rings.Since Tet-on systems carry out the expression of gene the accuracy controlling of time and tissue specificity, by more and more sections
Scholar is used as the tool of prepare transgenosis cell line or animal.The system studies certain key gene functions on cell model
When, the control gene expression of conditionability, there is apoptosis in cell after preventing gene knockout, to the later stage in body research gene function tool
There is important meaning.
Most early in being found in bacterial genomes, subsequent CRISPR-Cas9 systems are widely used CRISPR-Cas9 systems
In on various research models, it is increasingly becoming the powerful of functional genomics research.By CRISPR-Cas9 systems and Tet-on
Inducible expression is combined, and is realized CRISPR-Cas9 and is struck to the space-time of various kinds of cell model and animal model progress gene
It removes, knock in and modification, can be Animal Model and exploratory development full-length genome gene work(by researcher's wish and demand
It can analysis offer technical support.
Invention content
The characteristics of based on the above-mentioned system, induces the Chd1l- for knocking out Chd1l genes the present invention provides a kind of conditionity
EGFP liver cancer cell lines and its construction method.
In order to achieve the above-mentioned object of the invention, this invention takes following technical schemes：
A kind of conditionity induction knocks out the construction method for the liver cancer cell lines for being overexpressed Chd1l genes, includes the following steps：
1), liver cancer that Cas9 genes are transferred to overexpression Chd1l-EGFP by the transgenic method of lentivirus mediated is thin
In born of the same parents system, then adds Puro antibiotic and screened, the overexpression Chd1l- of conditionity induced expression Cas9 is obtained after 2 weeks
The liver cancer cell lines of EGFP；
2), the pLVX-mCherry-hU6-SgRNA carriers of structure expression red fluorescent protein
It is connected to by T2A behind the rice rhzomorph Blast genes that go out and builds polycistron expression vector, with SEQ ID No:1
With SEQ ID No:2 be primer, and PCR amplification obtains Blast-t2a segments, with SEQ ID No:3 and SEQ ID No:4 be to draw
Object, PCR amplification obtain t2a-mCherry segments, then with SEQ ID No:1 and SEQ ID No:4 be primer, Blast-t2a
Segment and t2a-mCherry segments are template, and PCR amplification obtains Blast-t2a-mCherry, then by Blast-t2a-
MCherry is connected into the pLVX-hU6-SgRNA carrier frameworks of removal Blast sequences by two restriction enzyme sites of SpeI and EcoRI
In, obtain pLVX-mCherry-hU6-SgRNA carriers；
3) pLVX-mCherry-hU6-Chd1l-SgRNA slow virus carriers, are built
With SEQ ID No:10 and SEQ ID No:11 be primer, and PCR amplification obtains hU6, with SEQ ID No:12 and draw
Object SEQ ID No:13 be primer, and PCR amplification obtains Chd1l-SgRNA segments, is then with hU6 and Chd1l-SgRNA segments
Template, with SEQ ID No:10 and SEQ ID No:13 be primer, and PCR amplification obtains hU6-Chd1l-SgRNA segments, then passes through
HU6-Chd1l-SgRNA segments are connected into the pLVX-mCherry- of step 2) acquisition by two restriction enzyme sites of XhoI and NheI
In hU6-SgRNA carriers, pLVX-mCherry-hU6-Chd1l-SgRNA slow virus carriers are obtained；
4), pLVX-mCherry-hU6-Chd1l-SgRNA carriers are transferred to by the transgenic method of lentivirus mediated
To conditionity induced expression Cas9 described in step 1) overexpression Chd1l-EGFP liver cancer cell lines in, then add Puro and
Two kinds of antibiotic of Blast are screened, the final Chd1l-EGFP liver cancer cell lines for obtaining conditionity and knocking out Chd1l genes.
In wherein some embodiments, the reaction system of PCR described in step 2) is：2×PrimeStar Buffer/
Premix 25μL；1.5 μ L of sense primer F；1.5 μ L of downstream primer R；Template 2 μ L and ddH2O 20μL。
In wherein some embodiments, response procedures described in step 2) are：95 DEG C of 3min of pre-degeneration；95℃30s、60℃
30s, 72 DEG C of 40s recycle 35；72 DEG C of 7min of last overall elongation.
In wherein some embodiments, the reaction system of PCR described in step 3) is：2×PrimeStar Buffer/
Premix 25μL；1.5 μ L of sense primer F；Downstream primer R1.5 μ L；Template 2 μ L and ddH2O 20μL。
In wherein some embodiments, response procedures described in step 3) are：95 DEG C of 3min of pre-degeneration；95℃30s、60℃
30s, 72 DEG C of 40s recycle 35；72 DEG C of 7min of last overall elongation.
In wherein some embodiments, a concentration of 1 ± 0.5 μ g/mL of Puro described in step 4), the Blast's is dense
Degree is 3 ± 0.5 μ g/mL.
The Chd1l- for knocking out Chd1l genes is induced the present invention also provides the conditionity obtained using above-mentioned construction method
EGFP liver cancer cell lines.
On the basis of above-mentioned Chd1l-EGFP liver cancer cell lines (7703) built, by using Tet-on-CRISPR-
The conditionity that Cas9 technologies structure is overexpressed Cas9 knocks out cell line, and the knockout cell line is before fortimicin (Dox) induction
Chd1l gene high expressions, Chd1l gene knockouts after induction.Compared with prior art, the present invention has the following advantages：
1, cell line of the invention is a kind of cell line being overexpressed and knockout has both, and is Chd1l before addition is drug-induced
The cell line of overexpression；And after addition Dox is drug-induced, 7703 liver cancer cell lines are then the cell line of Chd1l-KO.Early period
Result of study is shown, after Chd1l-KO, apoptosis occurs in cell, and Chd1l is prompted to play important work in hepatoma cell proliferation
Solved with, the invention can not be continued deeper into because of Apoptosis after Chd1l knockouts research just, predicament.By adding drug
Switch, under suitable condition induction knock out, and apoptosis related mechanism is studied after can carrying out knockout, are expected to clinically be used as one
The target spot of oncotherapy；
2, the present invention utilize gene editing technology and Tet-on technologies etc., establish one plant and meanwhile meet Chd1l be overexpressed with
The cell line of knockout, the cell line determine whether Chd1l expresses by drug switch；Meanwhile the foundation of the cell line can effectively subtract
Experimental error caused by changing due to epigenetics in few cells in vitro operating process, the method is easy to operate, contrastive is good；
Secondly, the successful structure of the cell line can also effectively reduce cell strain culture number, simplify research work, save experiment material,
It is the ideal tools of gene functional research.The present invention is not only that mechanism of action offer of the research Chd1l genes in tumour is important
Experiment material, and provide preferable reference role for functional genomics research.
Description of the drawings
Fig. 1 is the foundation for the liver cancer cell lines that the induction of 1 conditional of the embodiment of the present invention knocks out the Chd1l genes being overexpressed
The flow chart of method；
Fig. 2 be in the embodiment of the present invention 1 be overexpressed Chd1l genes 7703 liver cancer cell lines as a result, wherein A and B are
The selection result of white light and fluorescence；C is the testing result of Western-blotting；
The case where analysis of Fig. 3 plate clones is overexpressed the liver cancer cell lines cloning capacity and tumorigenesis ability of Chd1l genes, knot
The liver cancer cell lines cloning capacity that fruit display is overexpressed Chd1l genes is remarkably reinforced.A is that the liver cancer of overexpression Chd1l genes is thin
Born of the same parents are that B is control group.
Fig. 4 is that 1 conditional of the embodiment of the present invention induces 7703 cell lines of Cas9 to establish result, wherein A is screening
Steady 7703 cell masses for turning Cas9 obtained；B is the expression of Cas9 in the front and back detection cell of induction；
Fig. 5 is Blast-t2a segments (713bp), the t2a-mCherry segment expanded in the embodiment of the present invention 1
The electrophoretogram of (764bp) and Blast-t2a-mCherry segments (1457bp)；
Fig. 6 is the design of Chd1l-SgRNA and its position in the embodiment of the present invention 1；
Fig. 7 is Chd1l-SgRNA Sites Screening result figures in the embodiment of the present invention 1, wherein A：Expansion takes part hU6 to start
Subsequence (177bp)；B：HU6, Chd1l target sequence are left in amplification and SgRNA (85bp) segment is total (377bp)；C：Utilize overlapping
PCR obtains hU6-Chd1l-SgRNA segments (490bp) by template of hU6 and SgRNA sequences；
Fig. 8 is the structure result figure that the present invention is conditionity induction 7703 liver cancer cell lines of Chd1l-KO, wherein A：Sieve
The steady 7703- Cas9 cell masses for turning mCherry-hU6-Chd1l-SgRNA that choosing obtains；B：7703 liver cancer surely turn cell line
Passage and proliferation；C：After addition fortimicin (Dox) induces, 7703 liver cancer surely turn cell quantity and gradually decrease；D：It does not add
7703 liver cancer of Dox Fiber differentiations surely turn cell line；E：Sequencing analysis adds and does not add Dox, and Chd1l is cut in genome
The case where cutting；F, Western-blotting testing results.
Specific implementation mode
It is next in the following with reference to the drawings and specific embodiments that the invention will be further described.Experiment behaviour in following embodiment
Make, is this field customary technical operation unless otherwise specified.Experimental article unless otherwise specified, derives from commercially available.
The induction of 1 conditionity of embodiment knocks out the method for building up of the Chd1l-EGFP liver cancer cell lines of Chd1l genes
Referring to Fig. 1, knocking out the Chd1l-EGFP liver cancer cells of Chd1l genes for a kind of conditionity induction of the present embodiment
The method for building up of system, includes the following steps：
1) 7703 liver cancer cell lines for the Chd1l-EGFP for being overexpressed Chd1l genes, are established
According to Chd1l gene orders (NM_004284), structure CMV starts the expression vector for carrying EGFP, passes through transgenosis
7703 liver cancer cell lines of the Chd1l-EGFP of the overexpression Chd1l of expression EGFP are finally set up with drug screening (as schemed
2) it, analyzes after testing, which has following feature：Express EGFP fluorescence, oncogenicity increases ability and increases
(such as Fig. 3).
2), the structure of the Chd1l-7703 liver cancer cell lines of conditionity induced expression Cas9
Cas9 genes are transferred to the Chd1l- that established Chd1l is overexpressed by lentivirus mediated transgenic method
In 7703 liver cancer cell lines of EGFP, is screened by adding 1 μ g/mL Puro antibiotic (Sigma), finally obtain condition
7703 liver cancer cell lines (Fig. 3 A and 3B) of the Chd1l-EGFP of property induced expression Cas9；When adding fortimicin (Dox), carefully
Cas9 albumen starts to express in born of the same parents, does not add and does not express (such as Fig. 4).
3), the pLVX-mCherry-hU6-SgRNA carriers of structure expression red fluorescent protein
When being overexpressed Chd1l due to 7703 liver cancer cells, EGFP fluorescence is introduced, therefore
When building Chd1l-KO (knocking out Chd1l genes), other fluorescins are used instead in SgRNA carriers, therefore select
Red mCherry genes, pass through sgRNA-Blast-1F first：
AAGCCGCACGTCTCACTAGTACCCTCGCAGACGGAC(SEQ ID No:And sgRNA-t2a-1R 1)：
CGACGTCACCGCATGTTAGCAGACTTCCTCTGCCCTCGCCCTCC CACACATAACCAGAG(SEQ ID No:2) expand and take
Blast-t2a segments (template of the step is pLVX-hU6-SgRNA, is purchased from Addgene), with sgRNA-t2a-mCherry
F： GCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGC AAGGGCGAGGAGGATA(SEQ ID No:3)
With sgRNA-t2a-mCherry R： TCAAGATCTAGAATTCTTACTTGTACAGCTCGTCC(SEQ ID No:4) expand and take
T2a-mCherry segments (template of the step is pLVX-hU6-SgRNA, is purchased from Addgene), then pass through sgRNA-
Blast-1F(SEQ ID No:And sgRNA-t2a-mCherry 2R (SEQ ID No 1):4) it is primer, Blast-t2a segments
It is template with t2a-mCherry segments, Blast-t2a-mCherry segments (such as Fig. 5 A~C) is obtained by over-lap PCR, then
Blast-t2a-mCherry is connected into the pLVX-hU6- of removal Blast sequences by (SpeI-EcoRI) two restriction enzyme sites
In SgRNA carriers (Addgene50662) skeleton, pLVX-mCherry-hU6-SgRNA carriers are successfully obtained.
The reaction system (50 μ L) of the above PCR is：2×PrimeStar Buffer/Premix 25μL；Sense primer F
1.5μL；Downstream primer R1.5 μ L；Template 2 μ L and ddH2O 20 μL。
The response procedures of PCR are：95 DEG C of 3min of pre-degeneration；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 40s recycle 35；Most
72 DEG C of 7min of overall elongation afterwards.
The difference is that primer and template.
4) pLVX-mCherry-hU6-Chd1l-SgRNA slow virus carriers, are built
Website http is predicted according to SgRNA target sequences://tools.flycrispr.molbio.wisc.edu/
TargetFinder/index.php) and with reference to Chd1l gene orders (NM_004284), 5 Chd1l genes are predicted altogether
SgRNA target sequences (table 1 and Fig. 6).
Table 1Chd1l-SgRNA target spot prediction tables
Remarks：Atg：Initiator sequences；It is positive：Sense strand sequence；Reversely：Refer to the sequence of antisense strand.
By the target sequence of above-mentioned prediction, the highest SEQ ID No of scoring website prediction scoring are chosen:5 are building up to
In pLVX-mCherry-hU6-SgRNA carriers, using the primer table of table 2, hU6-Chd1l- is taken by overlapping PCR method expansion
SgRNA segments.
2 SgRNA vector construction primer tables of table
Remarks：F：Sense primer；R：Downstream primer；The sequence of lower horizontal line is target sequence.
The specific steps are：
With SEQ ID No:10 and SEQ ID No:11 be primer, with pLVX-hU6-SgRNA carriers
(Addgene50662) it is template, first time PCR expands to obtain hU6 (Fig. 7 A), with SEQ ID No:12 and primer SEQ ID
No:13 be primer, and with pLVX-hU6-SgRNA carriers (Addgene50662) for template, second of PCR amplification obtains Chd1l-
SgRNA segments (include wherein Chd1l target sequences, amplify to be building up in carrier, to reach the mesh of targeting Chd1l-KO
) (Fig. 7 B), then using hU6 and Chd1l-SgRNA segments as template, with SEQ ID No:10 and SEQ ID No:13 be to draw
Object, third time PCR amplification obtain hU6-Chd1l-SgRNA segments (Fig. 7 C), then will by two restriction enzyme sites of XhoI and NheI
HU6-Chd1l-SgRNA segments are connected into the pLVX-mCherry-hU6-SgRNA carriers of step 3) acquisition, obtain pLVX-
MCherry-hU6-Chd1l-SgRNA slow virus carriers；
The reaction system (50 μ L) of the above PCR is：2×PrimeStar Buffer/Premix 25μL；Sense primer F
1.5μL；Downstream primer R1.5 μ L；Template 2 μ L and ddH2O 20 μL。
The response procedures of PCR are：95 DEG C of 3min of pre-degeneration；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 40s recycle 35；Most
72 DEG C of 7min of overall elongation afterwards.
The difference is that primer and template.
5), the structure of the Chd1l-EGFP7703 liver cancer cell lines of conditionity induction Chd1l-KO
PLVX-mCherry-hU6-Chd1l-SgRNA is transferred to conditionity by the transgenic method of lentivirus mediated
In 7703 liver cancer cell lines of induced expression Cas9 and overexpression Chd1l-EGFP, passes through and add 1 μ g/mL Puro and 3 μ g/mL
Two kinds of antibiotic of Blast are screened, and final obtain surely turns mCherry-hU6-Chd1l-SgRNA (Fig. 8 A and B)；It integrates
7703 surely turn liver cancer cell lines (i.e. the Chd1l-EGFP liver cancer cell lines of conditionity induction knockout Chd1l genes)；The cell line
(mCherry) (such as Fig. 8 A and B) is marked with red fluorescence；When adding fortimicin (Dox) induction, test group cell is being trained
When supporting 1W, cell quantity gradually decreases, and cellular morphology deforms (Fig. 8 C)；And control group, when not adding Dox, cell growth is quick,
Refractivity is strong (Fig. 8 D)；By extracting cellular genome, Chd1l-KO sequencing analysis, sequencing result display addition Dox examinations are carried out
The Chd1l genes tested in group genome are cut；And control group does not occur to cut (Fig. 8 E), albumen variation is consistent (such as
Fig. 8 F).
The conditionity induction that test example 1 obtains knocks out 7703 liver cancer cell lines of Chd1l-EGFP of Chd1l genes
Western-bloting is analyzed
1, Western-blot immunoblottings measure required reagent
Bovine serum albumin(BSA), 40% acrylamide/methylene diacrylamide solution (40% Acr/bis), PMSF, 12
Sodium alkyl sulfate (SDS), persulfuric acid (AP), trishydroxymethylaminomethane (Tris), glycine are (public purchased from Shanghai life work biology
Department), proteinase inhibitor C ocktail, TEMED, dithiothreitol (DTT) (be purchased from Sigma companies), BCA protein quantifications kit,
ECL chemical luminescence for liquid (being purchased from Thermo Fisher Scientific companies of the U.S.), pvdf membrane are (public purchased from U.S. Bio-Rad
Department), RIPA lysates (Beijing Ding Guo companies).
2, Western-blot immunoblottings measure required solution and prepare
1) 1.5mol/L Tris.HCl (pH=8.8) solution：Tris 181.72g are dissolved in 800mL ultra-pure waters, adjust pH
It is settled to 1000mL after to 8.8, room temperature preservation after high pressure sterilization；
2) 0.5mol/L Tris.HCl (pH=6.8) solution：Tris 60.56g are dissolved in 800mL ultra-pure waters, and pH is extremely
It is settled to 1000mL after 6.8, room temperature preservation after high pressure sterilization；
3) 10% Ammonium Persulfate 98.5 (AP)：0.1g persulfuric acid is dissolved in 1mL ultra-pure waters, matching while using；
4) 10%SDS solution：It weighs in 10g SDS to 100mL ultra-pure waters, room temperature preservation after 55 DEG C of dissolvings；
5)10mmol/L PMSF：PMSF 0.174g are dissolved in 100mL isopropanols, packing, -20 DEG C of preservations；
6) 5 × SDS sample-loading buffers：0.5mol/L Tris.HCl (pH=6.8) 2.5mL, dithiothreitol (DTT) 0.39g,
SDS 0.5g, bromophenol blue 0.025g, glycerine 2.5mL, packing, 4 DEG C of preservations；
7) electrophoretic buffer：Tris 3.03g, glycine 18.77g, SDS 1g are dissolved in 1000 mL ultra-pure waters, room temperature
It preserves；
8) transfering buffering liquid：Tris 5.8g, glycine 2.9g, SDS 0.37g, 200 mL of methanol add ultra-pure water to be settled to
1000mL, room temperature preservation after dissolving；
9) 1mol/L Tris.HCl (pH=8.0) solution：Tris 121.14g are added in 800mL ultra-pure waters, adjust pH
To 8.0, it is settled to 1000mL, room temperature preservation；
10) TBST buffer solutions：800mL ultra-pure waters are added in 8.8 g of 1mol/L Tris.HCl (pH=8.0) 20mL, NaCl,
0.5mL Tween20 are added after dissolving, are settled to 1000mL, room temperature preservation after mixing；
11) 15% separation gel：Ultra-pure water 1.725mL, 40%Acr/Bic 1.875mL, 1.5 mol/L Tris.HCl (pH
=8.8) 1.3mL, 10%SDS 0.05mL, 10%AP 0.05mL, TMEMD 0.002mL.5% concentration glue：Ultra-pure water
1.48mL, 40%Acr/bis 0.25mL, 0.5mol/L Tris.HCl (pH=6.8) 0.5mL, 10%SDS 0.02mL, 10%
AP 0.02 mL、TMEMD 0.002mL；
3, Western-blotting immunoblottings determination step
1) cell protein extracts：The conditionity induction for collecting embodiment 1 knocks out 7703 liver cancer cells of Chd1l genes, uses
PBS is washed twice, and RIPA lysates (containing 110 μ L/mL PMSF and 5 μ L/mL protease inhibitors) are then added, crack on ice
30min, every 5min oscillation time, 12000rpm, 4 DEG C centrifuge and collect supernatant；
2) determination of protein concentration：It is measured using BCA protein quantification kits, operation in detail is carried out with reference to specification.
According to albumen concentration is measured, sample protein concentration is tuned into unanimously with RIPA lysates, is preserved in -80 DEG C of refrigerators after packing；
3) SDS-PAGE protein electrophoresis：Prepare 15% separation gel and 5% concentration glue.It is carried out at SDS denaturation before albumen loading
Reason, lysate sample is with 4：1 is added 5 × SDS sample-loading buffers in 95 DEG C of constant temperature oscillation 5min, and applied sample amount is 20 μ g；Deposition condition
For：Constant pressure 80V 40min, 120V 1h；
4) transferring film：Constant current 250mA-2.5h, wet turn on ice；
5) it closes：Room temperature closes 30min in 3%BSA solution；
6) primary antibody (rabbit source ACTB and rabbit source Chd1l polyclonal antibodies) is incubated antibody and is diluted with confining liquid, and 4 DEG C are incubated overnight
Afterwards, it is washed 4 × 5min/ times with 1 × TBST.
7) secondary antibody (IgG of goat-anti rabbit) is incubated：By corresponding IgG confining liquids with 1：5000 dilutions, are incubated at room temperature 1h,
It is washed 4 × 5min/ times with 1 × TBST.
8) development and gray analysis：It is imaged in chemiluminescence gel imaging system using ECL chemical illuminating reagents
Development, and picture editting and Quantityone (U.S. Bio-Rad are carried out using Imagelab (Bio-Rad companies of the U.S.) software
Company) software progress gray analysis.The tumorigenesis sexuality point of the liver cancer cell lines for the overexpression Chd1l-EGFP that test example 2 obtains
Analysis
Plate clone forms experiment
1. logarithmic growth phase be overexpressed Chd1l 7703 liver cancer cells, with 0.25% trypsin digestion and blow and beat at
Individual cells suspension, cell suspension is diluted, and every group of cell is inoculated with the density of 500 cells in every hole in six orifice plates respectively, and 37
DEG C pre-temperature culture solution adds 2mL culture mediums per hole, and gently rotates, and cell is made to be uniformly dispersed.Set 37 DEG C of 5%CO2And saturation is wet
It is cultivated 2~3 weeks in the cell incubator of degree, liquid is changed per 3-4d.
2. when occurring macroscopic clone in culture dish, culture is terminated.Liquid is discarded supernatant, carefully embathes 2 with PBS
It is secondary.4% paraformaldehyde is added to fix cell, fixed 15min.Then remove fixer, add appropriate GIMSA applications dyeing liquor dye 10~
Then 30min slowly washes away dyeing liquor with flowing water, be air-dried.
3. plate is inverted and is superimposed a transparent film with grid, clone is with the naked eye directly counted, or in microscope
(low power lens) counts clone's number more than 10 cells.Finally calculate cloning efficiency.Cloning efficiency=(clone's number/inoculation
Cell number) × 100%.
The results are shown in Figure 3, is overexpressed clone's quantity that Chd1l obviously increases 7703 liver cancer cells, and display Chd1l can promote
Into cell Proliferation.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
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Claims (8)
1. a kind of conditionity induction knocks out the construction method for the liver cancer cell lines for being overexpressed Chd1l genes, which is characterized in that including
Following steps：
1), Cas9 genes are transferred to the liver cancer cell lines for being overexpressed Chd1l-EGFP by the transgenic method of lentivirus mediated
In, it is screened using Puro antibiotic, the liver cancer cells of the Chd1l-EGFP of conditionity induced expression Cas9 is obtained after 2 weeks
System；
2), the pLVX-mCherry-hU6-SgRNA carriers of structure expression red fluorescent protein
It is connected to by T2A behind the rice rhzomorph Blast genes that go out and builds polycistron expression vector, with SEQ ID No:1 and SEQ
ID No:2 be primer, and PCR amplification obtains Blast-t2a segments, with SEQ ID No:3 and SEQ ID No:4 be primer, and PCR expands
Increasing obtains t2a-mCherry segments, then with SEQ ID No:1 and SEQ ID No:4 be primer, Blast-t2a segments and
T2a-mCherry segments are template, and PCR amplification obtains Blast-t2a-mCherry, then Blast-t2a-mCherry is passed through
Two restriction enzyme sites of SpeI and EcoRI are connected into the pLVX-hU6-SgRNA carrier frameworks of removal Blast sequences, obtain pLVX-
MCherry-hU6-SgRNA carriers；
3) pLVX-mCherry-hU6-Chd1l-SgRNA slow virus carriers, are built
With SEQ ID No:10 and SEQ ID No:11 be primer, and PCR amplification obtains hU6, with SEQ ID No:12 and primer SEQ
ID No:13 be primer, and PCR amplification obtains Chd1l-SgRNA segments, then using hU6 and Chd1l-SgRNA segments as template, with
SEQ ID No:10 and SEQ ID No:13 be primer, and PCR amplification obtains hU6-Chd1l-SgRNA segments, by XhoI and
HU6-Chd1l-SgRNA segments are connected into the pLVX-mCherry-hU6-SgRNA of step 2) acquisition by two restriction enzyme sites of NheI
In carrier, pLVX-mCherry-hU6-Chd1l-SgRNA slow virus carriers are obtained；
4) pLVX-mCherry-hU6-Chd1l-SgRNA carriers, are transferred to by step by the transgenic method of lentivirus mediated
1) in the liver cancer cell lines of the overexpression Chd1l-EGFP of the conditionity induced expression Cas9, Puro and two kinds of Blast is added
Antibiotic is screened, the final Chd1l-EGFP liver cancer cell lines for obtaining conditionity and knocking out Chd1l genes.
2. conditionity induction according to claim 1 knocks out the structure of the Chd1l-EGFP liver cancer cell lines of Chd1l genes
Method, which is characterized in that the reaction system of PCR described in step 2) is：2×PrimeStar Buffer/Premix 25μ
L；1.5 μ L of sense primer F；1.5 μ L of downstream primer R；Template 2 μ L and ddH2O 20μL。
3. conditionity induction according to claim 1 knocks out the structure of the Chd1l-EGFP liver cancer cell lines of Chd1l genes
Method, which is characterized in that response procedures described in step 2) are：95 DEG C of 3min of pre-degeneration；95℃30s、60℃30s、72℃
40s recycles 35；72 DEG C of 7min of last overall elongation.
4. conditionity induction according to claim 1 knocks out the structure of the Chd1l-EGFP liver cancer cell lines of Chd1l genes
Method, which is characterized in that the reaction system of PCR described in step 3) is：2×PrimeStar Buffer/Premix 25μ
L；1.5 μ L of sense primer F；Downstream primer R1.5 μ L；Template 2 μ L and ddH2O 20μL。
5. conditionity induction according to claim 1 knocks out the structure of the Chd1l-EGFP liver cancer cell lines of Chd1l genes
Method, which is characterized in that response procedures described in step 3) are：95 DEG C of 3min of pre-degeneration；95℃30s、60℃30s、72℃
40s recycles 35；72 DEG C of 7min of last overall elongation.
6. inducing the Chd1l-EGFP liver cancer cells for knocking out Chd1l genes according to Claims 1 to 4 any one of them conditionity
The construction method of system, which is characterized in that a concentration of 1 ± 0.5 μ g/mL of Puro described in step 4).
7. inducing the Chd1l-EGFP liver cancer cells for knocking out Chd1l genes according to Claims 1 to 4 any one of them conditionity
The construction method of system, which is characterized in that a concentration of 1 ± 0.5 μ g/mL of Puro described in step 4), the Blast's is a concentration of
3±0.5μg/mL。
8. the conditionity induction that claim 1~7 any one of them construction method is built, which knocks out, is overexpressed Chd1l genes
Liver cancer cell lines.
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| CN110055280A (en) * | 2019-03-19 | 2019-07-26 | 深圳大学 | A kind of cell line and its construction method and application of stable expression mCherry-tau |
| CN113308541A (en) * | 2021-03-29 | 2021-08-27 | 广州医科大学 | Application of c-Myc and Chd1l in tumor treatment and diagnosis |
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