CN110343752A - A method of Wnt signal and histone cooperate with target gene mechanism in research PGCs atomization - Google Patents
A method of Wnt signal and histone cooperate with target gene mechanism in research PGCs atomization Download PDFInfo
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of method that Wnt signal and histone cooperate with target gene mechanism in research PGCs atomization, it is related to field of biotechnology, whether the method has the function of the target gene of Wnt signal path by inside and outside experimental verification histone, analysis is in ESCs, tri- process histones of PGCs and SSCs whether target gene promoter region otherness be enriched with, detect whether histone methylated/demethylation influences response of the target gene to Wnt signal, detect influence of the histone demethylation modification enzyme to β-Catenin and the binding ability of target gene promoters binding site；Judged in PGCs atomization according to testing result, Wnt combined signal histone acts on the mechanism of action of target gene.Target gene is acted on to Wnt signal collaboration histone in PGCs forming process using the method for the invention to study, and is conducive to penetrate into the network mechanism for understanding PGCs formation.
Description
Technical field
The present invention relates to Wnt signal and histones in field of biotechnology more particularly to a kind of research PGCs atomization
The method for cooperating with target gene mechanism.
Background technique
It is well known that β-Catenin and TCFs mainly passes through change dye to the regulation that target gene is transcribed in Wnt signal path
Chromaticness state is realized, and chromatin is passed through by histone in nucleus with the nucleosome that DNA thereon is formed is closely wound
What multi-stage compression was formed.The dynamic embellishment (epigenetic modification) of histone and DNA affect chromatinic structural compactness,
The transcriptional state of gene is affected, therefore we have reason to believe that WNT signal may cooperate with epigenetic cytokine regulatory target gene
Transcription.But at present there is no to Wnt signal path and the epigenetic factor whether the research of coordinated regulation target gene transcription
Method.
Summary of the invention
The present invention provides whether coordinated regulation target gene is transcribed between a kind of pair of Wnt signal path and the epigenetic factor
Research method, the method for the invention links together Wnt signal with epigenetic modification, the network tune formed for PGCs
Control Mechanism Study provides new thinking.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides Wnt signals in a kind of research PGCs atomization and histone to cooperate with target gene mechanism
Method, comprising the following steps:
(1) target gene whether histone has the function of Wnt signal path is verified by vitro and in vivo experiments；
(2) whether the histone described in tri- processes of ESCs, PGCs and SSCs is analyzed in target gene by CHIP-seq
The enrichment of promoter region otherness；
(3) detect whether described histone methylated and demethylation influences response of the target gene to Wnt signal path；
(4) the histone demethylation modification enzyme is detected to turn of β-Catenin and the Wnt signal regulated and controled by histone
The factor is recorded in the influence of the binding ability of target gene promoters binding site, there are histone demethylation modifications to examine whether
Competitive binding relationship between enzyme and the Wnt signal transcription factor；
Judged in ESCs into PGCs atomization according to step (1)~(4) result, described in Wnt signal path joint
Histone acts on the mechanism of action of target gene.
Preferably, the method for step (1) described experiment in vitro, comprising the following steps:
A1, building histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier；
A2, histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier are distinguished
ESCs is infected, is broken up with BMP4 induction ESCs to PGCs after infecting 48h, the cell after collecting induction 2d, 4d, 6d, detection collection
Cell in expression conditions.
Preferably, the method for step (1) described experiment in vivo, comprising the following steps:
B1, building histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier；
B2, respectively by histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier
It is transferred into the body early embryo of 48~58h of hatching, continues after hatching 4.5d, measure the expression of target gene in the germinal tissue of embryo
Variation.
Preferably, if the result of step (2) is that the histone can be in target base during ESCs, PGCs and SSCs tri-
The promoter region otherness of cause is enriched with, then detects influence of the histone methylated and demethylation to target gene promoters area,
Method the following steps are included:
C1, building histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier；
C2, histone methylase slow virus interference carrier is transfected respectively in PGCs and histone demethylase is sick slowly
After malicious interference carrier, the variation of CHIP-qPCR detection target gene promoters district's groups albumen methylation state is utilized.
Preferably, detection method includes the following steps described in step (3):
The transcription factor of D1, building β-Catenin over-express vector, the Wnt signal path regulated and controled by the histone
Over-express vector, target gene promoters overall length carrier, histone methylase slow virus interference carrier and histone demethylation
Enzyme slow virus interference carrier
D2, by β-Catenin over-express vector, by the histone regulate and control Wnt signal path transcription factor mistake
The cell of expression vector and target gene promoters overall length carrier cotransfection to DF1 cell, after obtaining cotransfection；
D3, it is transferred to histone methylase slow virus interference carrier respectively into the cell after cotransfection or histone goes first
Base enzyme slow virus interference carrier detects the starting activity of target gene by luciferase reporter gene.
Preferably, detection method described in step (3) is further comprising the steps of:
The transcription factor of E1, building β-Catenin over-express vector, the Wnt signal path regulated and controled by the histone
Over-express vector, target gene promoters histone associated core promoter region carrier, histone methylase slow virus interference carrier
With histone demethylase slow virus interference carrier
E2, by β-Catenin over-express vector, by the histone regulate and control Wnt signal path transcription factor mistake
Expression vector and target gene promoters histone associated core promoter region carrier cotransfection are to DF1 cell, after obtaining cotransfection
Cell；
E3, it is transferred to histone methylase slow virus interference carrier respectively into the cell after cotransfection or histone goes first
Base enzyme slow virus interference carrier detects the starting activity of target gene by luciferase reporter gene.
Preferably, detection method includes the following steps described in step (4):
F1, detection histone demethylase and β-Catenin, by histone regulate and control Wnt signal path transcription because
Interaction between son；
F2, detection histone demethylase, β-Catenin transcription factor described in step F1 are in target gene promoters
Binding site whether there is competitive relation.
Preferably, the histone includes H3K4me2, and the target gene includes Lin28A/B, is regulated and controled by H3K4me2
The transcription factor of Wnt signal includes TCF7L2.
Preferably, the histone demethylase slow virus interference carrier includes LSD1 slow virus interference carrier, described
Histone methylase slow virus interference carrier includes MLL2 slow virus interference carrier.
Compared with prior art, beneficial effects of the present invention:
The present invention provides Wnt signals in a kind of research PGCs atomization and histone to cooperate with target gene mechanism
Method, comprising the following steps: by vitro and in vivo experiments verify histone whether have regulation Wnt signal path target base
The effect of cause, by CHIP-seq analyze the histone described in tri- processes of ESCs, PGCs and SSCs whether opening in target gene
Otherness enrichment in mover area detects whether described histone methylated and demethylation influences target gene to Wnt signal path
It responds, detect the histone demethylation modification enzyme to the transcription factor of β-Catenin and the Wnt signal regulated and controled by histone
In the influence of the binding ability of target gene promoters binding site, with examine whether there are histone demethylation modification enzyme with
Competitive binding relationship between the Wnt signal transcription factor；Judged in ESCs according to the result of above-mentioned detection to PGCs atomization
In, Wnt signal path combines the mechanism of action that the histone acts on target gene.Wnt signal and histone methylated wide
The general biological process for participating in many animals, not only plays a significant role in reproduction cell forming process, in mammal
Growth course is also essential.Wnt signal collaboration histone in PGCs forming process is made using the method for the invention
It is studied for target gene, is conducive to penetrate into the network mechanism for understanding PGCs formation.The method of the invention is easy to operate, cuts
Reality is feasible, is widely used.
As shown in the specific embodiment of the invention, according to the method described in the present invention to chicken ESCs into PGCs atomization
Wnt combined signal H3K4me2 histone acts on the mechanism of target gene Lin28, the results showed that, in the PGCs atomization of chicken
Wnt signal path and epigenetic modification act on the coordinated regulation that target gene is transcribed, and also demonstrate H3K4me2 in the process
Influence of the demethylation modification enzyme LSD1 of histone to β-Catenin and TCF7L2 binding ability.To chicken PGCs forming process
The research that middle Wnt signal collaboration H3K4me2 acts on target gene mechanism is more advantageous to the network mechanism for understanding PGCs formation in depth.
Detailed description of the invention
Fig. 1 is the mechanism of action figure that Wnt signal and histone H 3 K4me2 cooperate with target gene Lin28；
Fig. 2 is the expression that qRT-PCR detects group of cells Lin28A/B in Induction Process；Wherein, A is that induction is different
The expression variation diagram of time Lin28A；B is the expression variation diagram for inducing different time Lin28B；
Fig. 3 is the expression variation diagram that qRT-PCR detects internal Lin28A/B gene；
Fig. 4 be ChIP-qPCR detect PGCs in H3K4me2 Lin28A/B enrichment condition；Wherein, A is in PGCs
Enrichment condition of the H3K4me2 in Lin28A promoter region；B be PGCs in H3K4me2 Lin28B promoter region enrichment condition；
Fig. 5 is β-Catenin and the code area TCF7L2 amplified production electrophoretogram；Wherein, the code area-Catenin A: β expands
5000 marker of product electrophoretogram M:DL, 1: β-Catenin amplified band；The code area B:TCF7L2 amplified production electrophoretogram,
5000 marker of M:DL, 1:TCF7L2 amplified band；
Fig. 6 is oe β-Catenin and oeTCF7L2-Myc restriction enzyme digestion and electrophoresis analysis chart；Wherein, A:oe β-Catenin digestion electricity
Swimming analysis；M:DL5000 marker；1: non-digestion；2:BamHI+EcoRI double digestion；3:BamHI single endonuclease digestion；4:ddH2O；B:
The analysis of oeTCF7L2-Myc restriction enzyme digestion and electrophoresis；M:DL5000 marker；1: non-digestion 2:Xho I；Single endonuclease digestion；3:XhoI+EcoR I
Double digestion；4:ddH2O；
Fig. 7 is oe β-Catenin and oeTCF7L2-Myc carrier Sequencing chromatogram；Wherein, A:oe β-Catenin carrier is sequenced
Map；B:oeTCF7L2-Myc carrier Sequencing chromatogram；
Fig. 8 is that slow virus interference carrier plasmid and double-strand oligo sequencing detect；Wherein, A: β-Catenin-siRNA 1；
B: β-Catenin-siRNA 2；C: β-Catenin-siRNA 3；D: β-Catenin-siRNA 4；E:NC-siRNA；
Fig. 9 is the figure that slow virus interference carrier infects DF cell line (100 ×)；Note: 4 β-Catenin slow virus interference
After carrier and control vector transfect DF148h, stablize expression green fluorescence；
Figure 10 is the figure that qRT-PCR detects β-Catenin gene expression dose and jamming effectiveness；
Figure 11 is that Lin28A-PGL3-basic recombinant vector is identified and named；
Figure 12 is that Lin28B-PGL3-basic recombinant vector is identified and named；
Figure 13 is the building of carrier for expression of eukaryon pEGFP-Lin28A and pEGFP-Lin28B；Wherein, A, Lin28A starting
Sub- long segment amplification；M:5000bp Maker；1, Lin28A promoter Long fragment PCR product；B, Lin28B promoter long segment
Amplification；M:5000bp Marker；1, Lin28A promoter Long fragment PCR product；C:pEGFP-Lin28A carrier sequencer map；D:
PEGFP-Lin28B carrier sequencer map；
Figure 14 is Lin28A/B promoter function analysis (100 ×)；Note: pEGFP-N1 transfects DF1 cell and shows large area
Fluorescence；PEGFP-Lin28A and pEGFP-Lin28B transfection DF1 cell shows weaker green fluorescence；Blank does not show green
Fluorescence；
Figure 15 is each deletion fragment clone of Lin28A/B gene promoter and recombinant vector double digestion；Wherein, A Lin28A
Each deletion fragment PCR amplification；M:DL5000 Marker；1-4 is respectively promoter 2028bp, 1600bp, 1162bp, 684bp；B
For each deletion fragment PCR amplification of Lin28B；M:DL5000 Marker；1-4 be respectively promoter 1962bp, 1530bp,
1133bp,591bp；C is each deletion clone carrier digestion verification of Lin28A gene promoter；M:DL5000 Marker；1-4 points
It Wei not pLin28A/2028, pLin28A/1600, pLin28A/1096, pLin28A/684；D is that Lin28B gene promoter is each
Deletion clone carrier digestion verification；M:DL5000 Marker；1-4 be respectively pLin28B/1962, pLin28B/1530,
pLin28B/1133,pLin28B/591；
Figure 16 is pLin28A construction of recombinant vector sequencing result；
Figure 17 is pLin28B construction of recombinant vector sequencing result；
Figure 18 is transcription factor TCF7L2 binding site sequence chart；
Figure 19 is transcription factor TCF7L2 binding site deleted carrier digestion verification；Wherein, A pLin28A/684-mut
Carrier digestion gel electrophoresis figure；M:DL5000 Maker；1:pGL3.0 plasmid；2:KpnI single endonuclease digestion；III pair of enzyme of 3:KpnI, Hind
It cuts；B is pLin28B/1530-mut carrier digestion gel electrophoresis figure M:DL5000 Maker；1:pGL3.0 plasmid；2:KpnI is mono-
Digestion；III double digestion of 3:KpnI, Hind；
Figure 20 is Binding site for transcription factor deleted carrier Sequencing chromatogram；Wherein, A.pLin28A/684-mut Sequencing chromatogram
B.pLin28B/1530-mut Sequencing chromatogram；
Figure 21 is that double luciferase reporter genes detect Lin28A/B promoter activity under different disposal；Wherein A is not exist together
Manage lower Lin28A promoter activity；B is Lin28B promoter activity under different disposal；
Figure 22 is that double luciferase reporter genes detect Lin28A/B core promoter activity under different disposal；Wherein, A is not
With handling, lower Lin28A core promoter is active；B is Lin28B core promoter activity under different disposal；
Figure 23 is the interaction between co-immunoprecipitation detection LSD1 and β-Catenin；Note: Input: antibody is not used
Carry out COIP；IP: COIP is carried out using β-Catenin antibody or Flag antibody；
Figure 24 is the interaction between co-immunoprecipitation detection LSD1 and TCF7L2；Note: Input: with antibody into
Row COIP；IP: COIP is carried out using Myc antibody or Flag antibody；
Figure 25 is the interaction between co-immunoprecipitation detection LSD1, β-catenin and TCF7L2；Note: Input: do not have
Useful antibody carries out COIP；IP: COIP is carried out using Myc antibody or Flag antibody.
Specific embodiment
The present invention provides Wnt signals in a kind of research PGCs atomization and histone to cooperate with target gene mechanism
Method, comprising the following steps:
(1) target gene whether histone has the function of Wnt signal path is verified by vitro and in vivo experiments；
(2) whether the histone described in tri- processes of ESCs, PGCs and SSCs is analyzed in target gene by CHIP-seq
The enrichment of promoter region otherness；
(3) detect whether described histone methylated and demethylation influences response of the target gene to Wnt signal path；
(4) the histone demethylation modification enzyme is detected to turn of β-Catenin and the Wnt signal regulated and controled by histone
The factor is recorded in the influence of the binding ability of target gene promoters binding site, there are histone demethylation modifications to examine whether
Competitive binding relationship between enzyme and the Wnt signal transcription factor；
Judged in ESCs into PGCs atomization according to step (1)~(4) result, described in Wnt signal path joint
Histone acts on the mechanism of action of target gene.
In the present invention, the target base whether histone has regulation Wnt signal path is verified by vitro and in vivo experiments
The effect of cause, if result is that the histone has a regulating and controlling effect to the target gene of Wnt signal path, histone may can be with
Wnt signal adjusts expression of target gene jointly；If result is that the histone does not regulate and control to make to the target gene of Wnt signal path
With then there is no histone regulations for regulation of the Wnt signal to target gene.
In the present invention, the method for the experiment in vitro, comprising the following steps:
A1, building histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier；
A2, histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier are distinguished
ESCs is infected, is broken up with BMP4 induction ESCs to PGCs after infecting 48h, the cell after collecting induction 2d, 4d, 6d, detection collection
Cell in expression conditions.
In the present invention, the histone methylase slow virus interference carrier and histone demethylase slow virus are dry
The construction method of carrier is disturbed referring to paper " study on mechanism that histone H 3 K4me2 forms chicken SSCs " (He Nana group egg
Study on mechanism [D] .2018. that white H3K4me2 forms chicken SSCs) it constructs.
In the present invention, the culture medium that the BMP4 induction is used includes: to add 10% in DMEM high glucose medium
FBS, 2.5% chicken serum, 2mmol/L glutamine, 0.4 μm of ol/L MEM nonessential amino acid, 2mmol/L Sodium Pyruvate,
0.1mmol/L beta -mercaptoethanol, 5ng/mLhSCF, 10ng/ μ LbFGF, 10ng/ μ L mouse LIF, 100U/mL penicillin and 100U/
ML streptomysin, the BMP4 of 40ng/ μ l.In the present invention, the method detected in the step A2 is preferably qRT-PCR method.At this
In invention, the DMEM high glucose medium is preferably DMEM High Glucose (Gibco).
In the present invention, the method for the experiment in vivo, comprising the following steps:
B1, building histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier；
B2, respectively by histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier
It is transferred into the body early embryo of 48~58h of hatching, continues after hatching 4.5d, measure the expression of target gene in the germinal tissue of embryo
Variation.
In the present invention, the histone methylase slow virus interference carrier and histone demethylase slow virus are dry
The construction method of carrier is disturbed as shown in experiment in vitro part, details are not described herein.
In the present invention, negative control is arranged simultaneously in the step B2 to compare, the negative control refers to target base
The sequence being not present because in, referring to paper " study on mechanism that histone H 3 K4me2 forms chicken SSCs " (He Nana group
Study on mechanism [D] .2018. that albumen H3K4me2 forms chicken SSCs) it is designed.In the present invention, the step
The method detected in rapid B2 is preferably qRT-PCR method.
In the present invention, by CHIP-seq analyze the histone described in tri- processes of ESCs, PGCs and SSCs whether
The promoter region otherness of target gene is enriched with；If the result of the detection is histone in tri- processes of ESCs, PGCs and SSCs
The enrichment of target gene promoters area otherness, then show that histone may be able to adjust jointly expression of target gene with Wnt signal；If institute
It is not otherness enrichment in the target gene promoters area of tri- processes of ESCs, PGCs and SSCs that the result of detection, which is stated, as histone,
Then show that there is no histone regulations for regulation of the Wnt signal to target gene.In the present invention, otherness enrichment refer in ESCs and
The stage of significantly high enrichment in PGCs the and SSCs stage.
In the present invention, CHIP-seq analysis method specifically includes the following steps:
Step 1: cell is crosslinked and is crushed
The cell that each group reaches 1 × 107 is collected in 15ml centrifuge tube, 10ml culture medium is added to suspend, 625 μ L are added
16% formaldehyde mixes, and incubation at room temperature 10min is crosslinked；
Add 10 × Glycine of 1ml, mix, incubation at room temperature 5min terminates crosslinking, and 4 DEG C of 700g are centrifuged 5min, collect each group
Cell precipitation；
The PBS washing cell for adding 10ml to be pre-chilled, 4 DEG C of 700g are centrifuged 5min, abandon filtrate；Repeat (3) step；
Group of cells precipitating is resuspended in 1ml SDS Lysis Buffer (comprising 5 μ L Protease Inhibitor
Cocktail II)；
Prepare 1.5ml centrifuge tube, every solencyte cracks 300~400 μ L, with 30% power, is crushed 5s, interval 10s, altogether
Ultrasonic disruption under the conditions of 5min；4 DEG C of 15000g of breakdown products are centrifuged 10min, remove insoluble matter, by supernatant go to it is new from
In heart pipe.
Step 2: crosslinking protein/DNA immunoprecipitation
IP experimental group: each point 2 groups of each processing sample, respectively: negative control group (IgG group) and purpose antibody group (group
Protein groups), every group includes 100 μ L clasmatosis products, and places on ice；
Each IP needs to add 900 μ L Dilution Buffe (comprising 4.5 μ L Protease Inhibitor
Cocktail II)；
Add 60 μ L Protein GAgarose, 4 DEG C of rotations are incubated for 1h, the protein for pre cleaning non-specific binding
(noticing that Protein G Agarose will be mixed)；5000g is centrifuged 1min, precipitates agarose；
Supernatant is collected into new centrifuge tube, every group takes 10 μ L supernatants as Input group, saves at 4 DEG C；For step
Three；
Immunoprecipitating antibody is added in supernatant, 4 DEG C of rotations are incubated overnight, wherein 1 μ g/ group of IgG antibody；Histone
10 μ g/ group of antibody；
Add 60 μ L Protein G Agarose, 4 DEG C of rotations are incubated for 1h, for collecting antibody/DNA compound, 5000g from
Heart 1min removes supernatant fraction, leaves precipitating Protein G Agarose；
Low Salt Immune Complex Wash Buffer, 4 DEG C of rotations are incubated for 5min；
High Salt Immune Complex Wash Buffer, 4 DEG C of rotations are incubated for 5min；
LiCl Immune Complex Wash Buffer, 4 DEG C of rotations are incubated for 5min；
TE Buffer, 4 DEG C of rotations are incubated for 5min；It repeats (10).
Step protein iii/DNA compound elution
Prepare elution buffer Elution Buffer: for every group, the Elution Buffer for preparing 200 μ L includes 10
μ L 20%SDS, 20 μ L 1M NaHCO3 and 170 μ L ddH20, according to number of packet, it can configure mixed liquor；
It is each that 200 μ L Elution Buffer juxtapositions are added at room temperature for Input group, it is used for step 4；
For 100 μ L L Elution Buffer are added in each pipe containing antibody/agar saccharide complex IP group.It is logical
Cross and flick mixing, be incubated at room temperature 15min, 5000g, centrifugation 1min precipitates agarose, and by supernatant collection to newly from
Heart pipe；Step above-mentioned steps are repeated, it is 200 μ L that each group, which merges eluent to total volume,.
Step 4: protein/DNA compound and free DNA are solved and are crosslinked
8 μ L 5M Nacl are respectively added into all IP and Input, 65 DEG C are incubated overnight；
Respectively 1 μ L RNase A37 DEG C is added to be incubated for 30min in each pipe；
4 μ L 0.5M EDTA, 8 μ L 1M Tris-Hcl, 1 μ L Proteinase K, 45 DEG C of 2h are added in each pipe.
Step 5 centrifugal column purification and recovery DNA
1ml Bind Reagen A will respectively be added in all test tubes, is sufficiently mixed；
It moves to adsorption column (adsorption column is put into collecting pipe), 15000g is centrifuged 30s, abandons filtrate；
(2) step is repeated, until mixture is all filtered to adsorption column；
Sky is from 15000g is centrifuged 30s, and adsorption column is put into new centrifuge tube, dries；
50 μ L ddH are added among to adsorbed film20,15000g centrifugation 30s, can carry out ChIP-qPCR or -20 and save.
The DNA of ChIP enrichment is carried out to relative expression's situation of qPCR detection each group histone and β-Catenin promoter fragment.
In the present invention, if the result of CHIP-seq analysis is the histone in tri- processes of ESCs, PGCs and SSCs
It is middle to be enriched in the promoter region otherness of target gene, then histone methylated and demethylation is further preferably detected to target
The influence of gene promoter area, method the following steps are included:
C1, building histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier；
C2, histone methylase slow virus interference carrier is transfected respectively in PGCs and histone demethylase is sick slowly
After malicious interference carrier, the variation of CHIP-qPCR detection target gene promoters district's groups albumen methylation state is utilized.
In the present invention, the histone methylase slow virus interference carrier and histone demethylase slow virus are dry
The construction method of carrier is disturbed as shown in experiment in vitro part, details are not described herein.In the present invention, the CHIP-qPCR of step C2
Detection method is identical as step (2), and details are not described herein.
In the present invention, detect whether described histone methylated and demethylation influences target gene to Wnt signal path
Response；If testing result is the histone methylated response with demethylation influence target gene to Wnt signal path, illustrate
Histone methylated horizontal variation can influence response of the target gene to Wnt signal, histone methylated possible collaboration Wnt letter
Number adjust expression of target gene；If testing result is that histone methylated and demethylation does not influence target gene to Wnt signal path
Response, then the expression of target gene is not by histone methylated regulation.
In the present invention, the detection method it is preferred the following steps are included:
The transcription factor of D1, building β-Catenin over-express vector, the Wnt signal path regulated and controled by the histone
Over-express vector, target gene promoters overall length carrier, histone methylase slow virus interference carrier and histone demethylation
Enzyme slow virus interference carrier
D2, by β-Catenin over-express vector, by the histone regulate and control Wnt signal path transcription factor mistake
Expression vector and target gene promoters overall length carrier are with total plasmid: transfection reagent F μ gene=1 μ g:3 μ L transfection, while guaranteeing to carry
The mass ratio of body total amount and pRL-SV40 are 30:1.Cell of the cotransfection to DF1 cell, after obtaining cotransfection；
D3, it is transferred to histone methylase slow virus interference carrier respectively into the cell after cotransfection or histone goes first
Base enzyme slow virus interference carrier detects the starting activity of target gene by luciferase reporter gene.
In the present invention, the construction method of the β-Catenin over-express vector is preferred the following steps are included: according to β-
The gene order of Catenin carries out design of primers, carries out PCR amplification by template of the reproductive organs cDNA of tested animal, obtains
β-Catenin segment.β-Catenin the segment that amplification obtains is connected on plasmid vector, β-Catenin is obtained and is overexpressed load
Body.In the present invention, the condition of the PCR amplification preferably includes: 98 DEG C of 3min；98 DEG C of 25s, 64 DEG C of 30s, 72 DEG C of 1min,
35 circulations；72℃7min.
In the present invention, the construction method of the target gene promoters overall length carrier is the following steps are included: target gene is opened
Mover full-length gene is connect with expression vector, obtains target gene promoters overall length carrier.
In the present invention, the histone methylase slow virus interference carrier and histone demethylase slow virus are dry
The construction method of carrier is disturbed as shown in experiment in vitro part, details are not described herein.
It is further preferred that the detection method is further comprising the steps of:
The transcription factor of E1, building β-Catenin over-express vector, the Wnt signal path regulated and controled by the histone
Over-express vector, target gene promoters histone associated core promoter region carrier, histone methylase slow virus interference carrier
With histone demethylase slow virus interference carrier
E2, by β-Catenin over-express vector, by the histone regulate and control Wnt signal path transcription factor mistake
Expression vector and target gene promoters histone associated core promoter region carrier cotransfection are to DF1 cell, after obtaining cotransfection
Cell；
E3, it is transferred to histone methylase slow virus interference carrier respectively into the cell after cotransfection or histone goes first
Base enzyme slow virus interference carrier detects the starting activity of target gene by luciferase reporter gene.
In the present invention, in the step E1~E3, in addition to target gene promoters histone associated core promoter region carrier
Building outside, the condition of remaining step is identical as step D1~D3, and details are not described herein.
In the present invention, the construction method of target gene promoters histone associated core promoter region carrier preferably wraps
Include following steps:
G1, using luciferase reporter gene detection system screening target gene promoter nucleus；
G2, the genetic fragment of gained promoter nucleus is connect with carrier, obtains target gene promoters histone phase
Close core promoter region carrier.
In the present invention, the core space of the promoter with luciferase reporter gene detection system screening target gene
The method in domain, comprising the following steps:
The segment of 2000bp carries out design of primers, design missing different promoters piece before S1, prediction 5 ' UTR region of target gene
The upstream primer of section, redesigns a downstream primer, each upstream primer and downstream primer for being utilized respectively design are to animal gene
Group carries out PCR amplification, obtains the target gene promoters sequence of missing different fragments, is connected to containing luciferase reporter vector
On, obtain the target gene promoters carrier of missing different fragments；
S2, the target gene promoters carrier and pRL-SV40 cotransfection DF1 cell that different fragments will be lacked, after transfecting 48h,
Relative luciferase activity is detected, the activity height of the target gene promoters of missing different fragments is calculated, determines opening for target gene
The nucleus of mover TCF7L2 binding site.
In the present invention, described containing luciferase reporter vector is preferably pGL3-basic.In the present invention, the step
In rapid S2 when cotransfection, the target gene promoters carrier of deletion fragment and the mass ratio of pRL-SV40 are preferably 30:1.
In the present invention, in the step S2, the activity value of promoter is relative luciferase activity value, i.e., double fluoresceins
The ratio between firefly luciferase activity value and renilla luciferase activity value in enzyme reporter gene detection system.
In the present invention, the histone demethylation modification enzyme is detected to β-Catenin and the Wnt by histone regulation
The transcription factor of signal is in the influence of the binding ability of target gene promoters binding site, and to examine whether, there are histones to go first
Competitive binding relationship between base modification enzyme and the Wnt signal transcription factor；After adding histone demethylation modification enzyme, β-
Catenin reduces the starting activity of target gene, illustrates that histone methylated modification enzyme will affect β-Catenin to target gene
It adjusts, conversely, histone demethylase does not influence adjusting of the β-Catenin to target gene.
In the present invention, the detection method it is specific the following steps are included:
F1, detection histone demethylase and β-Catenin, by histone regulate and control Wnt signal path transcription because
Interaction between son；
F2, detection histone demethylase, β-Catenin transcription factor described in step F1 are in target gene promoters
Binding site whether there is competitive relation.
In the present invention, the method for detection described in step F1, it is preferred the following steps are included:
H1, combination of the transcription factor of the Wnt signal path regulated and controled by the histone in target gene promoters is detected
Location proximate, if the dynamic embellishment of histone occurs；
If H2, step H1 testing result are that the dynamic embellishment of histone occurs near the binding site, pass through CO-
IP is detected between the transcription factor and histone demethylase of β-Catenin, the Wnt signal path that the histone regulates and controls
Interaction；
If step H1 testing result is that the dynamic embellishment of histone does not occur near the binding site, illustrate Wnt
The adjusting of signal transcription factor pair target gene is not regulated and controled by histone.
In the present invention, the method for the detection of CO-IP described in step H2, it is preferred the following steps are included:
J1, building with flag label histone demethylase over-express vector, with Myc label by described group
The transcription factor over-express vector and β-Catenin over-express vector of the Wnt signal path of protein regulation；
J2, the step J1 histone demethylase over-express vector constructed and β-Catenin over-express vector are distinguished
It is transferred to DF1 cell jointly, after each cell culture 48h, extracts albumen, obtain histone demethylase antibody, β-Catenin
The compound antibody of antibody and histone demethylase and β-Catenin；
J3, Western Blot method detection histone demethylase antibody, β-Catenin antibody and histone demethyl
Whether the compound antibody for changing enzyme and β-Catenin being capable of its corresponding antigen co-precipitation；
J4, by the step J1 histone demethylase over-express vector constructed and transcription factor over-express vector respectively and
It is transferred to DF1 cell jointly, after each cell culture 48h, extracts albumen, it is anti-to obtain histone demethylase antibody, transcription factor
The compound antibody of body and histone demethylase and transcription factor；
J5, using CO-IP detection histone demethylase antibody, transcription factor antibody and histone demethylase with
Whether the compound antibody of transcription factor being capable of its corresponding antigen co-precipitation；
Turning for the Wnt signal path that β-Catenin, the histone regulate and control is judged according to the testing result of step J3 and J5
Record the interaction between the factor and histone demethylase.
In the present invention, F2, detection histone demethylase, β-Catenin transcription factor described in step F1 are in target
Binding site whether there is competitive relation on gene promoter.
In the present invention, the method for detection described in step F2, it is preferred the following steps are included:
K1, building with flag label histone demethylase over-express vector, with Myc label by described group
The transcription factor over-express vector and β-Catenin over-express vector of the Wnt signal path of protein regulation；
It is K2, the step K1 histone demethylase over-express vector constructed and β-Catenin over-express vector is common
It is transferred to DF1 cell, obtains cotransfection cells；
K3, β-Catenin over-express vector is transferred to cotransfection cells, it is thin that setting empty carrier is used as control to be transferred to cotransfection
Born of the same parents, each cell culture 48h extract albumen, obtain the compound antibody of histone demethylase Yu β-Catenin；
K4, detect histone demethylase whether can be corresponding with it to the compound antibody of β-Catenin using CO-IP
Antigen co-precipitation；
Judge what β-Catenin and histone demethylase regulated and controled in the histone according to the testing result of step K4
The mechanism of action of the binding site of the transcription factor of Wnt signal path.
Target gene of the present invention refers to that the transcription factor of the Wnt signal path regulated and controled by the histone to be studied exists
There is the target gene of corresponding binding site in target gene promoters area.
In the present invention, the histone is the histone for regulating and controlling epigenetic modification, and the histone includes but unlimited
In H3K4me2, the target gene includes but is not limited to Lin28A/B, and the transcription factor by the H3K4me2 Wnt signal regulated and controled includes
But it is not limited to TCF7L2.
In the present invention, the histone demethylase slow virus interference carrier preferably includes the interference of LSD1 slow virus
Carrier, the histone methylase slow virus interference carrier preferably include MLL2 slow virus interference carrier.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
Inventor studies have shown that Wnt/ β-catenin/Tcf7l2 target gene be Lin28A/B, based on this, research
Histone methylated participation in this adjustment process.
1.H3K4me2 regulates and controls the research of Lin28A/B transcription
(1) Validation in vitro H3K4me2 positive regulation Lin28A/B is transcribed
Inventor is studies have shown that histone methylase MLL2 (also known as Kmt2d) and histone demethylase LSD1
The methylation state that H3K4me2 can be regulated and controled, be utilized respectively build MLL2 and LSD1 slow virus interference carrier (shMLL2 and
ShLSD1 slow virus carrier) subsequent experimental is carried out, construction method is referring to referring to paper, " histone H 3 K4me2 forms chicken SSCs
Study on mechanism " (study on mechanism [D] .2018. that He Nana histone H 3 K4me2 forms chicken SSCs) side
Method is constructed, and slow virus packaging is completed by Shanghai Ji Man company.
The each group ESCs after infecting shMLL2 and shLSD1 slow virus carrier 48h will be stablized and carry out BMP4 induction (in DMEM
Addition 10%FBS, 2.5% chicken serum, 2mmol/L glutamine, 0.4 μm of ol/LMEM are nonessential in high glucose medium (Gibco)
Amino acid, 2mmol/L Sodium Pyruvate, 0.1mmol/L beta -mercaptoethanol, 5ng/mLhSCF, 10ng/ μ LbFGF, 10ng/ μ L mouse
LIF, 100U/mL penicillin and 100U/mL streptomysin, the BMP4 of 40ng/ μ l), the group of cells of induction 2d, 4d, 6d is collected, is adopted
With the expression conditions of qRT-PCR detection Lin28A and Lin28B.
The each group ESCs after infecting shMLL2 and shLSD1 slow virus carrier 48h will be stablized and carry out BMP4 induction, collection lures
The group of cells for leading 2d, 4d, 6d, using the expression conditions of qRT-PCR detection Lin28A and Lin28B.As a result such as Fig. 2 and
Shown in table 1: compared with BMP4 induction group, 0~4 day Lin28A and Lin28B expression quantity of H3K4me2 methylation (interference LSD1) is by 1
± 0 chronic up-regulation to 1.65 ± 0.057 and 1.829 ± 0.063, lowered, but is all remarkably higher than by 6dLin28B expression
BMP4 induction group；And H3K4me2 demethylation (interference MLL2), Lin28A and Lin28B expression are by 1 ± 0 chronic up-regulation to 1.31
± 0.017 and 1.152 ± 0.058, but it is substantially less than BMP4 induction group, illustrate that H3K4me2 in vitro can positive regulation
The transcription of Lin28A/B.
Lin28A/B gene expression qRT-PCR testing result in 1 different grouping of table
(2) verifying H3K4me2 positive regulation Lin28A/B transcription in vivo
Intravascular injection shMLL2 is aseptically carried out by the body early embryo (13-17HH) to hatching 48-58h in vivo
With shLSD1 slow virus carrier, and negative control sh is set (referring to the paper " effect that histone H 3 K4me2 forms chicken SSCs
Mechanism Study " (study on mechanism [D] .2018. that He Nana histone H 3 K4me2 forms chicken SSCs) be designed),
When chicken embryo normal incubation is to 4.5d, separate chicken embryo sex-ridge tissue, Trizol method extracts total tissue RNA, reverse transcription at cDNA,
With the expression variation of qRT-PCR detection each group Lin28A/B.
Intravascular injection shMM2 is aseptically carried out by the body early embryo (13-17HH) to 48~58h of hatching in vivo
With shLSD1 slow virus carrier, and negative control is set, when chicken embryo normal incubation is to 4.5d, separates chicken embryo sex-ridge tissue,
Trizol method extracts total tissue RNA, and reverse transcription is at cDNA, with the expression variation of qRT-PCR detection each group Lin28A/B.As a result such as
Shown in Fig. 3 and table 2: compared with control group (Blank and Control group), H3K4me2 demethylation (interference MLL2) extremely significant drop
The expression (0.53 ± 0.032 and 0.63 ± 0.041, P < 0.01) of low Lin28A and Lin28B, and H3K4me2 methylation (interference
It LSD1) can the extremely significant expression (2.44 ± 0.132 and 1.84 ± 0.081, P < 0.01) for improving Lin28A and Lin28B.In short,
The above result shows that H3K4me2 methylation ,/demethylation can promote/inhibits the transcription of Lin28, i.e. Lin28A/B be by
The target gene of H3K4me2 regulation.
Lin28A/B gene expression qRT-PCR testing result in 2 different grouping of table
2.H3K4me2 is enriched in the promoter region of Lin28A/B and otherness adjusts Lin28A/B transcription
The present invention carries out analysis by CHIP-seq result and finds the H3K4me2 energy during ESCs, PGCs, SSCs tri-
It is enriched in the promoter region otherness of Lin28.ChIP-qPCR is carried out after further transfecting shMLL2 and shLSD1 respectively in PGCs
Detect the variation of Lin28A/B promoter region H3K4me2 methylation state.
Inventor's early period during probing into function and mechanism of the H3K4me2 to germline, to shMLL2 and
The SSC of shLSD1 transfection carries out transcript profile sequencing, Analysis and Screening otherness target gene Lin28, and further rna level into
Verifying is gone；In addition, testing discovery H3K4me2 by CHIP-seq can be enriched in the promoter region otherness of Lin28.The present invention is same
Sample discovery is in ESCs into SSCs atomization, and H3K4me2 is capable of the mRNA expression of positive regulation Lin28A/B, in order to further
Verify H3K4me2 whether Lin28A/B promoter region be enriched with, this experiment by transfected respectively in PGCs shMLL2 and
ChIP-qPCR is carried out after shLSD1.
As a result as shown in Fig. 4 and table 3: compared with Control group, after interfering MLL2, H3K4me2 is rich in Lin28A promoter
The mean pole that catchments significantly is adjusted downward to 4.16 ± 0.827 and 5.79 ± 0.325 (P < 0.01), extremely aobvious in Lin28B promoter concentration level
Work is adjusted downward to 2.24 ± 0.041 and 0.32 ± 0.025 (P < 0.01)；After interfering LSD1, H3K4me2 is enriched in Lin28A promoter
19.29 ± 1.18 and 22.13 ± 2.01 (P < 0.05) are adjusted on level is significant, it is extremely significant in Lin28B promoter region concentration level
On be adjusted to 5.49 ± 0.013 and 10.96 ± 1.42 (P < 0.01).The result further proves that MLL2 and LSD1 can change
H3K4me2 illustrates that H3K4me2 may be by the starting of differential enrichment Lin28A/B in the concentration level of Lin28A/B promoter region
Sub-district, and then regulate and control the expression of these genes, the formation of PGCs is influenced, modification enzyme LSD1, MLL2 as H3K4me2 seem to send out
Wave indispensable role.
The ChIP-qPCR testing result that histone H 3 K4me2 is enriched in 3 different grouping of table
3.H3K4me2 methylation/demethylation influences response of the Lin28A/B to WNT signal
In order to probe into whether the H3K4me2 modification for the Lin28A/B promoter region that LSD1 and MLL2 is mediated influences Lin28A/B
Response to WNT signal, the present invention utilize the β-catenin of building early period, the promoter of TCF7L2 over-express vector, Lin28
The pGL3-baisc carrier of overall length and the pGL3-baisc carrier of core promoter region carry out subsequent experimental.
Vector construction (β-Catenin is overexpressed and TCF7L2 is overexpressed fusion vector)
(1) β-Catenin gene (NM_205081.1) the CDS sequence design β-Catenin clone provided according to NCBI draws
Object F1, R1 are for constructing β-Catenin over-express vector oe β-Catenin；TCF7L2 gene (the NM_ provided according to NCBI
001206510.4) CDS sequence design TCF7L2 cloning primer F2, R2 is used for wherein being mutated terminator codon in downstream primer
It constructs TCF7L2 fusion expression vector Myc-TCF7L2 (table 4)
4 vector construction design of primers of table
Note: underscore is restriction enzyme site, and lowercase is carrier homology arm
(2) using restriction enzyme BamHI, EcoR I and Xho I, EcoR I respectively to carrier for expression of eukaryon
PcDNA3.1 and pcDNA3.1-Myc carries out double digested.Digestion system: l carrier 1ug, restriction enzyme each 1 μ L, 10 ×
Buffer 2 μ L, ddH20 complements to 20 μ L.After mixing well, 37 DEG C of incubation 3h.Digestion products are through 1% agarose gel electrophoresis
Separation, linear carrier band is cut, and recycles linear carrier and gene clone products with DNA purification and recovery kit.
(3) target gene is attached with linear carrier: linked system: 2 × SoSoo Mix, 5 μ L, linear carrier
100ng；Target fragment 100ng, ddH2O complements to 10 μ L.50 DEG C of connection 15min.The connection product of 10 μ L is added to 50 μ L
It in competence DH5 α, mixes gently, ice bath 30min, 42 DEG C of thermal shock 90s, is put into 3min in ice rapidly, 1ml is added and is free of antibiosis
The LB liquid medium of element, is placed in shaking table 37 DEG C, 180r/min recovery 1h；It takes 100 μ L to be coated on addition and blocks that antibiotic
In solid LB media (detailed configuration is shown in annex) plate, 12-14h is cultivated in 37 DEG C of inversions.
(4) after growing bacterium colony in LB solid plate culture medium, sterile pipette tips picking single bacterium colony is placed in 1ml LB liquid
In culture medium (containing kanamycins), 37 DEG C of constant-temperature tables, 180rpm shakes bacterium 14-16h.According to the small extraction reagent kit specification of plasmid
Plasmid is extracted, and carries out double digestion identification.Digestion system is shown in step 2, and digestion products carry out agarose gel electrophoresis detection.Enzyme
It cuts and identifies that correct vector plasmid send Nanjing to hold up biology Co., Ltd, section and is sequenced.
(5) correct carrier bacterium solution will be sequenced, in the LB liquid medium of the 10ml added with the ratio of 1:1000,37 DEG C
Constant-temperature table, 180rpm shake bacterium 14-16h, collect bacterium solution.It is gone in endogenous toxic material to measure small extraction reagent kit specification extraction carrier according to Tiangeng
Plasmid, measurement concentration be placed on -20 DEG C it is spare.
As a result:
The area CDS of F1, R1 and F2, R2 primer clone β-Catenin and TCF7L2 is respectively adopted, length is respectively 2346bp
And 1794bp, PCR product are detected through agarose gel electrophoresis and found: nearby there is specific band, size and expection in 2000bp
It is consistent, shows that β-Catenin and TCF7L2 are cloned successfully.Clone products connect with pcDNA3.1 and pcDNA3.1-Myc carrier respectively
After connecing, convert, shaking bacterium, upgrading grain, through BamHI, EcoRI and XhoI, EcoRI double digestion, electrophoresis occur respectively 2346bp,
Two band of 5400bp and 1794bp and 5423bp.Sequencing result shows 2346bp clone products and β-Catenin in code area
5 ' flanking sequence matching degrees be 99%, 1794bp clone products and TCF7L2 in code area 5 ' then wing sequences match degree is 99%,
Show the success of pcDNA3.1- β-Catenin and pcDNA3.1-Myc-TCF7L2 vector construction, is respectively designated as oe β-Catenin
With oeTCF7L2-Myc (Fig. 5~7).
The building of β-Catenin slow virus interference carrier and activity verifying:
4 β-Catenin interference targets are separately designed according to chicken β-Catenin gene order (NM_205081.1) in NCBI
Point, while a negative control sequence is designed, each target spot clone is connected to piLenti-siRNA-GFP by target sequence such as table 5
In slow virus interference carrier.Interference carrier building service is completed by Zhenjiang Ai Bi dream Biotechnology Co., Ltd.
Successful interference carrier will be constructed and transfect DF1 cell respectively, the specific steps are as follows: mention the previous day with every hole 2 × 105
24 orifice plate of DF1 cell inoculation, when DF1 cell coverage rate reaches 50%~60%, by interference carrier with plasmid (Quality m):
FuGENE HD(Volume v) for the ratio transfection (for a hole) of 1:3: Opti-MEM dilutes 1 μ g plasmid to 50 μ L as A liquid；
Opti-MEM dilutes 3 μ L FuGENE HD to 50 μ L as B liquid.A, B liquid are mixed, gently blown and beaten, 37 DEG C of incubations 10~
15min.100 μ L of mixed liquor is slowly added into the hole containing 400 μ L complete mediums and is mixed, is trained in 37 DEG C, 5%CO2 constant temperature
It supports and is cultivated in case, puromycin screening is carried out after 48h, observation is taken pictures afterwards for 24 hours, is collected cell, is extracted by Trizol cracking process
Total serum IgE, and reverse transcription synthesizes cDNA, is changed using the expression that β-actin detects β-Catenin as internal reference, primer is shown in Table 3-2.PCR
Reaction system are as follows: 2 μ L, TB Green Premix Ex TaqII of cDNA 10 μ L, each 0.8 μ L of upstream and downstream primer (10uM),
6.4 μ L of ddH2O, 20 μ L of total volume.PCR response procedures are referring to Takara company's T B GreenTMPremix Ex TaqTMII is said
Bright book.
The primer sequence of 5 β-Catenin-siRNA interference carrier of table
6 qRT-PCR primer sequence of table and amplification length
The screening highest β-Catenin interference carrier of jamming effectiveness is detected by qRT-PCR, sending must dream biology to Zhenjiang love
Science and Technology Ltd. carries out slow virus package and titer determination, will wrap up successful β-Catenin interference carrier name are as follows: si β-
Catenin。
As a result:
Using piLenti-siRNA-GFP as slow virus interference carrier carrier framework, building β-Catenin slow virus interference is carried
Body, sequencing result show that β-Catenin slow virus interference carrier constructs successfully (Fig. 8).Finally successfully obtain 4 of connection correctly
A interference carrier and 1 negative control vector, are respectively designated as β-Catenin-siRNA 1, β-Catenin-siRNA 2, β-
Catenin-siRNA 3、β-Catenin-siRNA 4、NC-siRNA。
DF1 cell is transfected by interference carrier, interference carrier infects DF1 efficiency and can stably reach as the result is shown
70% or more (Fig. 9).To infect the DF1 cell line of NC-siRNA for control, qRT-PCR testing result shows β-Catenin-
SiRNA 3 and 4 slow virus interference carrier of β-Catenin-siRNA can the equal extremely significant tables for reducing β-Catenin gene mRNA of energy
Up to (P < 0.01), reduction amplitude is respectively 80% and 71% (Figure 10, table 7), and β-Catenin-siRNA 1 and β-Catenin-
SiRNA 2 has no significant effect the expression of β-Catenin, and wherein 3 interference carrier of β-Catenin-siRNA can efficiently be done
Disturb the expression of endogenous β-Catenin gene, thus by 3 interference carrier of β-Catenin-siRNA be sent to Zhenjiang love must dream biology have
Limit company carries out slow virus package, obtains titre 5 × 108Slow virus interference carrier, and be named as si β-Catenin.
The detection of 7 target gene β-Catenin gene jamming effectiveness of table
Note: different shoulder marks indicate significant difference
This research is thin with the pGL3-baisc carrier cotransfection DF1 of β-catenin, TCF7L2 and pLin28 promoter overall length
It is thin to be transferred to DF1 on this basis respectively by the basis that born of the same parents respond WNT signal as Lin28A/B for shMLL2 and shLSD1 carrier
In born of the same parents, the starting activity of Lin28A/B is detected by luciferase reporter gene.
Further, WNT is believed in Lin28A/B core promoter area H3K4me2 modification and Lin28A/B in the same way
Number the relationship of responding ability detected.In order to probe into the H3K4me2 for the Lin28A/B promoter region that LSD1 and MLL2 is mediated
Whether modification influences response of the Lin28A/B to WNT signal, and the present invention is with β-catenin, TCF7L2 and pLin28 associated deletion
The basis that carrier cotransfection DF1 cell responds WNT signal as Lin28A/B, respectively on this basis by shMLL2 and
ShLSD1 carrier is transferred in DF1 cell, and the starting activity of Lin28A/B is detected by luciferase reporter gene.
According to NCBI (https: //www.ncbi.nlm.nih.gov/) and UCSC (http: //
Genome.ucsc.edu/) the Lin28A/B sequence provided determines 2000bp genome before and after CDS sequence.According to starting daughter nucleus
Heart promoter original part TATA box and CAAT box are held in code area 5 ' determines promoter region and transcription initiation site.To transcribe
Beginning site is that+1 design primer expands respective segments.Design primer P0 simultaneously introduces restriction enzyme site AseI, SnaBI and HindIII
Cloning promoter longest segment (2028bp and 1962bp) simultaneously replaces the CMV promoter in pEGFP-N1 carrier.Fixed downstream is drawn
Object separately designs Pl, P2, P3, and P4 primer introduces restriction enzyme site KpnI, clones different length deletion fragment and is connected into PGL3-
In basic carrier.Primer is held up biology Co., Ltd, section by Nanjing and is synthesized.See Table 8 for details for specific primer sequence.
8 Lin28A/B promoter region different length segment PCR amplification primer of table
Note: underscore part is restriction enzyme site, and lowercase is carrier homology arm
Lin28A/B Assay of promoter activity
The building of pLin28A-EGFP, pLin28B-EGFP
(1) using chicken embryo genomic DNA as template, P0 primer clones Lin28A and Lin28B promoter region, reactant in table 8
System: Prime STAR Max DNA Polymerase 10 μ L, each l μ L of upstream and downstream primer, DNA profiling 1 μ L, ddH20 complements to
20μL.PCR reaction condition are as follows: 98 DEG C of initial denaturation 3min；98 DEG C of 25s, 62 DEG C of 30s, 72 DEG C of 90s, totally 35 recycle；72 DEG C last
Extend 7min.Amplified production is identified with 1% agarose gel electrophoresis.
(2) using restriction enzyme A seI, HindIII and SnaBI, HindIII respectively to carrier for expression of eukaryon
PEGFP-Nl carries out double digested.Digestion system: pEGFP-Nl carrier 1ug, restriction enzyme each 1 μ L, 10 × Buffer
2 μ L, ddH20 complements to 20 μ L.After mixing well, 37 DEG C of incubation 3h.Digestion products are separated through 1% agarose gel electrophoresis, will
Linear carrier band is cut, while by the promoter fragment of two genes of massive amplification, uses DNA purification and recovery kit respectively
Recycle linear carrier and target fragment.
(3) target gene is attached with linear carrier: linked system: 2 × SoSoo Mix, 5 μ L, linear carrier
100ng；Target fragment 100ng, ddH2O complements to 10 μ L.50 DEG C of connection 15min.The connection product of 10 μ L is added to 50 μ L
It in competence DH5 α, mixes gently, ice bath 30min, 42 DEG C of thermal shock 90s, is put into 3min in ice rapidly, 1ml is added and is free of antibiosis
The LB liquid medium of element, is placed in shaking table 37 DEG C, 180r/min recovery 1h；It takes 100 μ L to be coated on addition and blocks that antibiotic
In solid LB media (annex is shown in configuration) plate, 12-14h is cultivated in 37 DEG C of inversions.LB culture medium: Trypton 1.0g；
Yeast Extract 0.5g；NaCl 1.0g；Agar 1.5g (solid medium addition)；Deionized water dissolving adjusts pH=
7.2, it is settled to 1L, it is spare after high pressure sterilization.
(4) after growing bacterium colony in LB solid plate culture medium, sterile pipette tips picking single bacterium colony is placed in 1ml LB liquid
In culture medium (containing kanamycins), 37 DEG C of constant-temperature tables, 180rpm shakes bacterium 14-16h.According to the small extraction reagent kit specification of plasmid
Plasmid is extracted, and carries out double digestion identification.Digestion system is shown in step 2, and digestion products carry out agarose gel electrophoresis detection.Enzyme
It cuts and identifies that correct vector plasmid send Nanjing to hold up biology Co., Ltd, section and is sequenced.
(5) correct carrier bacterium solution will be sequenced, in the LB liquid medium of the 10ml added with the ratio of 1:1000,37 DEG C
Constant-temperature table, 180rpm shake bacterium 14-16h, collect bacterium solution.It is gone in endogenous toxic material to measure small extraction reagent kit specification extraction carrier according to Tiangeng
Plasmid, measurement concentration be placed on -20 DEG C it is spare.
Lin28A/B promoter difference deletion fragment PGL3-basic vector construction:
Referring to Rugao Huang chicken Lin28A/B gene promoter biological information credit analysis as a result, fixed downstream primer, no
It is disconnected to change upstream primer, 4 pairs of different size deletion fragment primers are devised, primer sequence is shown in Table 8.Specific method is referring to above-mentioned mistake
Expression vector establishment mode.
The identification of PGL3-basic recombinant vector and name
It is carried out using different deletion fragment PGL3-basic carriers of the restriction enzyme KpnI and HindIII to building double
Digestion, and Nanjing Qing Ke company is sent to be sequenced.Respective carrier is respectively designated as: pLin28A/684, pLin28A/1164,
PLin28A/1600, pLin28A/2028 and pLin28B/591, pLin28B/1133, pLin28B/1530, pLin28B/
1962 (Figure 11~12).
The screening of Lin28A/B promoter nucleus
With the starting activity of luciferase reporter gene detection system testing goal gene Lin28A/B promoter, specifically
Steps are as follows: with mass ratio being the ratio cotransfection of 30:1 by the recombinant plasmid comprising different promoters segment and pRL-SV40
DF1 cell, while negative control (the ratio corotation of pGL3-basic plasmid and pRL-SV40 plasmid with mass ratio for 30:1 is set
Contaminate DF1 cell).3 holes of transfection every time, are repeated 3 times.Cell is collected after transfection 48h, every pipe adds 70 μ L cell pyrolysis liquids, gently
It mixes, i.e. 70 μ L fluorescent liquids in equal volume is added in every hole cell, mix gently, are put into reading firefly fluorescent value in microplate reader,
Then 70 μ L STOP terminate liquids are added, gently piping and druming mixes, and places into reading sea pansy fluorescent value in microplate reader.Promoter activity
Value is the firefly luciferase activity value/sea relative luciferase activity value (Relative Luciferase activity) i.e.
Kidney uciferase activity value.The numerical value of luciferase activity is the " mean+SD of 3 repetition test results.Specific behaviour
Make referring to Promega company dual luciferase reporter gene detection kit specification.It is each by the detection of double luciferase reporter enzymes
Deletion fragment promoter activity height is to screen core promoter region.
As a result:
The building of carrier for expression of eukaryon pEGFP-Lin28A, pEGFP-Lin28B
Using chicken embryo genomic DNA as template, 8 primer of table expands Lin28A and Lin28B promoter gene fragment respectively, long
Degree is respectively 2028bp and 1962bp, and PCR product detects discovery 2000bp through agarose gel electrophoresis and specific item nearby occurs
Band, size are consistent (A of Figure 13, B) with expection, by clone products with corresponding linear pEGFP-N1 carrier connection, conversion, through surveying
PEGFP-Lin28A and pEGFP-Lin28B (C, D of Figure 13) are named as after sequence identification is correct.
Lin28A/B Assay of promoter activity
In order to detect Lin28A/B gene promoter long segment whether have starting activity, respectively by pEGFP-Lin28A,
PEGFP-Lin28B carrier transfects DF1 cell, observes under fluorescence inverted microscope after 48 hours, as a result, it has been found that: it is right with feminine gender
Compared according to Blank group, pEGFP-Lin28A, pEGFP-Lin28B group can expressing green fluorescent protein, but fluorescence intensity compared with
Positive control pEGFP-N1 group is weak.In short, this experimental result illustrates pEGFP-Lin28A and pEGFP-Lin28B carrier
Lin28A and Lin28B promoter all has the ability (Figure 14) of starting downstream gene transcription.
The screening of Lin28A/B promoter nucleus:
(1) Lin28A/B promoter difference deletion fragment PGL3-basic vector construction
Using chicken embryo genomic DNA as template, PCR expands the missing piece of Lin28A and Lin28B promoter different length respectively
Section 2028bp, 1600bp, 1162bp, 684bp and 1962bp, 1530bp, 1133bp, 591bp.PCR product is through 1.5% agarose
Detected through gel electrophoresis, PCR product clip size obtained is consistent with the result of desired design, illustrates that Lin28A and Lin28B are opened
The different length deletion fragment of mover clones successfully (Figure 15 A, B).Different deletion fragments are connected to line according to homologous recombination method
On property PGL3-basic carrier, it is inverted, screen, select positive colony after carry out shaking bacterium upgrading grain, to the difference built
The PGL3.0 recombinant vector of length carries out double digestion identification, and agarose gel electrophoresis results show that Lin28A/B promoter difference is long
Segment and carrier segments two clear bands (Figure 15 C, D) are spent, sequencing result is correct (Figure 16~17), illustrates vector construction success
It can be used for promoter nucleus screening experiment.
The building of the promoter Reporter gene vector of TCF7L2 binding site missing
According to Bioinformatics Prediction as a result, the binding site (Figure 18) of the promoter core space TCF7L2 to Lin28A/B
Carry out point mutation.It is specific: " ggagcctttgaaaaaac (SEQ ID NO.28) " series jump of Lin28A
"ggagcctgtgaaaaaac(SEQ ID NO.29)"；" ggaccatttgatgggct (SEQ ID NO.30) " sequence of Lin28B
Column sport " ggaccatgtgatgggct (SEQ ID NO.31) " point mutation vector construction service by Wuhan Jin Kairui biology section
Skill Co., Ltd completes, and the carrier of building is respectively designated as after digestion and sequencing are identified successfully: pLin28A/684-mut,
pLin28B/1530-mut。
As a result:
The building of the promoter Reporter gene vector of transcription factor TCF7L2 binding site mutation
Utilize The JASPAR database (http://jaspardev.genereg.net/) online database pair
Lin28A/B promoter region transcription factor TCF7L2 binding site predicted, discovery Lin28A core promoter area (- 584~+
There is 100bp) and TCF7L2 binding site in Lin28B core promoter area (- 1431bp~-1034bp), for the region point
It is other that the point mutation of TCF7L2 binding site is carried out to Lin28A and Lin28B core promoter area, and construct to pGL3.0-Basic and carry
It on body, is identified through double digestion, agarose gel electrophoresis results display discovery the specific band (A of Figure 19 occurs in 500bp or so
And B), sequencing result shows that Lin28A/B core promoter area Binding site for transcription factor is mutated really, former conservative " T " alkali
Base is sported " G " base (Figure 20), and vector construction success, we will construct successful carrier and are respectively designated as pLin28A/
684-mut and pLin28B/1530-mut.
9~10 institute of the active result of starting such as Figure 21 and table of Lin28A/B is detected by luciferase reporter gene
Show: for pLin28A/2028, compared with control group (4.99 ± 0.34), adding the extremely significant reduction pLin28A/2028 of shMLL2
Start active (2.16 ± 0.14, P < 0.01), and adds shLSD1 then extremely significant raising pLin28A/2028 starting activity
(13.16 ± 1.21, P < 0.01)；For pLin28B/1962, compared with control group (22.53 ± 2.04), addition shMLL2 is aobvious
Writing, which reduces pLin28B/1962, starts active (18.16 ± 1.64, P < 0.05), and adds shLSD1 then extremely significant raising
PLin28B/1962 starts active (35.32 ± 3.04, P < 0.01).These results suggest that H3K4me2 methylation can enhance
Response of the Lin28A/B to WNT signal, and H3K4me2 demethylation can reduce response of the Lin28A/B to WNT signal.
Lin28A promoter activity relative fluorescence under 9 different disposal of table
Lin28B promoter activity relative fluorescence under 10 different disposal of table
Further, to Lin28A/B core promoter area H3K4me2 modification and Lin28A/B to the responding ability of WNT signal
Relationship detected, as a result as shown in Figure 22 and table 11~12: for pLin28A/684, with control group (14.62 ±
1.24) it compares, the extremely significant reduction pLin28A/684 of addition shMLL2 starts active (9.44 ± 0.64, P < 0.01), and adds
Then extremely significant raising pLin28A/684 starts active (22.31 ± 2.41, P < 0.01) to shLSD1；For pLin28B/1530, with
Control group (22.80 ± 2.16) is compared, addition shMLL2 significant decrease pLin28B/1530 starting activity (16.80 ± 1.81, P <
0.05), adding shLSD1, then extremely significant raising pLin28B/1530 starts active (46.49 ± 3.89, P < 0.01).The above knot
Fruit, which illustrates that H3K4me2 methylates, can enhance response of the Lin28A/B core promoter area to WNT signal, and H3K4me2 demethyl
Change can reduce response of the Lin28A/B core promoter area to WNT signal.Lin28A/B core promoter is confirmed before this research
There are TCF7L2 binding sites in area, and the region is there are H3K4me2 modification, prompt modification enzyme LSD1, MLL2 of H3K4me2 with
Seem that there are certain connections between the β-catenin/TCF7L2 compound of WNT signal.
Lin28A core promoter activity relative fluorescence under 11 different disposal of table
Lin28B core promoter activity relative fluorescence under 12 different disposal of table
4. the transcription that β-catenin and LSD1 competition TCF7L2 binding site promotes Lin28A/B
Since the upper LSD1 of chicken does not have antibody, LSD1 gene (XM_417719.5) CDS sequence that the present invention is provided according to NCBI
The area CDS of LSD1 is cloned into pcDNA3.1-3Flag plasmid, constructs the fusion expression vector of LSD1, be used for by column, total 2541bp
Tape label is overexpressed LSD1, and vector construction service is completed by Zhenjiang Ai Bi dream Biotechnology Co., Ltd.The step can be by ready-made anti-
Body replaces.TCF7L2 in the above way with Myc tag fusion.
(1) LSD1 and TCF7L2 interaction rather than β-Catenin
According to the result of study of third portion, it is found that Lin28A/B promoter region TCF7L2 binding site nearby occurs
H3K4me2 dynamic embellishment, therefore, we guess that β-catenin/TCF7L2 may be by recruiting H3K4me2 modification enzyme, otherness
The H3K4me2 concentration level for adjusting the gene promoter area Lin28, to affect the transcription of target gene.
For this purpose, we detect the interaction between β-Catenin, TCF7L2 and LSD1 by CO-IP, by LSD1-
After flag, β-Catenin transfect DF1 cell line 48h individually and jointly, extract albumen, using antibody protein precipitation and its
The compound of interaction carries out Western Blot detection.As a result, it has been found that in cotransfection β-Catenn and LSD1-flag group,
Flag antibody cannot pull β-Catenn albumen, can not pull LSD1-flag equally using β-Catenn antibody, illustrate LSD1 not
It can be with β-Catenn interaction.
Using antibody protein precipitation and its compound of interaction, the method for carrying out Western Blot detection includes such as
Lower step:
(1) cell sample is collected, the culture medium in cell is removed, it is primary to clean cell with modified form Du Shi PBS.It will on ice
500 μ L IP cracking of pre-cooling/washing buffer is added in cell, is incubated for 5min on ice and periodically mixes cell pyrolysis liquid turn
It moves on in microcentrifugal tube ,~13000 × g is centrifuged 10min sedimentation cell fragment.Supernatant is transferred to a new microcentrifugation
Guan Zhong carries out determination of protein concentration.
(2) antibody immobilization: 20 μ LAminoLink binding resin resin grout liquids are added in centrifugal column, 1000 × g centrifugation
1min is discarded and is flowed through liquid；200 μ 1 × cross-linking buffers of L wash resin, and 1000 × g is centrifuged 1min, discards and flow through liquid, insert
Enter bottom cover；200 μ 1 × cross-linking buffers of L dilute 10 μ g antibody, are added in resin, and the sodium cyanoborohydride solution of 3 μ L is added；
Screw lid is covered, at room temperature in being incubated for 2h on rotator, it is ensured that slurries are in suspended state during incubation；1000
× g is centrifuged 1min, and preservation flows through liquid to verify antibody binding efficiency；Add 1 × cross-linking buffer of 200 μ L, 1000 × g centrifugation
1min is discarded and is flowed through liquid；Repetitive operation is primary.Add 200 μ L quenching buffers to centrifugal column, 1000 × g is centrifuged 1min, discards stream
Liquid is worn, 200 μ L quenching buffers are added into resin, 3 μ L sodium cyanoborohydride solutions simultaneously cover screw-cap, gently shake up, incubate
Educate 15min；Bottom plug is unloaded, screw-cap is unscrewed, centrifugal column is put back in collecting pipe, 1000 × g is centrifuged 1min, discards and flows through liquid
Body；Resin is washed twice with 200 μ 1 × cross-linking buffers of L, and 1000 × g is centrifuged 1min, discards and flows through liquid；It is washed with 150 μ L
Buffer washs resin six times, and 1000 × g is centrifuged 1min, discards and flows through liquid；
(3) using control agarose resin pretreatment cell lysate: taking 80 μ L control agarose resin slurries, (40 μ L are solid
Phase resin) into centrifugal column, 1000 × g is centrifuged 1min, removal storage storage buffer；100 μ 1 × cross-linking buffers of L are added,
1000 × g is centrifuged 1min, discards and flows through liquid；The cell pyrolysis liquid of 1mg is added in the centrifugal column containing resin, 4 DEG C of rotations are incubated
1h is educated, 1000 × g is centrifuged 1min, discards the centrifugal column containing resin, and reservation flows through liquid, and as crucial point manages sample.
(4) co-immunoprecipitation: washing resin twice with IP cracking/washing buffer of 200 μ L, and 1000 × g is centrifuged 1min,
It discards and flows through liquid, pat the bottom of centrifugal column on paper handkerchief to remove remaining liquid, be inserted into bottom cover；Processing sample is added to
In resin antibody-containing, screw lid is covered, 4 DEG C overnight；Remove bottom cover, unclamp screw lid, centrifugal column is put into collecting pipe,
1000 × g is centrifuged 1min, discards and flows through liquid；Screw lid is removed, centrifugal column is put into a new collecting pipe, adds 200 μ L
IP cracking/washing buffer and be centrifuged；IP cracking/washing buffer of 200 μ L washes sample twice, and 1000 × g is centrifuged 1min,
It discards and flows through liquid；
(5) co-immunoprecipitation elute: centrifugal column is placed in a new collecting pipe, add 10 μ L elution buffers and from
The heart；It keeps centrifugal column in collecting pipe, adds 50 μ L elution buffers, be stored at room temperature 5min；1000 × g is centrifuged 1min, collects stream
Wear liquid；
The sample preparation of SDS-PAGE analysis: 5 × swimming lane label sample-loading buffer is balanced to room temperature.By overturning 5-10
It is secondary to be gently mixed sample buffer；Be added into sample 5 × sample-loading buffer to final concentration of 1 ×；It is incubated at 95-100 DEG C
Sample about 5min carries out SDS-PAGE electrophoresis.
Next LSD1-Flag and TCF7L2-Myc individually and are jointly transfected into DF1 cell, carry out CO-IP detection,
As a result as shown in Figure 23~24, the TCF7L2-Myc interaction protein compound under co-immunoprecipitation is carried out in Myc antibody
In, in the case where individually transfecting LSD1-Flag or TCF7L2-Myc, the expression of LSD1 albumen, only LSD1- is not detected
After Flag and TCF7L2-Myc cotransfection, the expression of LSD1 albumen is detected；Equally, it is carried out under co-immunoprecipitation in Flag antibody
In the LSD1 interaction protein compound come, in the case where individually transfecting LSD1-Flag or TCF7L2-Myc, it is not detected
The expression of TCF7L2-Myc albumen detects TCF7L2-Myc albumen only after LSD1-Flag and TCF7L2-Myc cotransfection
Expression illustrates there is interaction between LSD1 albumen and TCF7L2 albumen.In short, the above result shows that LSD1 is mutual with TCF7L2
Make rather than β-Catenin.
(2) β-catenin and LSD1 competes TCF7L2 binding site
This method finds LSD1 and TCF7L2 interaction rather than β-Catenin, and TCF7L2 is same as β-Catenin mutual
Make, therefore it is proposed that assumes that β-catenin may compete TCF7L2 binding site with LSD1.
In order to verify this conjecture, we choose the good DF1 cell of growth conditions, in LSD1-Flag and TCF7L2-
On the basis of Myc cotransfection DF1, it is divided to two groups, one group of addition β-Catenin, one group of addition pcDNA3.1 pass through as compareing
CO-IP verifies our guess.As the result is shown: in the LSD1 interaction albumen that Flag antibody carries out under co-immunoprecipitation, together
When transfection LSD1-Flag and TCF7L2-Myc group in be capable of detecting when LSD1-Flag and TCF7L2-Myc protein expression, however,
In the group for adding β-Catenin on this basis, without the expression of TCF7L2-Myc and β-Catenin, illustrate β-Catenin's
In the presence of preventing the formation of LSD1/TCF7L2 complex；With Myc antibody carry out immunoprecipitation come TCF7L2 interaction albumen
In, while transfecting and being capable of detecting when LSD1-Flag and TCF7L2-Myc protein expression in LSD1-Flag and TCF7L2-Myc group,
However, on this basis add β-Catenin group in, without the expression of LSD1-Flag albumen, but exist TCF7L2-Myc and
The expression of β-Catenin illustrates that β-Catenin replaces LSD1 and TCF7L2 to form complex at this time.In short, result above table
Bright β-catenin and LSD1 competes TCF7L2 binding site.
In conclusion how to combine the expression for adjusting Lin28A/B for research Wnt combined signal H3K4me2, pass through first
QRT-PCR detects H3K4me2 and studies to the transcriptional regulatory of Lin28A/B, then by Chip-qPCR (bindingsite assay method)
Enrichment of the H3K4me2 in the promoter region of Lin28A/B.On this basis, it is studied by Dual-luciferase reportor systerm
Whether H3K4me2 influences response of the Lin28A/B to Wnt signal.Wnt signal is often with core effector β-catenin/ simultaneously
TCF7L2 complex form adjusts the expression of target gene, under the premise of being proved to be successful early period, passes through Co-IP experimental verification mistake herein
Influence of the H3K4me2 histone demethylation modification enzyme LSD1 to β-catenin and TCF7L2 binding ability in journey.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Yangzhou University
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gggtgaactc acgtcagaac 20
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<400> 11
ctgtgatctc cttctgcatc c 21
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ggcaatcaag aaagtaagc 19
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tcgactgcag aattcgaagc ttgtgctctt ctgctttaac ccaacatc 48
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Claims (9)
1. a kind of method that Wnt signal and histone cooperate with target gene mechanism in research PGCs atomization, including with
Lower step:
(1) target gene whether histone has the function of Wnt signal path is verified by vitro and in vivo experiments；
(2) by CHIP-seq analyze the histone described in tri- processes of ESCs, PGCs and SSCs whether target gene starting
The enrichment of sub-district otherness；
(3) detect whether described histone methylated and demethylation influences response of the target gene to Wnt signal path；
(4) detect the histone demethylation modification enzyme to the transcription of β-Catenin and the Wnt signal regulated and controled by histone because
Son the binding ability of target gene promoters binding site influence, with examine whether there are histone demethylation modification enzyme with
Competitive binding relationship between the Wnt signal transcription factor；
Judged in ESCs into PGCs atomization according to step (1)~(4) result, Wnt signal path combines described group of egg
The white mechanism of action for acting on target gene.
2. the method according to claim 1, wherein the method for step (1) described experiment in vitro, including following step
It is rapid:
A1, building histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier；
A2, histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier are infected respectively
ESCs, is broken up with BMP4 induction ESCs to PGCs after infecting 48h, and the cell after collecting induction 2d, 4d, 6d detects the thin of collection
Expression conditions in born of the same parents.
3. the method according to claim 1, wherein the method for step (1) described experiment in vivo, including following step
It is rapid:
B1, building histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier；
B2, histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier be transferred to respectively
Into the body early embryo of 48~58h of hatching, continue after hatching 4.5d, the expression for measuring target gene in the germinal tissue of embryo becomes
Change.
4. the method according to claim 1, wherein if the result of step (2) be the histone ESCs,
It can be enriched in the promoter region otherness of target gene during PGCs and SSCs tri-, then detect histone methylated and demethyl
Change influence to target gene promoters area, method the following steps are included:
C1, building histone methylase slow virus interference carrier and histone demethylase slow virus interference carrier；
C2, to transfect histone methylase slow virus interference carrier and histone demethylase slow virus respectively in PGCs dry
After disturbing carrier, the variation of CHIP-qPCR detection target gene promoters district's groups albumen methylation state is utilized.
5. the method according to claim 1, wherein step (3) is described, detection method includes the following steps:
D1, building β-Catenin over-express vector, by the histone regulate and control Wnt signal path transcription factor cross table
It is slow up to carrier, target gene promoters overall length carrier, histone methylase slow virus interference carrier and histone demethylase
Viral interference carrier
D2, by β-Catenin over-express vector, by the histone regulate and control Wnt signal path transcription factor overexpression
The cell of carrier and target gene promoters overall length carrier cotransfection to DF1 cell, after obtaining cotransfection；
It is transferred to histone methylase slow virus interference carrier respectively in cell after D3, Xiang Suoshu cotransfection or histone goes first
Base enzyme slow virus interference carrier detects the starting activity of target gene by luciferase reporter gene.
6. according to the method described in claim 5, it is characterized in that, detection method described in step (3) is further comprising the steps of:
E1, building β-Catenin over-express vector, by the histone regulate and control Wnt signal path transcription factor cross table
Up to carrier, target gene promoters histone associated core promoter region carrier, histone methylase slow virus interference carrier and group
Albumen demethylase slow virus interference carrier；
E2, by β-Catenin over-express vector, by the histone regulate and control Wnt signal path transcription factor overexpression
The cell of carrier and target gene promoters histone associated core promoter region carrier cotransfection to DF1 cell, after obtaining cotransfection；
E3, histone methylase slow virus interference carrier or histone demethylation are transferred to respectively into the cell after cotransfection
Enzyme slow virus interference carrier detects the starting activity of target gene by luciferase reporter gene.
7. the method according to claim 1, wherein step (4) is described, detection method includes the following steps:
F1, detection histone demethylase and β-Catenin, by histone regulate and control Wnt signal path transcription factor it
Between interaction；
F2, detection histone demethylase, β-Catenin transcription factor described in step F1 combine in target gene promoters
Site whether there is competitive relation.
8. method described in any one according to claim 1~7, which is characterized in that the histone includes H3K4me2, institute
Stating target gene includes Lin28A/B, and the transcription factor by the H3K4me2 Wnt signal regulated and controled includes TCF7L2.
9. according to method described in claim 2~6 any one, which is characterized in that the histone demethylase is sick slowly
Malicious interference carrier includes LSD1 slow virus interference carrier, and the histone methylase slow virus interference carrier includes MLL2 sick slowly
Malicious interference carrier.
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| CN111826430A (en) * | 2020-07-24 | 2020-10-27 | 扬州大学 | Method for researching co-regulation target gene of lncRNA and histone methylase in chicken PGCs |
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