CN110066803A - The method and the cytotropic method of target of nestin gene expression in a kind of regulation neural stem cell - Google Patents

Abstract

The invention discloses the methods and the cytotropic method of target of nestin gene expression in a kind of regulation neural stem cell, it is related to gene expression regulation technical field, including the following steps successively carried out, the gRNA of nestin gene is first designed near the transcription initiation of nestin gene, the building of carrier is carried out again, and the gRNA of nestin gene can be any one in the nestin-i gRNA of guidance Transcription inhibition and the nestin-a gRNA of guidance transcription activator.This method is simple, and off-target rate is low, and can stablize expression in cell strain.The drawbacks of overcoming with conventional method building stable cell line, can obtain in short term stable cell line.

Description

The method of nestin gene expression and target are cytotropic in a kind of regulation neural stem cell Method

Technical field

The present invention relates to gene expression regulation technical fields, in particular in a kind of regulation neural stem cell The cytotropic method of method and target of nestin gene expression.

Background technique

The encoding histone product of nestin gene is NESTIN.NESTIN is the 6th class intermediate filament protein, also referred to as nest egg White, molecular size range 220KDa-250KDa is located in No. 1 chromosome.Nestin gene is included in structure by 3 Son and 4 exon compositions.NESTIN albumen is broadly divided into N-terminal (1-7aa), alpha helical region (8-313aa) and C in structure It holds (314-1621aa), it is 1A, 1B, 2A and 2B respectively that wherein alpha helical region, which is subdivided into 4 parts,.

Wide expression is presented in nestin gene in embryo development procedure, and expression earliest is outside the unicellular nerve of embryo On germinal layer.In addition, nestin is also expressed in various kinds of cell, such as neovascular endothelium cell, middle interstitial cell, but in mind Expression through nestin in system is the most abundant.Development of central nervous system is one during mammal early development Highly important event, the process show as the migration of cell and the variation of cellular morphology, and NESTIN is as a kind of intermediate filament Albumen plays a very important role the self-renewing of neural stem cell by the mark molecule as neural stem cell.To the greatest extent Pipe Nestin plays an important role in nervous system development, but its specific influence and mechanism of action are still not clear, Therefore molecule mechanism of the Nestin in nervous system development is illustrated to seem very necessary.

Overexpression or silencing of target genes in cell are common in for the research of gene, observe target gene in cell fate The effect played in change, and realizing the overexpression of gene or silencing mainly has transient expression and stablizes two classes of expression.Wherein, Exogenous DNA will not be integrated into genome in transient expression, but transient expression can only maintain several days short times；Stablize table Up to middle foreign gene and genome conformity, can in cell continuous expression.Traditional stable cell line of establishing usually passes through wink When the mode that transfects and the resistant gene on genophore is combined screen and final to obtain stable cell line, such mode There are problems that longevity of service, low efficiency, additionally due to Nestin Gene sequence comparison is long, is established by way of traditional steady It is relatively difficult to determine overexpressing cell strain, it is therefore necessary to establish the gRNA target of a kind of novel Nestin gene overexpression or silencing Point sequence.

CRISPR/Cas9 is a kind of technology of operator group, and under the guidance of guidance RNA, Cas9 being capable of target gene Specific region and DNA is cut in group, to cause DNA double-strand break.CRISPR/Cas9 be usually used in genome into Edlin.

In consideration of it, the present invention is specifically proposed.

Summary of the invention

The purpose of the present invention is to provide a kind of method of nestin gene expression in regulation neural stem cell, this method letters Single, off-target rate is low, and can stablize expression in cell strain.The drawbacks of overcoming with conventional method building stable cell line, energy It is enough to obtain stable cell line in short term.This would be even more beneficial to biological function of the research Nestin gene in nervous system development And its specific mechanism of action, the life rule for recognizing neural stem cell for us have important directive significance.

Another object of the present invention is to provide a kind of cytotropic method of target, this method can accurately target cell, right Cell transcribed before gene regulation.

The present invention is implemented as follows:

A kind of method of nestin gene expression in regulation neural stem cell first exists including the following steps successively carried out The transcription initiation of nestin gene nearby designs the gRNA of nestin gene, then carries out the building of carrier, nestin gene GRNA can be appointing in the nestin-i gRNA of guidance Transcription inhibition and the nestin-a gRNA of guidance transcription activator It anticipates one kind.

In the present invention using in preferred embodiment, the sequence of above-mentioned nestin-i gRNA as shown in sequence 1-12, As shown in sequence 13-24, the sequence of nestin-i gRNA can be as shown in sequence 1-12 the sequence of nestin-a gRNA The combination of any one or more, the sequence of nestin-a gRNA can be as shown in sequence 13-24 any one or more Combination.

In the present invention using in preferred embodiment, the sequence that targets of above-mentioned nestin-i gRNA is located at First Exon Within the scope of the 1-112bp of transcription initiation site downstream, the sequence that targets of nestin-a gRNA is located at First Exon transcription initiation Within the scope of site upstream 1-450bp.

In the present invention using in preferred embodiment, the above method further includes that viral packaging, tool are carried out using 293T cell Body is evenly dispersed including first carrying out to 293T cell, PEI mixed liquor is added in plasmid mixed liquor, plasmid-PEI mixing is made Liquid, then plasmid-PEI mixed liquor is added in the culture medium containing 239T cell and is co-cultured, virus is collected in concentration.

In the present invention using in preferred embodiment, the sense of cell is carried out after the above-mentioned viral packaging using the progress of 293T cell Dye and screening, specifically comprise the following steps: first to prepare viral mixed liquor, then be added into the good neural stem cell of growth conditions The virus liquid mixed liquor prepared co-cultures.

In the present invention using in preferred embodiment, the expression of nes gene is carried out after the infection of above-mentioned cell respectively The detection of detection and NESTIN protein level, wherein the primer of the expression detection of nes gene detection nes gene used As shown in sequence 26-27.

In the present invention using in preferred embodiment, the above method include be overexpressed in neural stem cell nestin gene or Nestin gene in silencing neural stem cell.

A kind of supporting molecular includes the gRNA of nestin gene, the sequence of the gRNA of nestin gene such as sequence 1-24 Shown in any one or a variety of combinations.

A kind of cytotropic method of target, including contact cell with supporting molecular, supporting molecular includes nestin gene GRNA, supporting molecular include catalytically inactive structural domain dCas9, the sequence of dCas9 is as shown in sequence 25, nestin gene GRNA sequence as shown in sequence 1-24, cell is neural stem cell, in neovascular endothelium cell and middle interstitial cell Any one.

In the present invention using in preferred embodiment, above-mentioned supporting molecular also includes antibiotic, and antibiotic is that purine is mould Element, hygromycin, roxithromycin, spiramvcin, azithromycin, clarithromycin, vancomycin, streptomysin, gentamicin, Fourth Ring Element, Doxycycline, minocycline, kanamycins, the combination of any one or more in ampicillin and chloramphenicol；It is preferred that , antibiotic is puromycin, hygromycin.

The invention has the following advantages:

The present invention provides a kind of methods of nestin gene expression in regulation neural stem cell, and this method can be used for interior Source property is overexpressed or the gRNA target sequence of silencing nestin gene, overcomes the disadvantage with conventional method building stable cell line End, and off-target rate is low, can obtain in short term stable cell line.This would be even more beneficial to research Nestin gene and sends out in nervous system Biological function and its specific mechanism of action in educating have the life rule that we recognize neural stem cell important Directive significance.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is the structure chart of the position gRNA and nestin gene；

Fig. 2 be gRNA-a group neural stem cell nestin gene mRNA qRT-PCR result figure (Control group representative surely Surely lenti-EF1a-dCas9-VPR-Puro and lentiGuide-Hygro-mTagBFP2 cell is expressed；GRNAa1 represents steady Surely lenti-EF1a-dCas9-VPR-Puro and lentiGuide-Hygro-mTagBFP2-gRNAa1 cell is expressed； GRNAa2, which is represented, stablizes expression lenti-EF1a-dCas9-VPR-Puro and lentiGuide-Hygro-mTagBFP2- GRNAa2 cell；GRNAa3, which is represented, stablizes expression lenti-EF1a-dCas9-VPR-Puro and lentiGuide-Hygro- MTagBFP2-gRNAa3 cell；GRNAa4 represent stablize expression lenti-EF1a-dCas9-VPR-Puro and LentiGuide-Hygro-mTagBFP2-gRNAa4 cell；GRNAa5, which is represented, stablizes expression lenti-EF1a-dCas9-VPR- Puro and lentiGuide-Hygro-mTagBFP2-gRNAa5 cell；GRNAa6, which is represented, stablizes expression lenti-EF1a- DCas9-VPR-Puro and lentiGuide-Hygro-mTagBFP2-gRNAa6 cell；It is examined with Student ' s t-test Test the comparison for carrying out group difference, p < 0.01 * p < 0.05, * *)；

Fig. 3 is qRT-PCR result (the Control group representative stabilization of gRNA-i group neural stem cell nestin gene mRNA Express lenti-EF1a-dCas9-KRAB-Puro and lentiGuide-Hygro-mTagBFP2 cell；GRNAi1 represents steady Surely lenti-EF1a-dCas9-KRAB-Puro and lentiGuide-Hygro-mTagBFP2-gRNAi1 cell is expressed； GRNAi2, which is represented, stablizes expression lenti-EF1a-dCas9-KRAB-Puro and lentiGuide-Hygro-mTagBFP2- GRNAi2 cell；GRNAi3, which is represented, stablizes expression lenti-EF1a-dCas9-KRAB-Puro and lentiGuide- Hygro-mTagBFP2-gRNAi3 cell；GRNAi4 represent stablize expression lenti-EF1a-dCas9-KRAB-Puro and LentiGuide-Hygro-mTagBFP2-gRNAi4 cell；GRNAi5, which is represented, stablizes expression lenti-EF1a-dCas9- KRAB-Puro and lentiGuide-Hygro-mTagBFP2-gRNAi5 cell；GRNAi6, which is represented, stablizes expression lenti- EF1a-dCas9-KRAB-Puro and lentiGuide-Hygro-mTagBFP2-gRNAi6 cell；With Student ' s t- Test examines the comparison for carrying out group difference, and * p < 0.05, * * p < 0.01, #p < 0.001, n.s are no significant difference)；

Fig. 4 is that nestin gene stablizes the immune-blotting method result figure for being overexpressed NESTIN in neural stem cell (in Fig. 4 Caption it is identical as the caption in Fig. 2, correspondence represent identical substance)；

Fig. 5 is the immune-blotting method result figure of NESTIN in nestin gene stabilization checking neural stem cell (in Fig. 5 Caption is identical as the caption in Fig. 3, and correspondence represents identical substance).

Specific embodiment

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

Feature and performance of the invention are described in further detail with reference to embodiments.

Embodiment 1

A kind of method of nestin gene expression in regulation neural stem cell, specifically includes the following steps successively carried out:

The gRNA of nestin gene is designed first.

Shown in the cDNA sequence canonical sequence 30 of nestin gene, 31 institute of amino acid sequence canonical sequence of NESTIN albumen Show, for the sequence of nestin gene, 12 couples of gRNA, including 6 pairs of guidance are devised near the transcription initiation of nestin gene The nestin-a gRNA, wherein nestin-i gRNA of the nestin-i gRNA of Transcription inhibition and 6 pairs of guidance transcription activators Sequence as shown in sequence 1-12, the sequence of nestin-a gRNA is as shown in sequence 13-24.Nestin-i gRNA is for drawing Transcription inhibition is led in conjunction with the target site in nestin gene First Exon transcription initiation site downstream.nestin-a GRNA is for guiding transcription activator in conjunction with the target site of nestin gene First Exon transcription initiation site upstream. The structure chart of the 12 pairs of gRNA and nestin genes designed near the transcription initiation of nestin gene is shown referring to Fig.1, figure A1 in 1, A2, A3, A4, A5 and A6 are located at the 5 ' area UTR upstreams, respectively represent 6 couples of nestin-a gRNA；I1, I2, I3 in Fig. 1, I4, I5 and I6 are located at the 5 ' area UTR downstreams, respectively represent 6 couples of nestin-i gRNA.

Carrier construction.

Carrier construction specifically comprises the following steps:

1. the plasmid for the use of Addgene company production model being 99374: lentiGuide-Hygro-mTagBFP2 plasmid Digestion is carried out, in 37 DEG C of digestion 30min, digestion system is as follows:

2. the gRNA sequence of the good lentiGuide-Hygro-mTagBFP2 carrier of digestion and annealing is attached, item Part is 4 DEG C of connection reactions overnight.

3. above-mentioned connection product is converted into DH5 α competent cell, specific steps are as follows:

(1) competent cell DH5 α is taken out from -80 DEG C, is slowly thawed on ice；

(2) connection product is added in competent cell DH5 α, and gently pressure-vaccum mixes, and is put in 30min on ice；

(3) mixture in step 2 is taken out, carries out heat shock, heat shock 90s in 42 DEG C of water-baths rapidly；

(4) heat shock terminates to be put in rapidly stands 5min on ice；

(5) the LB culture medium of 900 μ l preheating is added, is cultivated 1 hour under 37 DEG C of shaking table 180g revolving speeds；

(6) 4000rpm is centrifuged 3min at room temperature, discards supernatant remaining 100 μ l culture mediums and is resuspended；

(7) bacterium solution of resuspension is uniformly coated on the LB plate containing hygromycin resistance, overnight incubation in 37 DEG C of incubators.

4. extracting plasmid

Use the extraction for going the small extraction reagent kit of endotoxin plasmid to carry out plasmid, the specific steps are as follows:

1) it selects the monoclonal in above-mentioned steps 3 on LB plate to be inoculated into the LB culture medium containing hygromycin resistance, shake Bed overnight incubation；

2) bacterium solution is centrifuged, collects thallus, centrifugal condition is that 5000rpm is centrifuged 10min；

3) the Solution I that 500 μ l have added RNase A is added into thallus, mixes well resuspension thallus；

4) 500 μ l Solution II are added into the thallus being resuspended, and lightly turn upside down for several times until solution becomes It is transparent, be put in room temperature 3min；

5) N3Buffer of 250 μ l pre-cooling is added into pipe, and lightly turns upside down for several times, under the conditions of room temperature 13000g It is centrifuged 10min；

6) supernatant that previous step obtains is transferred in another centrifuge tube, the ETR Solution of 0.1 volume is added, It turns upside down mixing, is put in and stands 10min on ice；

7) it is put in 42 DEG C of water-baths after standing and is incubated for 5min, be centrifuged 3min under the conditions of room temperature 12000g；

8) supernatant is transferred in new centrifuge tube, the dehydrated alcohol of 0.5 volume is added, is lightly mixed by inversion, room Temperature stands 2min；

9) mixed liquor that previous step obtains is added into adsorption column, is centrifuged 1min under the conditions of room temperature 13000g, discard from Heart liquid；

10) it is added into adsorption column under the conditions of 500 μ l HBC Buffer, room temperature 13000g and is centrifuged 1min, discard centrifugation Liquid；

11) it is added into adsorption column under the conditions of 700 μ l DNA Wash Buffer, room temperature 13000g and is centrifuged 1min, discarded Centrifugate；

12) it will be centrifuged 1min under the conditions of suction attached column 13000g, and upper layer suction attached column is transferred in new centrifuge tube, 100 μ l ddH are added into adsorption column₂O is stored at room temperature 5min；

13) room temperature 13000g is centrifuged 1min, and containing in the liquid after centrifugation has purpose plasmid, carries out concentration to purpose plasmid Measurement, be put in -20 DEG C of preservations.

5. the identification of recombinant plasmid

Recombinant plasmid is sent to sequencing company to be sequenced, determines whether plasmid constructs success.

Prepare 293T cell

1) culture medium in culture dish is inhaled and is abandoned, wherein culture has 293T cell in culture dish, and 293T cell is conventional Vehicles cells；

2) cell surface is cleaned with 1ml PBS, and discards PBS, without purging when operation；

3) 1ml 0.25% trypsase-EDTA (1x) vitellophag is added in every ware cell, shaken up and down, Yu Xian Micro- microscopic observation digests situation.Digestion can be terminated after 293T cell rounding, preferably as early as possible, the time is unsuitable too long for this process；

4) digestion that 1ml DMEM+10%FBS terminates pancreatin is added in every ware cell, and piping and druming mixes and is transferred to 15ml centrifugation Guan Zhong；

5) it draws 2ml PBS to wash culture dish one time, and is transferred in 15ml centrifuge tube；

6) 15ml centrifuge tube is put into a centrifuge, 1000rpm, supernatant is abandoned in 5min centrifugation；

7) 1ml DMEM+10%FBS is added, cell is resuspended, pressure-vaccum mixes；

8) cell suspension of the step of taking a centrifuge tube, the trypan blue and 10 μ l of 10 μ l is added into centrifuge tube (7), fills After dividing pressure-vaccum to mix, 10 μ l are taken every time and carry out cell count twice；

9) Tissue Culture Dish of 10cm is got out, is marked, each DMEM+10%FBS that 8ml is added；

10) 8,000,000 cells are added in every ware, and piping and druming mixes, and shakes up culture dish by " cross method ", and cell is made to be scattered in culture In ware；

11) it avales, is slowly steadily put into spare in incubator to cell.

Virus packaging

(1) cell is taken out from cell incubator, in microscopically observation growing state.If cell is in order, can Carry out cell transfecting step.

(2) before cell transfecting, prepare following mixed liquor:

Plasmid mixed liquor is prepared before transfection, the plasmid and psPAX2 and VSV-G carrier that step 4 is extracted are according to table 1 In adding proportion mixing after, be added in Opti-MEM culture medium, gently mixed under pressure-vaccum 2-3, the static 5min of room temperature.Table 1 Middle Insert is the plasmid that step 4 is extracted, and Packing and Envelop respectively indicate the additive amount of psPAX2 and VSV-G. For the purchase of psPAX2 carrier from Addgene company, production model is plasmid 12260, and VSV-G carrier is bought from Addgene company, Production model is plasmid 8454.Opti-MEM culture medium is bought from Gibco company, production model 31985-070.

In table 1, lenti-EF1a-dCas9-VPR-Puro and lenti-EF1a-dCas9-KRAB-Puro carrier by Addgene company is commercially available, the former production model is 99373, and the production model of the latter is 99372.

PEI mixed liquor: PEI (polyetherimide) (1mg/ml) is added in Opti-MEM culture medium, gently pressure-vaccum 2- 3 lower mixings, the static 5min of room temperature.

1 10cm culture dish packaging plasmid dosage of table

3) PEI mixed liquor is added in plasmid mixed liquor after static 5min, is added dropwise, centrifugation is shaken in edged mixing in side Pipe avoids precipitating from occurring, the static 15min of room temperature.

4) plasmid-PEI mixed liquor is even added to dropwise in not antibiotic culture medium after standing 15min, is being trained It supports and replaces culture medium after cultivating 6h in case (culture medium used in whole experiment process is free of antibiotic).

5) plasmid-PEI mixed liquor and 293T mixing with cells transfect 48h, collect vial supernatant for the first time, after transfecting 72h Second collects vial supernatant and by 0.45 μm of membrane filtration of all vial supernatants；

6) vial supernatant of collection is subjected to centrifugal concentrating, centrifugal condition is 4 DEG C, and 19400rpm is centrifuged 2h, centrifugation knot Supernatant is discarded after beam and the resuspension of DMEM basal medium is added, final vial supernatant is concentrated 20 times, the virus that will be concentrated 1.5ml EP pipe is dispensed into save in -80 DEG C.

Cell infection and screening

1. cell infection:

1) on the day before carrying out virus infection, by the good source of people neural stem cell of growth conditions according to the cell in the hole 50K/ Density is seeded in 24 orifice plates, and the people's derived neural stem cell is conventional neural stem cell；

2) prepare viral mixed liquor, lenti-EF1a-dCas9-VPR-Puro or lenti- is added into centrifuge tube respectively EF1a-dCas9-KRAB-Puro vial supernatant and polybrene (Polybrene), final concentration of 5 μ that Polybrene is used G/ml, it is spare；

Polybrene is mainly used for improving the efficiency of retroviral infection cell in cell culture, by neutralizing cell table Electrical charge rejection between the sialic acid and virion in face is realized.

3) culture medium in 24 orifice plates is inhaled and is abandoned, the viral mixed liquor prepared is added into cell；

4) orifice plate is centrifuged 30min under the conditions of 25 DEG C, 2250rpm；

5) cell is put back into 37 DEG C of incubator cultures after being centrifuged, replacement fresh culture continues to cultivate afterwards for 24 hours.

2. cell screening:

1) after virus infected cell 72h, culture medium is inhaled and is abandoned, the culture medium containing puromycin is added into cell and carries out Culture；

2) fresh culture containing puromycin is replaced every three days；

3) it after 7 days, is changed to containing the culture for halving drug concentration using the culture medium culture cell containing puromycin Base maintains culture, obtains stable expression lenti-EF1a-dCas9-KRAB-Puro, lenti-EF1a-dCas9- respectively at this time The cell strain of VPR-Puro；

4) stablizing expression lenti-EF1a-dCas9-KRAB-Puro's and lenti-EF1a-dCas9-VPR-Puro LentiGuide-Hygro-mTagBFP2-gRNA-I and lentiGuide-Hygro- is carried out on cell strain respectively The infection of mTagBFP2-gRNA-A virus, infection step are carried out according to step described in above-mentioned cell infection；

5) after virus infected cell 72h, culture medium is inhaled and is abandoned, the culture medium containing hygromycin B is added into cell and carries out Culture；

6) fresh culture containing hygromycin B is replaced every three days；

7) it after 7 days, is changed to containing the culture medium for halving drug concentration using the culture medium culture cell containing hygromycin B Culture is maintained, obtains the cell strain stablized silencing or be overexpressed nes gene at this time；

8) detection of expression of nestin gene is carried out to the cell strain of acquisition.

Embodiment 2

Detection to nestin gene expression dose.

1. extracting the total serum IgE of cell strain, specifically comprise the following steps:

1) it inhales the culture medium abandoned in culture dish and 2ml PBS rinsing cell is added, 1ml TRIZOL is added after discarding PBS Solution is incubated for 10min on ice；

2) solution for being added with TRIZOL in step (1) is transferred in centrifuge tube, the chloroform and vortex of 200 μ l is added Concussion mixes, and is put in and is incubated for 5min on ice；

3) centrifuge tube is centrifuged 15min under the conditions of 4 DEG C of 13000g, after centrifugation carefully by upper strata aqueous phase be transferred to it is another from In heart pipe；

4) isopropanol of equivalent volumes is added into centrifuge tube, centrifuge tube is put in -80 DEG C after mixing and precipitates 30min；

5) centrifuge tube is centrifuged 15min under the conditions of 4 DEG C of 13000g, precipitates, wants at this time small in centrifuge tube bottom visible white Heart, which inhales supernatant, abandons；

6) into centrifuge tube be added 1ml pre-cooling 75% ethanol solution, be mixed by inversion and under the conditions of 4 DEG C of 13000g from Heart 5min inhales and abandons supernatant；

7) it is primary that step (6) are repeated；

8) centrifuge tube pipe lid opening is put in and is stored at room temperature 3~5min, allow extra ethyl alcohol to volatilize clean, at this time centrifuge tube Tube bottom white precipitate presents colorless and transparent；

9) the DEPC water containing RNase inhibitor is added into centrifuge tube and dissolves RNA, and carry out the survey of concentration and purity It is fixed.

The synthesis of the first chain of 2.cDNA

1) following reaction system is prepared in the removal of genomic DNA on ice:

2) it sets following procedure in PCR instrument to be reacted: 37 DEG C of 30min, 75 DEG C of 10 min, 4 DEG C of of short duration preservations；

3) 10 μ l RNA solutions are taken out from step (2) into centrifuge tube, prepare following reaction system:

4) centrifuge tube is put in 65 DEG C of water-baths and reacts 5min, reaction terminates to be put in cooled on ice rapidly；

5) by after centrifuge tube brief centrifugation, following components is added into centrifuge tube on ice:

6) by step (5) centrifuge tube in 37 DEG C of water-bath 2min；

7) 1 μ l M-MLV reverse transcriptase is added into centrifuge tube；

8) it sets following procedure in PCR instrument to be reacted: 25 DEG C of 10min → 37 DEG C, 50 min → 70 DEG C 15min → 4 DEG C Of short duration preservation；

9) the product taking-up of step (8) is put in -20 DEG C and is saved.

The expression of 3.qRT-PCR detection nes mRNA.

1) the cDNA ddH that will be prepared₂O is used as reaction template after diluting 6 times；

2) following reaction system is prepared on ice, wherein shown in the sequence canonical sequence 26-27 of nestin primer, internal reference Shown in the primer sequence canonical sequence 28-29 of gene GAPDH:

3) mixed reaction solution in step (2) is transferred in qRT-PCR plate and carries out sealing plate；

4) in the qRT-PCR instrument that qRT-PCR plate is put into the production of Bio-Rad company, using the standard two-step method in software It brings into operation with solubility curve program.

Embodiment 3

The detection of NESTIN protein level

The total protein for extracting cell first, then carries out Western Blot.Specifically comprise the following steps:

1) after rinsing the cell strain collected in embodiment 1 with PBS, protein lysate is added in cracking 15min on ice；

2) mixture in step (1) is subjected to ultrasonication, abundant lytic cell using Ultrasound Instrument；

3) centrifuge tube is centrifuged 15min under the conditions of 4 DEG C of 13000g, supernatant protein is transferred in new centrifuge tube；

4) the Bradford determination of protein concentration kit produced using Solarbio company is measured protein concentration, The product type of Bradford determination of protein concentration kit is PC0010；

5) it takes 50 μ g total proteins to be added in 20 μ l 2xSDS loading buffer, adds ddH₂O makes total volume 40 μ l, heat 10min in boiling water；

6) sample that 35 μ l have been denaturalized in step (5) is taken to carry out SDS-PAGE electrophoresis；

7) albumen in gel is gone on pvdf membrane by wet process transferring film；

8) antibody incubation and PDVF Membrane cleaning；

9) develop toward dropwise addition ECL on the PDVF film on step (8).

Embodiment 4

The qRT-PCR result of neural stem cell nestin gene mRNA is referring to shown in table 2 and Fig. 2.

It can be obtained referring to table 2 and Fig. 2, be control, control group with the expression quantity of cellular control unit nestin mRNA (control) plasmid vector being inserted into is lentiGuide-Hygro-mTagBFP2, other experimental group relative comparison groups Nestin MRNA expression is respectively as follows: 5.2 times that Nestin mRNA expression in gRNAa1 group cell is cellular control unit, Nestin mRNA expression is 1.77 times of cellular control unit, Nestin in gRNAa3 group cell in gRNAa2 group cell MRNA expression is 1.93 times of cellular control unit, and Nestin mRNA expression is that control group is thin in gRNAa4 group cell 1.86 times of born of the same parents, Nestin mRNA expression is 2.37 times of cellular control unit, gRNAa6 group cell in gRNAa5 group cell Middle Nestin mRNA expression is 1.18 times of cellular control unit.Wherein, gRNAa1 is to neural stem cell nestin gene Transcriptional activation it is most strong.

The qRT-PCR result of 2 gRNA-a group neural stem cell nestin gene mRNA of table

Control	gRNAa1	gRNAa2	gRNAa3	gRNAa4	gRNAa5	gRNAa6
							1.00	5.20	1.77	1.93	1.86	2.37	1.18

It can be obtained referring to table 3 and Fig. 3, be control, control group (control) with cellular control unit Nestin mrna expression amount The plasmid vector of insertion is lentiGuide-Hygro-mTagBFP2, other experimental group relative comparison group Nestin mRNA expression Level is respectively as follows: 0.87 times that Nestin mRNA expression in gRNAi1 group cell is cellular control unit, gRNAi2 group cell Middle Nestin mRNA expression is 0.40 times of cellular control unit, and Nestin mRNA expression is in gRNAi3 group cell 0.27 times of cellular control unit, Nestin mRNA expression is 0.46 times of cellular control unit in gRNAi4 group cell, Nestin mRNA expression is 0.91 times of cellular control unit, Nestin in gRNAi6 group cell in gRNAi5 group cell MRNA expression is 0.95 times of cellular control unit.Wherein, Transcription inhibition of the gRNAi3 to neural stem cell nestin gene It acts on most strong.

The qRT-PCR result of 3 gRNA-i group neural stem cell nestin gene mRNA of table

Control	gRNAi1	gRNAi2	gRNAi3	gRNAi4	gRNAi5	gRNAi6
							1.00	0.87	0.40	0.27	0.46	0.91	0.95

Embodiment 5

For the nestin gene expression results for further verifying embodiment 4, respectively to the albumen of neural stem cell NESTIN Expression is detected, as a result referring to shown in Fig. 4 and Fig. 5.GRNAa1-a6, which can stablize, as the result is shown is overexpressed in neural stem cell NESTIN, wherein gRNAa1 is most strong to the transcriptional activation of neural stem cell nestin gene, NESTIN expressing quantity Highest.NESTIN in gRNAi1-i6 energy stabilization checking neural stem cell, wherein gRNAi3 is to neural stem cell nestin base The transcripting suppressioning action of cause is most strong.The test result of embodiment 5 and the experimental result of embodiment 4 are fitted, and therefore, the present invention is mentioned The method of nestin gene expression can be used for endogenous overexpression or silencing nestin in a kind of regulation neural stem cell supplied The gRNA target sequence of gene.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

SEQUENCE LISTING

<110>Zhuhai Le Wei regenerative medicine Science and Technology Ltd.

<120>it is a kind of regulation neural stem cell in nestin gene expression method and the cytotropic method of target

<160> 31

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213>artificial sequence

<400> 1

caccggcaga atgagctgct cagcg 25

<210> 2

<211> 25

<212> DNA

<213>artificial sequence

<400> 2

aaaccgctga gcagctcatt ctgcc 25

<210> 3

<211> 25

<212> DNA

<213>artificial sequence

<400> 3

caccggaccg acggggacaa tgacg 25

<210> 4

<211> 25

<212> DNA

<213>artificial sequence

<400> 4

aaaccgtcat tgtccccgtc ggtcc 25

<210> 5

<211> 25

<212> DNA

<213>artificial sequence

<400> 5

caccggagac cgacggggac aatga 25

<210> 6

<211> 25

<212> DNA

<213>artificial sequence

<400> 6

aaactcattg tccccgtcgg tctcc 25

<210> 7

<211> 25

<212> DNA

<213>artificial sequence

<400> 7

caccgggagg agtcgtttca gatgt 25

<210> 8

<211> 25

<212> DNA

<213>artificial sequence

<400> 8

aaacacatct gaaacgactc ctccc 25

<210> 9

<211> 25

<212> DNA

<213>artificial sequence

<400> 9

caccggtggg agctcaatcg gcgcc 25

<210> 10

<211> 25

<212> DNA

<213>artificial sequence

<400> 10

aaacggcgcc gattgagctc ccacc 25

<210> 11

<211> 25

<212> DNA

<213>artificial sequence

<400> 11

caccggacgg ggacaatgac ggggc 25

<210> 12

<211> 25

<212> DNA

<213>artificial sequence

<400> 12

aaacgccccg tcattgtccc cgtcc 25

<210> 13

<211> 25

<212> DNA

<213>artificial sequence

<400> 13

caccgggttc cactctccag caccc 25

<210> 14

<211> 25

<212> DNA

<213>artificial sequence

<400> 14

aaacgggtgc tggagagtgg aaccc 25

<210> 15

<211> 25

<212> DNA

<213>artificial sequence

<400> 15

caccgccatg gtacagcttc ttctt 25

<210> 16

<211> 25

<212> DNA

<213>artificial sequence

<400> 16

aaacaagaag aagctgtacc atggc 25

<210> 17

<211> 25

<212> DNA

<213>artificial sequence

<400> 17

caccgcccag gccttaaccc cttag 25

<210> 18

<211> 25

<212> DNA

<213>artificial sequence

<400> 18

aaacctaagg ggttaaggcc tgggc 25

<210> 19

<211> 25

<212> DNA

<213>artificial sequence

<400> 19

caccggaagc tgtaccatgg agaat 25

<210> 20

<211> 25

<212> DNA

<213>artificial sequence

<400> 20

aaacattctc catggtacag cttcc 25

<210> 21

<211> 25

<212> DNA

<213>artificial sequence

<400> 21

caccggcaca gaactgagcc tgtgg 25

<210> 22

<211> 25

<212> DNA

<213>artificial sequence

<400> 22

aaacccacag gctcagttct gtgcc 25

<210> 23

<211> 25

<212> DNA

<213>artificial sequence

<400> 23

caccggactg cactccctcc caggc 25

<210> 24

<211> 25

<212> DNA

<213>artificial sequence

<400> 24

aaacgcctgg gagggagtgc agtcc 25

<210> 25

<211> 4101

<212> DNA

<213>artificial sequence

<400> 25

gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc 60

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg 720

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080

tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260

attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440

gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560

aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400

gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccacatc 2520

gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580

aaggcccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000

cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080

ctgtctcagc tgggaggcga c 4101

<210> 26

<211> 19

<212> DNA

<213>artificial sequence

<400> 26

ctggagcagg agaaacagg 19

<210> 27

<211> 20

<212> DNA

<213>artificial sequence

<400> 27

tgggagcaaa gatccaagac 20

<210> 28

<211> 19

<212> DNA

<213>artificial sequence

<400> 28

acccactcct ccacctttg 19

<210> 29

<211> 19

<212> DNA

<213>artificial sequence

<400> 29

ctcttgtgct cttgctggg 19

<210> 30

<211> 5568

<212> DNA

<213>artificial sequence

<400> 30

agtcgctcag gctactccca ccccgccccg ccccgtcatt gtccccgtcg gtctcttttc 60

tcttccgtcc taaaagctct gcgagccgct cccttctccc ggtgccccgc gtctgtccat 120

cctcagtggg tcagacgagc aggatggagg gctgcatggg ggaggagtcg tttcagatgt 180

gggagctcaa tcggcgcctg gaggcctacc tggcccgggt caaggcgctg gaggagcaga 240

atgagctgct cagcgcggag ctcggggggc tccgggcaca atccgcggac acctcctggc 300

gggcgcatgc cgacgacgag ctggcggccc tgcgggccct cgttgaccaa cgctggcggg 360

agaagcacgc ggccgaggtg gcgcgcgaca acctggctga agagctggag ggcgtggcag 420

gccgatgcca gcagctgcgg ctggcccggg agcggacgac ggaggaggta gcccgcaacc 480

ggcgcgccgt cgaggcagag aaatgcgccc gggcctggct gagtagccag gtggcagagc 540

tggagcgcga gctagaggct ctacgcgtgg cgcacgagga ggagcgcgtc ggcctgaacg 600

cgcaggctgc ctgtgccccc cgctgccccg cgccgccccg cgggcctccc gcgccggccc 660

cggaggtaga ggagctggca aggcgactgg gcgaggcgtg gcgcggggca gtgcgcggct 720

accaggagcg cgtggcacac atggagacgt cgctgggcca ggcccgcgag cggctgggcc 780

gggcggtgca gggtgcccgc gagggccgcc tggagctgca gcagctccag gctgagcgcg 840

gaggcctcct ggagcgcagg gcagcgttgg aacagaggtt ggagggccgc tggcaggagc 900

ggctgcgggc tactgaaaag ttccagctgg ctgtggaggc cctggagcag gagaaacagg 960

gcctacagag ccagatcgct caggtcctgg aaggtcggca gcagctggcg cacctcaaga 1020

tgtccctcag cctggaggtg gccacgtaca ggaccctcct ggaggctgag aactcccggc 1080

tgcaaacacc tggcggtggc tccaagactt ccctcagctt tcaggacccc aagctggagc 1140

tgcaattccc taggacccca gagggccggc gtcttggatc tttgctccca gtcctgagcc 1200

caacttccct cccctcaccc ttgcctgcta cccttgagac acctgtgcca gcctttctta 1260

agaaccaaga attcctccag gcccgtaccc ctaccttggc cagcaccccc atccccccca 1320

cacctcaggc accctctcct gctgtagatg cagagatcag agcccaggat gctcctctct 1380

ctctgctcca gacacagggt gggaggaaac aggctccaga gcccctgcgg gctgaagcca 1440

gggtggccat tcctgccagc gtcctgcctg gaccagagga gcctgggggc cagcggcaag 1500

aggccagtac aggccagtcc ccagaggacc atgcctcctt ggcaccaccc ctcagccctg 1560

accactccag tttagaggct aaggatggag aatccggtgg gtctagagtg ttcagcatat 1620

gccgagggga aggtgaaggg caaatctggg ggttggtaga gaaagaaaca gccatagagg 1680

gcaaagtggt aagcagcttg cagcaggaaa tatgggaaga agaggatcta aacaggaagg 1740

aaatccagga ctcccaggtt cctttggaaa aagaaaccct gaagtctctg ggagaggaga 1800

ttcaagagtc actgaagact ctggaaaacc agagccatga gacactagaa agggagaatc 1860

aagaatgtcc gaggtcttta gaagaagact tagaaacact aaaaagtcta gaaaaggaaa 1920

ataaagagct attaaaggat gtggaggtag tgagacctct agaaaaagag gctgtaggcc 1980

aacttaagcc tacaggaaaa gaggacacac agacattgca atccctgcaa aaggagaatc 2040

aagaactaat gaaatctctt gaaggtaatc tagagacatt tttatttcca ggaacggaaa 2100

atcaagaatt agtaagttct ctgcaagaga acttagagtc attgacagct ctggaaaagg 2160

agaatcaaga gccactgaga tctccagaag taggggatga ggaggcactg agacctctga 2220

caaaggagaa tcaggaaccc ctgaggtctc ttgaagatga gaacaaagag gcctttagat 2280

ctctagaaaa agagaaccag gagccactga agactctaga agaagaggac cagagtattg 2340

tgagacctct agaaacagag aatcacaaat cactgaggtc tttagaagaa caggaccaag 2400

agacattgag aactcttgaa aaagagactc aacagcgacg gaggtctcta ggggaacagg 2460

atcagatgac attaagaccc ccagaaaaag tggatctaga accactgaag tctcttgacc 2520

aggagatagc tagacctctt gaaaatgaga atcaagagtt cttaaagtca ctcaaagaag 2580

agagcgtaga ggcagtaaaa tctttagaaa cagagatcct agaatcactg aagtctgcgg 2640

gacaagagaa cctggaaaca ctgaaatctc cagaaactca agcaccactg tggactccag 2700

aagaaataaa tcagggggca atgaatcctc tagaaaagga aattcaagaa ccactggagt 2760

ctgtggaagt gaaccaagag acattcagac tcctggaaga ggagaatcag gaatcattga 2820

gatctctggg agcatggaac ctggagaatt tgagatctcc agaggaggta gacaaggaaa 2880

gtcaaaggaa tctggaagag gaagagaacc tgggaaaggg agagtaccaa gagtcactga 2940

ggtctctgga ggaggaggga caggagctgc cgcagtctgc agatgtgcag aggtgggaag 3000

atacggtgga gaaggaccaa gaactggctc aggaaagccc tcctgggatg gctggagtgg 3060

aaaatgagga tgaggcagag ctgaatctga gggagcagga tggcttcact gggaaggagg 3120

aggtggtaga gcagggagag ctgaatgcca cagaggaggt ctggatccca ggcgaggggc 3180

acccagagag ccctgagccc aaagagcaga gaggcctggt tgagggagcc agtgtgaagg 3240

gaggggctga gggcctccag gaccctgaag ggcaatcaca acaggtgggg gccccaggcc 3300

tccaggctcc ccaggggctg ccagaggcga tagagcccct ggtggaagat gatgtggccc 3360

cagggggtga ccaagcctcc ccagaggtca tgttggggtc agagcctgcc atgggtgagt 3420

ctgctgcggg agctgagcca ggcccggggc agggggtggg agggctgggg gacccaggcc 3480

atctgaccag ggaagaggtg atggaaccac ccctggaaga ggagagtttg gaggcaaaga 3540

gggttcaggg cttggaaggg cctagaaagg acctagagga ggcaggtggt ctggggacag 3600

agttctccga gctgcctggg aagagcagag acccttggga gcctcccagg gagggtaggg 3660

aggagtcaga ggctgaggcc cccaggggag cagaggaggc gttccctgct gagaccctgg 3720

gccacactgg aagtgatgcc ccttcacctt ggcctctggg gtcagaggaa gctgaggagg 3780

atgtaccacc agtgctggtc tcccccagcc caacgtacac cccgatcctg gaagatgccc 3840

ctgggcctca gcctcaggct gaagggagtc aggaggctag ctggggggtg caggggaggg 3900

ctgaagccct ggggaaagta gagagcgagc aggaggagtt gggttctggg gagatccccg 3960

agggccccca ggaggaaggg gaggagagca gagaagagag cgaggaggat gagctcgggg 4020

agacccttcc agactccact cccctgggct tctacctcag gtcccccacc tcccccaggt 4080

gggaccccac tggagagcag aggccacccc ctcaagggga gactggaaag gagggctggg 4140

atcctgctgt cctggcttcc gagggccttg aggccccacc ctcagaaaag gaggaggggg 4200

aggagggaga agaggagtgt ggccgtgact ctgacctgtc agaagaattt gaggacctgg 4260

ggactgaggc accttttctt cctggggtcc ctggggaggt ggcagaacct ctgggccagg 4320

tgccccagct gctactggat cctgcagcct gggatcgaga tggggagtcc gatgggtttg 4380

cagatgagga agaaagtggg gaggagggag aggaggatca ggaggagggg agggagccag 4440

gggctgggcg gtgggggcca gggtcttctg ttggcagcct ccaggccctg agtagctccc 4500

agagagggga attcctggag tctgattctg tgagtgtcag tgtcccctgg gatgacagct 4560

tgaggggtgc agtggctggt gcccccaaga ctgccctgga aacggagtcc caggacagtg 4620

ctgagccttc tggctcagag gaagagtctg accctgtttc cttggagagg gaggacaaag 4680

tccctggccc tctagagatc cccagtggga tggaggatgc aggcccaggg gcagacatca 4740

ttggtgttaa tggccagggt cccaacttgg aggggaagtc acagcatgtg aatgggggag 4800

tgatgaacgg gctggagcag tctgaggaag tggggcaagg aatgccgcta gtctctgagg 4860

gagaccgagg gagccccttt caggaggagg aggggagtgc tctgaagacc tcttgggcag 4920

gggctcctgt tcacctgggc cagggtcagt tcctgaagtt cactcagagg gaaggagata 4980

gagagtcctg gtcctcaggg gaggactagg aaaagaccat ctgcccggca ctggggactt 5040

aggggtgcgg ggaggggaag gacgcctcca agcccgctcc ctgctcagga gcagcactct 5100

taacttacga tctcttgaca tatggtttct ggctgagagg cctggcccgc taaggtgaaa 5160

aggggtgtgg caaaggagcc tactccaaga atggaggctg taggaatata acctcccacc 5220

ctgcaaaggg aatctcttgc ctgctccatc tcataggcta agtcagctga atcccgatag 5280

tactaggtcc ccttccctcc gcatcccgtc agctggaaaa ggcctgtggc ccagaggctt 5340

ctccaaaggg agggtgacat gctggctttt gtgcccaagc tcaccagccc tgcgccacct 5400

cactgcagta gtgcaccatc tcactgcagt agcacgccct cctgggccgt ctggcctgtg 5460

gctaatggag gtgacggcac tcccatgtgc tgactccccc catccctgcc acgctgtggc 5520

cctgcctggc tagtccctgc ctgaataaag taatgcctcc gcttcaaa 5568

<210> 31

<211> 1621

<212> PRT

<213>artificial sequence

<400> 31

Met Glu Gly Cys Met Gly Glu Glu Ser Phe Gln Met Trp Glu Leu Asn

1 5 10 15

Arg Arg Leu Glu Ala Tyr Leu Ala Arg Val Lys Ala Leu Glu Glu Gln

20 25 30

Asn Glu Leu Leu Ser Ala Glu Leu Gly Gly Leu Arg Ala Gln Ser Ala

35 40 45

Asp Thr Ser Trp Arg Ala His Ala Asp Asp Glu Leu Ala Ala Leu Arg

50 55 60

Ala Leu Val Asp Gln Arg Trp Arg Glu Lys His Ala Ala Glu Val Ala

65 70 75 80

Arg Asp Asn Leu Ala Glu Glu Leu Glu Gly Val Ala Gly Arg Cys Gln

85 90 95

Gln Leu Arg Leu Ala Arg Glu Arg Thr Thr Glu Glu Val Ala Arg Asn

100 105 110

Arg Arg Ala Val Glu Ala Glu Lys Cys Ala Arg Ala Trp Leu Ser Ser

115 120 125

Gln Val Ala Glu Leu Glu Arg Glu Leu Glu Ala Leu Arg Val Ala His

130 135 140

Glu Glu Glu Arg Val Gly Leu Asn Ala Gln Ala Ala Cys Ala Pro Arg

145 150 155 160

Cys Pro Ala Pro Pro Arg Gly Pro Pro Ala Pro Ala Pro Glu Val Glu

165 170 175

Glu Leu Ala Arg Arg Leu Gly Glu Ala Trp Arg Gly Ala Val Arg Gly

180 185 190

Tyr Gln Glu Arg Val Ala His Met Glu Thr Ser Leu Gly Gln Ala Arg

195 200 205

Glu Arg Leu Gly Arg Ala Val Gln Gly Ala Arg Glu Gly Arg Leu Glu

210 215 220

Leu Gln Gln Leu Gln Ala Glu Arg Gly Gly Leu Leu Glu Arg Arg Ala

225 230 235 240

Ala Leu Glu Gln Arg Leu Glu Gly Arg Trp Gln Glu Arg Leu Arg Ala

245 250 255

Thr Glu Lys Phe Gln Leu Ala Val Glu Ala Leu Glu Gln Glu Lys Gln

260 265 270

Gly Leu Gln Ser Gln Ile Ala Gln Val Leu Glu Gly Arg Gln Gln Leu

275 280 285

Ala His Leu Lys Met Ser Leu Ser Leu Glu Val Ala Thr Tyr Arg Thr

290 295 300

Leu Leu Glu Ala Glu Asn Ser Arg Leu Gln Thr Pro Gly Gly Gly Ser

305 310 315 320

Lys Thr Ser Leu Ser Phe Gln Asp Pro Lys Leu Glu Leu Gln Phe Pro

325 330 335

Arg Thr Pro Glu Gly Arg Arg Leu Gly Ser Leu Leu Pro Val Leu Ser

340 345 350

Pro Thr Ser Leu Pro Ser Pro Leu Pro Ala Thr Leu Glu Thr Pro Val

355 360 365

Pro Ala Phe Leu Lys Asn Gln Glu Phe Leu Gln Ala Arg Thr Pro Thr

370 375 380

Leu Ala Ser Thr Pro Ile Pro Pro Thr Pro Gln Ala Pro Ser Pro Ala

385 390 395 400

Val Asp Ala Glu Ile Arg Ala Gln Asp Ala Pro Leu Ser Leu Leu Gln

405 410 415

Thr Gln Gly Gly Arg Lys Gln Ala Pro Glu Pro Leu Arg Ala Glu Ala

420 425 430

Arg Val Ala Ile Pro Ala Ser Val Leu Pro Gly Pro Glu Glu Pro Gly

435 440 445

Gly Gln Arg Gln Glu Ala Ser Thr Gly Gln Ser Pro Glu Asp His Ala

450 455 460

Ser Leu Ala Pro Pro Leu Ser Pro Asp His Ser Ser Leu Glu Ala Lys

465 470 475 480

Asp Gly Glu Ser Gly Gly Ser Arg Val Phe Ser Ile Cys Arg Gly Glu

485 490 495

Gly Glu Gly Gln Ile Trp Gly Leu Val Glu Lys Glu Thr Ala Ile Glu

500 505 510

Gly Lys Val Val Ser Ser Leu Gln Gln Glu Ile Trp Glu Glu Glu Asp

515 520 525

Leu Asn Arg Lys Glu Ile Gln Asp Ser Gln Val Pro Leu Glu Lys Glu

530 535 540

Thr Leu Lys Ser Leu Gly Glu Glu Ile Gln Glu Ser Leu Lys Thr Leu

545 550 555 560

Glu Asn Gln Ser His Glu Thr Leu Glu Arg Glu Asn Gln Glu Cys Pro

565 570 575

Arg Ser Leu Glu Glu Asp Leu Glu Thr Leu Lys Ser Leu Glu Lys Glu

580 585 590

Asn Lys Glu Leu Leu Lys Asp Val Glu Val Val Arg Pro Leu Glu Lys

595 600 605

Glu Ala Val Gly Gln Leu Lys Pro Thr Gly Lys Glu Asp Thr Gln Thr

610 615 620

Leu Gln Ser Leu Gln Lys Glu Asn Gln Glu Leu Met Lys Ser Leu Glu

625 630 635 640

Gly Asn Leu Glu Thr Phe Leu Phe Pro Gly Thr Glu Asn Gln Glu Leu

645 650 655

Val Ser Ser Leu Gln Glu Asn Leu Glu Ser Leu Thr Ala Leu Glu Lys

660 665 670

Glu Asn Gln Glu Pro Leu Arg Ser Pro Glu Val Gly Asp Glu Glu Ala

675 680 685

Leu Arg Pro Leu Thr Lys Glu Asn Gln Glu Pro Leu Arg Ser Leu Glu

690 695 700

Asp Glu Asn Lys Glu Ala Phe Arg Ser Leu Glu Lys Glu Asn Gln Glu

705 710 715 720

Pro Leu Lys Thr Leu Glu Glu Glu Asp Gln Ser Ile Val Arg Pro Leu

725 730 735

Glu Thr Glu Asn His Lys Ser Leu Arg Ser Leu Glu Glu Gln Asp Gln

740 745 750

Glu Thr Leu Arg Thr Leu Glu Lys Glu Thr Gln Gln Arg Arg Arg Ser

755 760 765

Leu Gly Glu Gln Asp Gln Met Thr Leu Arg Pro Pro Glu Lys Val Asp

770 775 780

Leu Glu Pro Leu Lys Ser Leu Asp Gln Glu Ile Ala Arg Pro Leu Glu

785 790 795 800

Asn Glu Asn Gln Glu Phe Leu Lys Ser Leu Lys Glu Glu Ser Val Glu

805 810 815

Ala Val Lys Ser Leu Glu Thr Glu Ile Leu Glu Ser Leu Lys Ser Ala

820 825 830

Gly Gln Glu Asn Leu Glu Thr Leu Lys Ser Pro Glu Thr Gln Ala Pro

835 840 845

Leu Trp Thr Pro Glu Glu Ile Asn Gln Gly Ala Met Asn Pro Leu Glu

850 855 860

Lys Glu Ile Gln Glu Pro Leu Glu Ser Val Glu Val Asn Gln Glu Thr

865 870 875 880

Phe Arg Leu Leu Glu Glu Glu Asn Gln Glu Ser Leu Arg Ser Leu Gly

885 890 895

Ala Trp Asn Leu Glu Asn Leu Arg Ser Pro Glu Glu Val Asp Lys Glu

900 905 910

Ser Gln Arg Asn Leu Glu Glu Glu Glu Asn Leu Gly Lys Gly Glu Tyr

915 920 925

Gln Glu Ser Leu Arg Ser Leu Glu Glu Glu Gly Gln Glu Leu Pro Gln

930 935 940

Ser Ala Asp Val Gln Arg Trp Glu Asp Thr Val Glu Lys Asp Gln Glu

945 950 955 960

Leu Ala Gln Glu Ser Pro Pro Gly Met Ala Gly Val Glu Asn Glu Asp

965 970 975

Glu Ala Glu Leu Asn Leu Arg Glu Gln Asp Gly Phe Thr Gly Lys Glu

980 985 990

Glu Val Val Glu Gln Gly Glu Leu Asn Ala Thr Glu Glu Val Trp Ile

995 1000 1005

Pro Gly Glu Gly His Pro Glu Ser Pro Glu Pro Lys Glu Gln Arg

1010 1015 1020

Gly Leu Val Glu Gly Ala Ser Val Lys Gly Gly Ala Glu Gly Leu

1025 1030 1035

Gln Asp Pro Glu Gly Gln Ser Gln Gln Val Gly Ala Pro Gly Leu

1040 1045 1050

Gln Ala Pro Gln Gly Leu Pro Glu Ala Ile Glu Pro Leu Val Glu

1055 1060 1065

Asp Asp Val Ala Pro Gly Gly Asp Gln Ala Ser Pro Glu Val Met

1070 1075 1080

Leu Gly Ser Glu Pro Ala Met Gly Glu Ser Ala Ala Gly Ala Glu

1085 1090 1095

Pro Gly Pro Gly Gln Gly Val Gly Gly Leu Gly Asp Pro Gly His

1100 1105 1110

Leu Thr Arg Glu Glu Val Met Glu Pro Pro Leu Glu Glu Glu Ser

1115 1120 1125

Leu Glu Ala Lys Arg Val Gln Gly Leu Glu Gly Pro Arg Lys Asp

1130 1135 1140

Leu Glu Glu Ala Gly Gly Leu Gly Thr Glu Phe Ser Glu Leu Pro

1145 1150 1155

Gly Lys Ser Arg Asp Pro Trp Glu Pro Pro Arg Glu Gly Arg Glu

1160 1165 1170

Glu Ser Glu Ala Glu Ala Pro Arg Gly Ala Glu Glu Ala Phe Pro

1175 1180 1185

Ala Glu Thr Leu Gly His Thr Gly Ser Asp Ala Pro Ser Pro Trp

1190 1195 1200

Pro Leu Gly Ser Glu Glu Ala Glu Glu Asp Val Pro Pro Val Leu

1205 1210 1215

Val Ser Pro Ser Pro Thr Tyr Thr Pro Ile Leu Glu Asp Ala Pro

1220 1225 1230

Gly Pro Gln Pro Gln Ala Glu Gly Ser Gln Glu Ala Ser Trp Gly

1235 1240 1245

Val Gln Gly Arg Ala Glu Ala Leu Gly Lys Val Glu Ser Glu Gln

1250 1255 1260

Glu Glu Leu Gly Ser Gly Glu Ile Pro Glu Gly Pro Gln Glu Glu

1265 1270 1275

Gly Glu Glu Ser Arg Glu Glu Ser Glu Glu Asp Glu Leu Gly Glu

1280 1285 1290

Thr Leu Pro Asp Ser Thr Pro Leu Gly Phe Tyr Leu Arg Ser Pro

1295 1300 1305

Thr Ser Pro Arg Trp Asp Pro Thr Gly Glu Gln Arg Pro Pro Pro

1310 1315 1320

Gln Gly Glu Thr Gly Lys Glu Gly Trp Asp Pro Ala Val Leu Ala

1325 1330 1335

Ser Glu Gly Leu Glu Ala Pro Pro Ser Glu Lys Glu Glu Gly Glu

1340 1345 1350

Glu Gly Glu Glu Glu Cys Gly Arg Asp Ser Asp Leu Ser Glu Glu

1355 1360 1365

Phe Glu Asp Leu Gly Thr Glu Ala Pro Phe Leu Pro Gly Val Pro

1370 1375 1380

Gly Glu Val Ala Glu Pro Leu Gly Gln Val Pro Gln Leu Leu Leu

1385 1390 1395

Asp Pro Ala Ala Trp Asp Arg Asp Gly Glu Ser Asp Gly Phe Ala

1400 1405 1410

Asp Glu Glu Glu Ser Gly Glu Glu Gly Glu Glu Asp Gln Glu Glu

1415 1420 1425

Gly Arg Glu Pro Gly Ala Gly Arg Trp Gly Pro Gly Ser Ser Val

1430 1435 1440

Gly Ser Leu Gln Ala Leu Ser Ser Ser Gln Arg Gly Glu Phe Leu

1445 1450 1455

Glu Ser Asp Ser Val Ser Val Ser Val Pro Trp Asp Asp Ser Leu

1460 1465 1470

Arg Gly Ala Val Ala Gly Ala Pro Lys Thr Ala Leu Glu Thr Glu

1475 1480 1485

Ser Gln Asp Ser Ala Glu Pro Ser Gly Ser Glu Glu Glu Ser Asp

1490 1495 1500

Pro Val Ser Leu Glu Arg Glu Asp Lys Val Pro Gly Pro Leu Glu

1505 1510 1515

Ile Pro Ser Gly Met Glu Asp Ala Gly Pro Gly Ala Asp Ile Ile

1520 1525 1530

Gly Val Asn Gly Gln Gly Pro Asn Leu Glu Gly Lys Ser Gln His

1535 1540 1545

Val Asn Gly Gly Val Met Asn Gly Leu Glu Gln Ser Glu Glu Val

1550 1555 1560

Gly Gln Gly Met Pro Leu Val Ser Glu Gly Asp Arg Gly Ser Pro

1565 1570 1575

Phe Gln Glu Glu Glu Gly Ser Ala Leu Lys Thr Ser Trp Ala Gly

1580 1585 1590

Ala Pro Val His Leu Gly Gln Gly Gln Phe Leu Lys Phe Thr Gln

1595 1600 1605

Arg Glu Gly Asp Arg Glu Ser Trp Ser Ser Gly Glu Asp

1610 1615 1620

Claims (10)

1. a kind of method of nestin gene expression in regulation neural stem cell, which is characterized in that as follows including what is successively carried out Step: first designing the gRNA of nestin gene near the transcription initiation of nestin gene, then carry out the building of carrier, described The gRNA of nestin gene can be the nestin-i gRNA of guidance Transcription inhibition and the nestin-a of guidance transcription activator Any one in gRNA.

2. the method for nestin gene expression in regulation neural stem cell according to claim 1, which is characterized in that described For the sequence of nestin-i gRNA as shown in sequence 1-12, the sequence of the nestin-a gRNA is described as shown in sequence 13-24 The sequence of nestin-i gRNA can be as shown in sequence 1-12 the combination of any one or more, the nestin-a The sequence of gRNA can be as shown in sequence 13-24 the combination of any one or more.

3. the method for nestin gene expression in regulation neural stem cell according to claim 1, which is characterized in that described The sequence that targets of nestin-i gRNA is located within the scope of First Exon transcription initiation site downstream 1-112bp, described The sequence that targets of nestin-a gRNA is located within the scope of First Exon transcription initiation site upstream 1-450bp.

4. the method for nestin gene expression in regulation neural stem cell according to claim 1, which is characterized in that also wrap It includes and carries out viral packaging using 293T cell, specifically include and first 293T cell is carried out evenly dispersed, PEI mixed liquor is added to Plasmid-PEI mixed liquor is made in plasmid mixed liquor, then plasmid-PEI mixed liquor is added in the culture medium containing 239T cell altogether Virus is collected in culture, concentration.

5. the method for nestin gene expression in regulation neural stem cell according to claim 4, which is characterized in that use 293T cell carries out the infection and screening of progress cell after viral packaging, specifically comprises the following steps: first to prepare viral mixed liquor, The virus liquid mixed liquor prepared is added into the good neural stem cell of growth conditions again, co-cultures.

6. the method for nestin gene expression in regulation neural stem cell according to claim 5, which is characterized in that cell Infection after carry out respectively nes gene expression detection and NESTIN protein level detection, wherein the table of nes gene The primer of the detection nes gene used up to horizontal detection is as shown in sequence 26-27.

7. the method for nestin gene expression in regulation neural stem cell according to claim 1, which is characterized in that described Method includes nestin gene in nestin gene in overexpression neural stem cell or silencing neural stem cell.

8. a kind of supporting molecular, which is characterized in that it include the gRNA of nestin gene, the sequence of the gRNA of the nestin gene Column any one or a variety of combinations as shown in sequence 1-24.

9. a kind of cytotropic method of target, which is characterized in that including contacting cell with supporting molecular, the supporting molecular includes There is the gRNA of nestin gene, the supporting molecular includes the dCas9 of catalytically inactive structural domain, and the sequence of the dCas9 is such as Shown in sequence 25, for the sequence of the gRNA of the nestin gene as shown in sequence 1-24, the cell is neural stem cell, newborn Any one in vascular endothelial cell and middle interstitial cell.

10. the cytotropic method of target according to claim 9, the supporting molecular also includes antibiotic, the antibiosis Element is puromycin, and hygromycin, roxithromycin, spiramvcin, azithromycin, clarithromycin, vancomycin, streptomysin, celebrating is greatly Mycin, tetracycline, Doxycycline, minocycline, kanamycins, in ampicillin and chloramphenicol any one or more Combination；Preferably, the antibiotic is puromycin, hygromycin.