CN103937745B - People A375 stable cell strain and the construction method of long-chain non-coding GAS5 defect - Google Patents

Abstract

The invention belongs to Medical Molecular Biology field. the people A375 stable cell strain of long-chain non-coding GAS5 defect of the present invention is that F chain 5 '-GATCCTGGCACAAGAAGATTAAAACTCGAGTTTTAATCTTCTTGTGCCATTTTTG-synthetic design 3 ' and R chain 5 '-AATTCAAAAATGGCACAAGAATATTAAAACTCGAGTTTTAATCTTCTTGTGCCAG-3 ' are formed to DNA double chain through annealing, then insert in slow virus carrier pLVX-shRNA2, infect again A375 cell, this DNA double chain is incorporated in the genome of A375 cell, target strikes and subtracts A375 cell endogenous GAS5 and express more than 90% stable cell strain. compared with traditional antisense technology, the present invention has the advantages such as high specificity, with strong points, cascade amplification.

Description

People A375 stable cell strain and the construction method of long-chain non-coding GAS5 defect

Technical field

The invention belongs to Medical Molecular Biology field.

Background technology

The protein coding gene of 20,000 left and right of the mankind only accounts for whole genomic 2%, and more than 90% peopleGenoid group all occurred to transcribe, and this just means that a considerable amount of transcripts are coded proteins not,This part transcript is called as " non-coding RNA " (non-codingRNA, ncRNA). They are considered to beforeTranscribe " noise ", but increasing research shows that ncRNA is in processes such as Growth of Cells, differentiation and metabolismMiddlely bringing into play important biological function. Be divided into two classes according to the length of ncRNA, length is at 200 coresBelow thuja acid, be called short chain non-coding RNA, length more than nucleotides is referred to as the non-volume of long-chain at 200Code RNA (longnon-codingRNA, lncRNA). Short chain non-coding RNA comprises that length is at 21-23ntBetween Microrna (microRNAormiRNA), small molecules interference RNA (smallinterferenceRNA,SiRNA), microRNA (smallnucleolarRNA, snoRNA) and mutually doing with piwi albumen in coreWith RNA (piwi-interactingRNAs, piRNAs) etc. The research of lncRNA is relatively less, but nearOver year, find that lncRNA extensively joins in epigenetics level, transcriptional level, post-transcriptional level and translation skillRegulation and control with gene expression. LncRNA except with systemic loupus erythematosus, Alzheimer and cardiovascular and cerebrovascular diseaseOutside the Pass the sick generation development mutually that waits various diseases, also close with being related of kinds of tumors, wherein partLncRNA and melanomatous generation development are in close relations.

Long-chain non-coding RNA retarded growth specific transcriptional basis 5 (GrowthArrestSpecifictranscript5,GAS5) the assignment of genes gene mapping, in 1q25.1, comprises 12 extrons and 11 intrones, and its full length gene is4087bp, ripe GAS5 transcript is 651nt. GAS5 is to growth the earliest research in 1988While blocking responsive gene, find, the expression of this transcript has tissue specificity, at the cell of active growthMiddle expression is extremely low, but obviously raises in the cells level of retarded growth. In recent years find GAS5The retarded growth under normal circumstances of regulation and control T lymphocyte, and at mammal rapamycin target body albumenThe T lymphocyte differentiation process of inhibition that (mammaliantargetofRapamycin, mTOR) antagonist producesIn, need to rely on the participation of GAS5. Mourtada etc. think that GAS5 performance function is mainly by its introneMicroRNA (smallnucleolarRNA, snoRNA) in the core of coding. GAS5 has different transcribingThis, different transcripts function in different cell lines differs greatly, GAS51B, GAS52B wherein,GAS53A, GAS54A can increase the sensitiveness of cell to apoptosis after crossing in different clone and expressing,GAS5-01 is starkly lower than other transcripts in breast cancer.

Still do not find that there is at present the document that closes long-chain non-coding RNA GAS5 and melanoma generation development relationReport.

Melanoma is the malignant tumour that betides dermal melanin cell, and grade malignancy is high, and poor prognosis is sent outSick rate presents rising trend in the world. The data of issuing according to American Cancer Society 2013, greatlyIn the situation that most of malignant tumor incidence and the death rate all decline, the melanomatous incidence of disease still but presentsThe trend of liter, annual growth is between 2.5%-3.9%. Annual melanoma new cases 199627 examples in the whole world,Dead number of cases is 46372 examples. Although melanoma is lower at China's incidence of disease, be doubled and redoubled in recent years,2000 annual morbidity statistics are only 0.2/10 ten thousand, 2005-2007 China incidence of disease approximately 1,/10 ten thousand, new every yearMorbidity example approximately 20,000 people. Melanoma has become one of disease seriously jeopardizing China's people ' s health. Although haveScholar proposes, and melanomatous is the coefficient result of nature-nurture, as easy formation mole andToo much be exposed to sunlight inferior, still the molecular mechanism of morbidity is illustrated not yet, understands melanoma in depth and occurs to send outExhibition mechanism and the current problem demanding prompt solution of the effective methods for the treatment of of searching.

Summary of the invention

Object of the present invention aims to provide a kind of people A375 stable cell strain of long-chain non-coding GAS5 defect, doesFor model is for studying the correlation of GAS5 and tumour and possible mechanism thereof.

Another object of the present invention is to provide the construction method of this cell line.

The people A375 stable cell strain of long-chain non-coding GAS5 defect of the present invention is by F synthetic designChain 5 '-GATCCTGGCACAAGAAGATTAAAACTCGAGTTTTAATCTTCTTGTGCCATTTTTG-3 'With R chain

5’-AATTCAAAAATGGCACAAGAATATTAAAACTCGAGTTTTAATCTTCTTGTGCCAG-3’Form DNA double chain through annealing, then insert in slow virus carrier pLVX-shRNA2, then it is thin to infect A375Born of the same parents, are incorporated in the genome of A375 cell this DNA double chain, and target strikes that to subtract A375 cell endogenousProperty GAS5 expresses more than 90% stable cell strain.

Above-mentioned A375 cell line is purchased from Shanghai Inst. of Life Science, CAS cell resource center. This is thinBorn of the same parents' strain derives from the women of 54 years old, is the strain in a series of cell lines of setting up of the people such as D.J.Giard.This cell belongs to epithelial cell, and adherent growth has oncogenicity, and it is in ATG (immunosupressAgent), form the hypodermic tumour of the rapid growth that is similar to chromoma in the NIH Switzerland mouse of serum processing.Show that through CYTOGENETIC ANALYSIS OF ONE this cell is to contain 62 chromosomal hypo-triploid cells, wherein 9 marksChromosome, the STR site on its DNA has: Amelogenin:X; CSF1PO:11,12; D13S317:11,14;D16S539:9; D5S818:12; D7S820:9; THO1:8; TPOX:8,10 and vWA:16,17. Of the present inventionThe people A375 stable cell strain of long-chain non-coding GAS5 defect be that above-mentioned DNA double chain fragment is inserted and containedThe pLVX-shRNA2 expression vector of ZsGreen1 green fluorescence protein gene, then proceed to A375 cell line,Cells siRNA, through positive cell clone screening, the quantitative detection display siRNA of qRT-PCR target is heavyThe expression of endogenous GAS5 of having write from memory reaches more than 90%, and through the cultivation of repeatedly going down to posterity, fluorescence intensity is stable and disturbEfficiency does not reduce.

The construction method of the people A375 stable cell strain of long-chain non-coding GAS5 defect of the present invention by belowStep composition:

One, shRNA sequences Design is with synthetic

Interference sequence is by the online programming of TBI, according to interference sequence design complementary series, through adding BamHIBe prepared into targeted human with loop structure hair fastener ring and the protectiveness base of EcoRI restriction enzyme site, 6 nucleotidesThe F chain of the shRNA of GAS5, according to the synthetic R chain of base complementrity pairing;

Two, build shRNA-Lentiviral

The shRNAF chain designing and R chain strand equal-volume are mixed, boil, after annealing, form double-stranded;BamHI and EcoRI double digestion slow virus carrier pLVX-shRNA2 make it to become linear plasmid, and shRNA is twoChain and linear plasmid are connected to form recombinant plasmid, recombinant plasmid transformed E.coli under the effect of T4DNA ligaseDH5 α competence bacterium, containing the dull and stereotyped coating of LB solid medium of ampicillin, overnight incubation, selects sunProperty bacterium colony amplification cultivation, extract plasmid order-checking qualification, shRNA-Lentiviral successfully constructs;

Three, slow virus packaging and production

Virus packaging plasmid and recombinant plasmid pLVX-shRNA2-GAS5-shRNA pass through lipofectamine2000Cotransfection 293T cell, takes out culture dish after 6h, discard culture medium, and 1 × PBS cleans cell 3 times, softGround adds the 293T culture medium that 8ml is fresh, continues to cultivate; After cell transfecting 36h, collect culture medium supernatantLiquid is as slow virus liquid, and every 3h collects once afterwards, collects 3-4 time altogether; Each viral supernatant of collecting is usedThe syringe needle filter of 0.45 μ m filters, to remove 293T cell;

Four, slow-virus infection A375 cell

Determine that according to preliminary experiment the required infection multiplicity of cell and virus, on cell state and the impact of going down to posterity, infect multipleNumber is defined as the ratio of viral while infection and cell quantity, before virus infected cell, uses trypsinization in growthThe A375 cell of logarithmic phase, the centrifugal rear culture medium re-suspended cell with antibiotic-free, and carry out cell count; Get5×10⁵Individual A375 cell is inoculated in culture dish, after cell attachment, in culture dish, adds after the bath of 2ml temperatureViral supernatant, adding final concentration is the polybrene Polybrane of 2 μ g/ml; Cultivate 3h, discard culture medium,Change 2ml virus supernatant and continue to cultivate, repeat 2 times;

Five, A375-GAS5 △ stable cell strain screening and identification

A375 cell infects after 3 times through viral supernatant, to the culture medium that adds antibiotic-free in culture dish, trainingSupport after 48h, discard culture medium, use instead containing the culture medium of ampicillin Ampicillin and screen positive cell;The next day observation of cell, discard former culture medium and dead cell, 1 × PBS cleans cell 3 times, changes resistance culture baseThe cultured cell of sobbing, continues screening 7 days; Treat that cell covers with, vitellophag, is inoculated in 10mm culture dishAmplification cultivation, goes down to posterity 3 times; Screening is placed on fluorescence microscopy Microscopic observation, and all cells all presents highlightedGreen fluorescence; Further row qRT-PCR detects the expression of GAS5, and result shows A375-GAS5 △ cell lineThe expression of middle GAS5 has declined 93.74% compared with A375 cell, and jamming effectiveness reaches expection, long-chain non-codingThe people A375 stable cell strain of GAS5 defect successfully constructs.

In domestic and foreign literature, there is not yet at present Humanmachine tumour A375 stable cell strain and the phase thereof of GAS5 defectClose the report of research. The table of the reticent A375 cell of the application's application shRNA-slow virus system endogenous GAS5Reach, built the A375 stable cell strain (A375-GAS5 △) of GAS5 defect, and table to its GAS5Reach and detect. This model can be used for probing into correlation and the machine thereof of long-chain non-coding RNA GAS5 and tumourReason; For the mechanism of further investigation long-chain non-coding RNA GAS5 and melanoma generation development is established materialBasis.

The method of the A375 cell of the GAS5 defect that the application builds is different from prior art. RNAi on the one handTechnology is the gene silent technology that fast development is got up in recent years, and its mechanism is: in biological cell,Exogenous or endogenic double-stranded RNA (double-strandedRNA, dsRNA) causes homologous mRNASpecific explanation, thereby the technology of inhibition destination gene expression. Compared with traditional antisense technology, toolThere are the advantages such as high specificity, with strong points, cascade amplification. On the other hand, by buildingPLVX-shRNA2-GAS5-shRNA recombinant vector also generates slow virus particle infection A375 cell, will carryThe DNA profiling of GAS5-shRNA is incorporated into A375 cellular genome with viral genome. Due toPLVX-shRNA2 carrier has U6 promoter and can start the expression of GAS5-shRNA, GAS5-shRNA fromFigure becomes hairpin structure, and the GAS5-shRNA expressing in nucleus is transported to the work at Dicer enzyme after cytoplasmWith under be cut into GAS5-siRNA, via the compound-mediated reticent target gene specifically of RISC. Institute is usedPLVX-shRNA2 carrier there is amicillin resistance, can be used for monoclonal cell and select.

Brief description of the drawings

Fig. 1 is the physical map of plasmid pLVX-shRNA2.

Fig. 2 is the sequencing result figure of interference sequence.

Fig. 3 is that qRT-PCR detects jamming effectiveness result figure.

Fig. 4 be the micro-Figure 40 of virus infected cell ×.

Fig. 5 is the expression that qRT-PCR detects GAS5 in stable cell strain.

Detailed description of the invention

1. select the synthetic shRNA template DNA of interference carrier and design

Choose PLVX-shRNA2 as interference carrier, 3 interference sequences of GAS5 (NR_002578.2) and nothingClose control sequence by TBI online database design (http://RNA.tbi.univie.ac.at/cgi-bin/RNAxs), warpAdd the F chain that restriction enzyme site, hair fastener ring and protectiveness base are prepared into the siRNA of targeted human GAS5, foundationBase complementrity pair principle synthesizes R chain.

2.shRNA template DNA insertion vector

After equal-volume mixes respectively with R chain by the F chain of the GAS5shRNA designing, in boiling water bath, boil 5Min, then 72 DEG C keep 15min, naturally cool to room temperature, and about 60min is for subsequent use. Carrier double digestion,At aseptic 0.2mLEP reaction tube, get pLVX-shRNA2 carrier 15 μ L, with BamHI and EcoRIDouble digestion, enzyme is cut system and is prepared to specifications, after mixing, more than 37 DEG C of reaction 4h, solidifying with DNAGlue reclaims kit recovery enzyme and cuts product, finally obtains the DNA eluting. By the GAS5 preparingShRNA1, the each 4 μ L of 2,3 fragments and carrier 1 μ L, and T4DNALigase etc. is equipped with reaction system 16DEG C connect 2h, as shown in Figure 1.

3. restructuring interference carrier transforms DH5 α competence bacterium qualification

Connect the conversion of product, in ice bath, 5 μ L are connected to product and be added to respectively 50 μ LDH5 α competent cellsIn, rotation mixes gently, ice bath 30min, and 42 DEG C of water-bath heat shock 90min, are transferred to ice by pipe fastIn bath, after ice bath 2min, add 200 μ LLB culture mediums, mix, 37 DEG C, 200rpm shaken cultivation 1h,In superclean bench, bacterium liquid is evenly coated to the LB flat board containing ampicillin (Amp) (100 μ g/mL)Upper, under room temperature, place, be inverted flat board, be transferred to 37 DEG C of biochemical cultivation case incubated overnight. Observe LB next dayCulture medium turbidity, gets 600 μ l bacterium liquid in super-clean bench, add 280 μ l glycerine to mix ,-20 DEG C of preservations, all the otherBacterium liquid adopts with the high pure plasmid of purification column and extracts in a small amount kit, to specifications operation. Weight after extractionGroup interference carrier send Hua Da gene to check order, and order-checking structure shows construction of recombinant vector success, as shown in Figure 2.

4. slow virus packaging and production

293T cell, through recovery, goes down to posterity, and can be used for transfection in the time that cell density reaches 70%-80%. Before transfection2 hours, cell culture medium is replaced by without dual anti-complete medium to prepare dna precipitated liquid. By 40 μ LLipofectamine2000 and 1.46ml complete medium join in 1.5mlEP pipe and (are labeled as A pipe);In another 1.5mlEP pipe (being labeled as B pipe), add DNA precipitated liquid, recombinant plasmid and packaging plasmid are altogether1.5ml slow virus packaging plasmid (pGag/Pol, pRev, pVSV-G) mixes according to 1:1:1 ratio. B is managedIn DNA precipitated liquid gently dropwise join in A pipe, mix gently. Leave standstill after a period of time in room temperature,Precipitation mixture is dropwise evenly joined in 10cm Tissue Culture Dish, rock gently, put into 37 DEG C, 5%CO2In incubator, cultivate. Transfection morning next day, discards old culture medium, and every ware adds the fresh complete medium of 10ml (to contain1% is dual anti-). Collect virus, collect respectively transfection 24h and 48h containing Virus culture supernatant, 1000rpmCentrifugal 5min, supernatant filters with 0.45 μ m syringe needle filter, to remove 293T cell; 4 DEG C, 50000g are highThe centrifugal 2h of speed, abandons supernatant, and virus precipitation is fully dissolved with 1mlPBS, then uses 0.22 μ m syringe needle filterFilter, (general 0.5 μ of bacterium diameter m), takes out-80 DEG C of preservations of part virus and gives over to remove possible bacteriumAfter use.

5. slow-virus infection A375 cell

Determine infection multiplicity according to preliminary experiment: prepare 2 aseptic 1.5mLEp pipes, in each pipe, add 45The conventional medium of μ L, draws 1 × 10 of 5 μ L⁸The virus (melting on ice from-80 DEG C of taking-ups in advance) of TU/mLJoin in first pipe, softly mix, do not make to produce foam. The same virus of drawing 5 μ L from the first pipeIn second pipe, mix, the virus titer in first pipe is 1 × 10⁷TU/mL, and in second pipeTitre is 1 × 10⁶TU/mL; The virus of tri-different gradients of 10 μ L is added in the respective aperture of each group. AddVirus quantity is respectively 1 × 10⁶TU、1×10⁵TU、1×10⁴TU, the number of cell is 1 × 10⁴Individual, three holesMOI be respectively 100,10,1; Pat gently in the horizontal direction culture plate, make the reagent such as culture medium and virusFully mix, then cell plates are put back to incubator and hatch; Cultivate 8～12h observation of cell state later, discardCell conditioned medium liquid, is replaced by fresh culture. Infect after 2～3 days, observe luciferase expression situation, by observingCell infection effect, confirms that the best MOI of infection of object cell is 100. Getting density is 1 × 10⁶The A375 of/mlCell 5ml, adding titre is 1 × 10⁸The virus 5 ml of TU/mL, puts back to CO after fully mixing₂Incubator is hatched;After 12 hours, change fresh culture; After 72 hours, observe luciferase expression situation, find that most cells presentGreen fluorescence.

Note: MOI: be the abbreviation of " MultiplicityofInfection ", Chinese is that infection multiplicity or multiple infection refer toNumber, the ratio of virus and cell quantity when implication is infection.

6. the jamming effectiveness of 3 recombinant vectors after fluorescence quantitative PCR detection transient transfection

In cell, total RNA extracts: turn rear cell wink and be incubated in 6 orifice plates, when cell degree of converging reaches approximately 80%Time, discard culture medium, add 1 × PBS solution 1ml of precooling to clean cell 1 time, add RNAisoPlus1ml/well, the piping and druming of rifle head, makes the abundant cracking of cell; Room temperature is transferred to 1.5mlEP pipe (DEPC after placing 5minProcess) in, add chloroform 0.2ml, firmly jolting 15s. Room temperature leaves standstill after 5min, centrifugal (4 DEG C, 12000rpm,15min). Centrifugal rear liquid is divided into three layers (colourless supernatant, the red organic layers of middle level white protein layer and bottom).The careful supernatant of drawing is also transferred in another EP pipe, adds equal-volume isopropyl alcohol, mixes, quiet at-20 DEG CPut 30min, centrifugal (4 DEG C, 12000rpm, 10min). Abandoning supernatant, adds 1ml75% ethanol, gentlenessVibration suspends and precipitates, and centrifugal (4 DEG C, 12000rpm, 5min) discard ethanol as far as possible, are deposited in super-clean bench in pipeMiddle air blast standing and drying 3-5min. Rear every pipe adds 20-50 μ l water (DEPC processing) to dissolve.

Reverse transcription synthesizes cDNA: get total RNA2ng-2 μ g, reverse with ReverseTranscriptaseM-MLVRecord enzyme, by the synthetic cDNA of explanation. Concrete steps: (concentration is first to add 2 μ l random primers in total RNA25 μ M), add water (DEPC processing) complements to 12 μ l, mix, after 70 DEG C of incubation 10min, ice bath chilling 2min.Add successively RNA enzyme inhibitor 0.5 μ l, 5 × M-MLVBuffer4 μ l, RTaseM-MLV (200U/ μ l) 0.5μ l, dNTPMixture (each 10mM) 1 μ l, add water (DEPC processing) complements to 20 μ l, 30 DEG C of reaction 10min,42 DEG C of insulation 1h again, cooled on ice after last 70 DEG C of insulation 15min deactivation reverse transcriptase.

Quantitative fluorescent PCR: by specification configuration reaction system, upper machine carries out pcr amplification and detection. Reaction is totalSystem is 20 μ l, specifically 2 × MixSYBRGreenI fluorescence reaction liquid, 10 μ l, upstream and downstream primer (10 μ M)Each 0.25 μ l, sample template 1 μ l, supplies 20 μ l systems with aqua sterilisa. 95 DEG C of 2min denaturations of reaction condition,The interior 95 DEG C of 30s sex change that circulate, 60 DEG C of annealing 35s, PCR reaction arranges 40 circulations, and in each circulationExtend end and collect fluorescence signal, draw amplification curve. After 40 circulations, arrange (95 DEG C of 15s, 60 DEG C of 30s,95 DEG C of 15s) reactions steps, and 60 DEG C to 95 DEG C temperature-rise periods are carried out to omnidistance fluorescence signal collection, drawMelt curve analysis. Carry out data analysis with Bio-RadCFXManagerversion1.6 software, as shown in Figure 3.

7. the A375-GAS5 △ stable cell strain of the screening of restructuring interference carrier jamming effectiveness and defect GAS5 builds

Filter out according to fluorescence quantitative PCR detection result virus corresponding to recombinant vector that jamming effectiveness is the highestGrain carries out follow-up test. By repeated multiple times this virion infection A375 cell, concrete operations are: A375-WTCell infects after 3 times through viral supernatant, in culture dish, adds the DMEM (10%FBS) of antibiotic-free to trainSupport base, 37 DEG C, under 5%CO2 condition, cultivate after 48h, discard culture medium, change with Ampicillin (0.5μ g/ml) medium culture with screening positive clone. In the time that cell covers with, vitellophag, is inoculated in 10mmAmplification cultivation in culture dish, the next day observation of cell, discard former culture medium and dead cell, 1 × PBS cleans cell 3Inferior, change DMEM (10%FBS, 0.5 μ g/mlAmpicillin) culture medium and continue cultured cell, go down to posterity 3 timesAfter in fluorescence microscopy Microscopic observation, under the visual field all cells all with green fluorescence, the success of prompting slow virus carrierInfect A375 cell, as shown in Figure 4.

The screening of A375-GAS5 △ stable cell strain and the qualification of 8.GAS5 defect

Quantitative fluorescent PCR qualification A375-GAS5 △ stable cell strain: fluorescence quantitative PCR detectionA375-GAS5 △ surely turns GAS5 △ mrna expression level in cell. Total RNA extracts, reverse transcription is syntheticCDNA and quantitative fluorescent PCR process are the same, as shown in Figure 5.

F chain 5 '-GATCCTGGCACAAGAAGATTAAAACTCGAGTTTTAATCTTCTTGTGCCATTTTTG-3 '

R chain 5 '-AATTCAAAAATGGCACAAGAATATTAAAACTCGAGTTTTAATCTTCTTGTGCCAG-3 '

Claims (2)

1. a people A375 stable cell strain for long-chain non-coding GAS5 defect, it is characterized in that design to closeThe F chain becoming

5’-GATCCTGGCACAAGAAGATTAAAACTCGAGTTTTAATCTTCTTGTGCCATTTTTG-3 ' and R chain

5’-AATTCAAAAATGGCACAAGAATATTAAAACTCGAGTTTTAATCTTCTTGTGCCAG-3 ' forms DNA double chain through annealing, then inserts in slow virus carrier pLVX-shRNA2, thenInfect A375 cell, this DNA double chain is incorporated in the genome of A375 cell, target strikes and subtractsA375 cell endogenous GAS5 expresses more than 90% stable cell strain.

2. the structure side of the people A375 stable cell strain of long-chain non-coding GAS5 defect as claimed in claim 1Method, is characterized in that being made up of following steps:

One, shRNA sequences Design is with synthetic

Interference sequence is by the online programming of TBI, according to interference sequence design complementary series, through adding BamHIBe prepared into targeted human with loop structure hair fastener ring and the protectiveness base of EcoRI restriction enzyme site, 6 nucleotidesThe F chain of the shRNA of GAS5, according to the synthetic R chain of base complementrity pairing;

Two, build shRNA-Lentiviral

The shRNAF chain designing and R chain strand equal-volume are mixed, boil, after annealing, form double-stranded;BamHI and EcoRI double digestion slow virus carrier pLVX-shRNA2 make it to become linear plasmid, shRNADouble-stranded and linear plasmid is connected to form recombinant plasmid, recombinant plasmid transformed under the effect of T4DNA ligaseE.coliDH5 α competence bacterium, containing the dull and stereotyped coating of LB solid medium of ampicillin, overnight incubation,Select positive bacterium colony amplification cultivation, extract plasmid order-checking qualification, shRNA-Lentiviral successfully constructs;

Three, slow virus packaging and production

Virus packaging plasmid and recombinant plasmid pLVX-shRNA2-GAS5-shRNA pass throughLipofectamine2000 cotransfection 293T cell, takes out culture dish after 6h, discard culture medium, and 1 × PBS is clearWash cell 3 times, gently add the 293T culture medium that 8ml is fresh, continue to cultivate; After cell transfecting 36h,Collect medium supernatant as slow virus liquid, every 3h collects once afterwards, collects 3-4 time altogether; Each receiptsThe viral supernatant of collection filters with the syringe needle filter of 0.45 μ m, to remove 293T cell;

Four, slow-virus infection A375 cell

Determine that according to preliminary experiment the required infection multiplicity of cell and virus, on cell state and the impact of going down to posterity, infectPlural number is defined as the ratio of viral while infection and cell quantity, before virus infected cell, uses trypsinization in lifeThe A375 cell of long logarithmic phase, the centrifugal rear culture medium re-suspended cell with antibiotic-free, and carry out cell count;Get 5 × 10⁵Individual A375 cell is inoculated in culture dish, after cell attachment, in culture dish, adds 2ml temperatureViral supernatant after bath, adding final concentration is the polybrene Polybrane of 2 μ g/ml; Cultivate 3h, discardCulture medium, changes 2ml virus supernatant and continues to cultivate, and repeats 2 times;

Five, A375-GAS5 △ stable cell strain screening and identification

A375 cell infects after 3 times through viral supernatant, to the culture medium that adds antibiotic-free in culture dish,Cultivate after 48h, discard culture medium, use instead containing culture medium the screening of ampicillin Ampicillin positive thinBorn of the same parents; The next day observation of cell, discard former culture medium and dead cell, 1 × PBS cleans cell 3 times, changes resistance trainingSupport base and continue cultured cell, continue screening 7 days; Treat that cell covers with, vitellophag, is inoculated in 10mm trainingSupport amplification cultivation in ware, go down to posterity 3 times; Screening is placed on fluorescence microscopy Microscopic observation, and all cells all presentsHighlighted green fluorescence; Further row qRT-PCR detects the expression of GAS5, and result shows A375-GAS5 △In cell line, the expression of GAS5 has declined 93.74% compared with A375 cell, and jamming effectiveness reaches expection, longThe people A375 stable cell strain of chain non-coding GAS5 defect successfully constructs.