CN103911346A - Method of knocking out spinal muscular atrophy SMN genes and cell model - Google Patents

Method of knocking out spinal muscular atrophy SMN genes and cell model Download PDF

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Publication number
CN103911346A
CN103911346A CN201410120625.1A CN201410120625A CN103911346A CN 103911346 A CN103911346 A CN 103911346A CN 201410120625 A CN201410120625 A CN 201410120625A CN 103911346 A CN103911346 A CN 103911346A
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smn
cell
gene knockout
smn gene
zfn
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CN103911346B (en
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陈实
姜东旭
黎寒
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JIANGSU XIONGMING MEDICAL TECHNOLOGY Co Ltd
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JIANGSU XIONGMING MEDICAL TECHNOLOGY Co Ltd
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Abstract

The invention relates to a method of knocking out spinal muscular atrophy SMN genes and a cell model. The cell model is constructed by adopting the following steps: (1) constructing and designing zinc finger nuclease plasmids for knocking out the SMN genes; (2) transferring the constructed ZFN plasmids for knocking out the SMN genes into mammalian cells by using a lipidosome method; (3) detecting the effectiveness of the ZFN gene knockout by using a genome PCR product of a ZFN peculiar knockout site and DNA sequence determination; (4) specific to the cells, the ZFN genes of which are effectively knocked out, carrying out monoclonal separation and identifying cell knockout types one by one; (5) carrying out mRNA level identification on gene knockout cell strains; (6) carrying out protein expression level identification on the gene knockout cell strains. The spinal muscular atrophy pathology cell model can be used for research of SMA pathogenesis; medicines can be screened by detecting the level of protein expression of SMN compounds in the cell model.

Description

A kind of knockout technique and cell model of spinal muscular atrophy SMN gene
Technical field
The present invention relates to genetically engineered field, be specifically related to a kind of knockout technique and cell model of spinal muscular atrophy SMN gene.
Background technology
Spinal cord muscular dystrophy (Spinal Muscular Atrophy, SMA) be a kind of neuromuscular disease of autosomal recessive inheritance, pathological characters is shown as anterior horn motor neurons sex change, unable and the atrophy of limbs of patient near-end and muscle of trunk, finally causes respiratory insufficiency even dead.Its sickness rate in life birth baby is 1/6000 ~ 1/10000, and Population carriage is 1/40 ~ 1/60, occupies the second of lethality autosomal recessive inheritance disease, is only second to cystic fibrosis.The generation of this disease, brings huge body and mind misery to patient, also brings white elephant to family simultaneously.
Along with molecular biology and genetic development, research is now found: motor neuron survival (SMN) gene is the main Disease-causing gene of SMA.This assignment of genes gene mapping is in 5q11.2-13.3.In human chromosomal group, SMN gene has multiple copies: a SMN1(is positioned at telomere side SMN) and multiple SMN2(kinetochore side SMN).The total length transcript fl-SMN of the SMN1 gene a kind of full length protein that contains 294 amino acid whose function-stables (smn protein) of can encoding.And SMN2 exists the variation of (840C → T) of a single base compared with SMN1 to recently seeing coding region, this change causes the transcript forming that optionally jumps of cell shearing mechanism to lack 7 exons, form the mRNA isomer " Δ 7SMN " of a brachymemma, " the truncated protein matter " that coding does not have biological function and degrades rapidly, only have the SMN2 premessenger RNA of 10%-20% that normal expression SMN (SMN-fl) albumen, this compensating action that is SMN2 sheared occurs.Think at present, it is the basic reason that causes SMA morbidity that SMN1 gene alteration causes smn protein horizontal down-regulation, and SMN2 is generally considered the modifying factor of SMA, and its copy number is relevant to the severity of disease with montage efficiency.Approximately 95% patient causes SMN1 disappearance for SMN1 homozygous deletion or SMN1 are converted into SMN2.
Suffer from SMA disease for why causing ventricornu ɑ motor neuron sex change after SMN1 genetically deficient, mechanism is still not clear at present.And that SMA transgenic models is made so far technology is immature and spend huge.Therefore, knock out SMN1 gene if energy applying gene knocks out technology specificity, external structure SMA pathological cells model, can be the SMA cause of disease and pathogenetic further investigation and the new platform of high flux screening treatment SMA drug provision.
Zinc finger nuclease (zinc finger nuclease, ZFN) be a kind of gene Knockout of rising in recent years, it is a kind of engineered endonuclease, formed by an artificial zinc finger dna differential threshold and a non-specific endonuclease, zinc fingers can be specifically in conjunction with DNA sequence dna, each zinc refers to identify three continuous DNA bases, and non-specific endonuclease is exercised shearing function, and both are in conjunction with just fixing a point to rupture at DNA specific site.The non-specific endonuclease being connected with zinc finger protein group is sheared territory from the DNA of 96 amino-acid residue compositions of FokI carboxyl terminal.FokI is a kind of restriction enzyme from Flavobacterium okeanokoites, only in the time of dimer state, just there is enzyme to cut activity, each FokI monomer is connected with a zinc finger protein group and forms a ZFN, identify specific site, when two recognition sites are during at a distance of appropriate distance (6 ~ 8 bp), two monomer ZFN interactions produce enzyme and cut function, thereby reach DNA target cutting object.For 8 ~ 10 Zinc finger domains of target sequence design, these zinc structural domains are connected on DNA nuclease, just can realize the double-strand break (Double strand break, DSB) of target sequence.When two ZFN cutting target sites, after producing double-strand break, the repair mechanism of cell is activated, and comprises homologous recombination repair and non-homologous end joining (Non-homologous end-joining, NHEJ).Be that nonhomologous end connects if DNA repairs, will have about 70% probability by deleting at random or adding the base that can cause phase shift mutation, or nonsense mutation causes protein length variations, thereby cause gene knockout.Up to the present, show from the pertinent data retrieval including Chinese patent, utilize ZFNs technology to knock out SMN gene, set up the cell model of SMA disease, there is not yet relevant report.
Summary of the invention
The object of this invention is to provide a kind of for by the Zinc finger nuclease of spinal muscular atrophy SMN gene knockout, and plasmid vector, and the cell model obtaining.The present invention's application ZFN technology knocks out SMN gene, adopt liposome transfection method, realize the cell model of setting up spinal muscular atrophy (SMA) disease, can be applied in the research of spinal muscular atrophy pathogenesis, simultaneously also for the high flux screening of anti-spinal muscular atrophy medicine provides cell model.
The invention provides a kind of plasmid vector of the expression Zinc finger nuclease for SMN gene knockout, refer to the plasmid that is inserted with respectively base sequence as shown in SEQ ID NO.2 and SEQ ID NO.3.
Further, described plasmid refers to pAVX.
Further, the downstream of described base sequence is also connected with FokI base sequence.
On the other hand, the invention provides the Zinc finger nuclease that above-mentioned plasmid vector gives expression in mammalian cell.
On the other hand, the invention provides one for SMN gene knockout method, comprise the steps:, by above-mentioned plamid vector transfection mammalian cell, SMN gene to be carried out to pcr amplification, then order-checking detects the cell that SMN gene knocks out to amplified production.
Described mammalian cell is any in Hela, K562, HEK293, A549, MCF-7 or LNCap.
In described gene knockout method, be to adopt liposome Lipofectamine2000 reagent to carry out transfection.
On the other hand, the invention provides the SMA pathological cells model being knocked by the preparation-obtained SMN gene of above-mentioned SMN gene knockout method.
Further, above-mentioned cell model refers to the mammalian cell of SMN1 homozygous mutant gene, SMN2 genetic heterozygosis disappearance.
beneficial effect
Spinal muscular atrophy (SMA) cell model that the present invention sets up, provide strong instrument for research SMN gene at the mechanism of action of SMA disease incidence, by take this cell model as mediation, and then be correlated with micromolecular compound and the screening of genetically engineered recombinant protein, acquisition can significantly increase micromolecular compound and the genetically engineered recombinant proteins medicine of smn protein expression amount.Screening method is simple, with low cost, has satisfactory stability and reliability.
Accompanying drawing explanation
Fig. 1 is the collection of illustrative plates of the plasmid vector for expressing Zinc finger nuclease.
Fig. 2 is that the cell after transfection carries out the product electrophorogram after pcr amplification.
Fig. 3 is that the cell after transfection carries out the product order-checking collection of illustrative plates after pcr amplification, and order-checking peak figure produces cover peak near ZFN cleavage site.
Fig. 4 is Hela cell strain HS-66 and the HS-1-7 WB detected result obtaining in embodiment 1.
Fig. 5 is the cell fluorescence figure of accumulation mark test in the SMN mixture core of Hela, HS-66 and HS-1-7 cell strain in embodiment 4.
Embodiment
Below embodiments of the invention are elaborated by reference to the accompanying drawings.The present embodiment is implemented under take technical solution of the present invention as prerequisite, has provided embodiment and specific operation process, but protection scope of the present invention is not limited to following embodiment.All any this areas of carrying out according to the disclosure of invention are equal to replacement, all belong to protection scope of the present invention.
Main agents and detection kit: Hela cell, ZFN knocks out SMN gene plasmid, purchased from sigma company; Transfection reagent lipofectin2000 is purchased from Invitrogen company; DMEM. foetal calf serum FBS is purchased from Hyclone company; Total RNA extraction reagent Trizol is Invitrogen product; The anti-SMN(H-195 of rabbit) monoclonal antibody is santa cruz company of U.S. product, two anti-Anti-Rabbit IgG are GE company of U.S. product; Reverse transcription test kit is import and domestic analytical reagent purchased from other reagent bacterium of Tian Gen biochemical technology company limited.The normal experiment operation stepss such as in embodiment, enzyme is cut, connected, recovery, conversion, pcr amplification refer to " molecular cloning (third edition) ".
knocking out of embodiment 1 SMN gene
one, SMN gene ZFN plasmid knocks out design & formulation
According to ZFN know-why, design dna target recognition site.This site design is at the total First Exon of SMN1.SMN2 transcript place, and through a large amount of repetition tests, definite target sequence information is:
TCCGTGCTGTTCCGGCGCggcac AGGCCAGGTGAGGTCGCA(SEQ ID NO.1)。Two ends line part is ZFN land, and middle lowercase is ZFN cutting area.Its plasmid construction is completed and is identified by Sigma company of the U.S..Its ZFN plasmid specifying information as shown in Figure 1.
The concrete construction process of described artificial Zinc finger nuclease expression vector, expression vector skeleton is that this carrier of pAVX (Sigma) contains pUC oir and starts this plasmid copying in prokaryotic cell prokaryocyte, has kalamycin resistance gene; Contain a wide spectrum promotor CMV sequence in multiple clone site upstream, can make to be cloned into this multiple clone site gene efficient expression.Utilize the method for external chemosynthesis to build zinc finger protein Expression element (EcoRI-Triple Flag-NLS-ZFP1-BamHI and EcoRI-Triple Flag-NLS-ZFP2-BamHI), the overall sequence of Expression element is respectively as shown in SEQ ID NO. 2 and SEQ ID NO. 3, EcoRI and BamHI restriction enzyme site are contained respectively in synthetic zinc finger protein Expression element both sides, can utilize EcoRI and BamHI restriction enzyme site respectively zinc finger protein to be expressed to territory (ZFP1 and ZFP2) and be cloned in the multiple clone site of carrier pAVX; NLS is to be nuclear localization signal, can guide restructuring ZFP to enter core district; Triple Flag detects for the WB of expression of recombinant proteins, and the ZFP1 in these two kinds of elements and ZFP2 territory are for expressing zinc finger protein, are combined respectively with left arm and the right arm of target sequence.Equally, synthesize in vitro again BamHI-FokI-XhaI gene, utilize BamHI and XhaI restriction enzyme site FokI endonuclease Expression element to be cloned into the downstream position of ZFP element on this carrier, like this zinc finger protein DNA is mutually combined in conjunction with territory and FokI DNA cutting territory, formed a chimeric restriction endonuclease.Obtain two kinds of plasmids with this, be respectively used to express being combined in the left arm of target sequence and the zinc finger protein enzyme of right arm, target sequence can be knocked out.
The ZFN having identified is knocked out to plasmid amplification and cultivate, extract plasmid, purity requirement reaches OD260/280=1.8 ~ 2.0 in order to transfection.
two, SMN gene ZFN knocks out plasmid transfection
(1) inoculating cell.Transfection is seeded to Hela cell in six orifice plates the day before yesterday.Cell inoculation quantity, depending on its speed of growth, is generally 1 × 10 6individual cell.When transfection, requiring cell degree of converging is 80~90%.
(2) the plasmid vector DNA 3 μ g that get respectively the ZFN left arm that obtains in embodiment 1 and right arm being added to OPTI-MEM substratum, to make its final volume be 250 μ l, mixes, and obtains DNA suspension.
(3) Lipofectamine2000 is positioned over to 4 ℃ of ice chest shelfs or on ice, before using, the slow speed of revolution is centrifugal, the Lipofectamine2000 that gets 10 μ l adds in the OPTI-MEM of other 240 μ l, mix gently, this step operation wants rapidly in order to avoid at room temperature long impact of open-assembly time transfection efficiency below of liposome then leaves standstill 5 minutes by mixture room temperature.
(4) by DNA suspension (250 μ l) and Lipofectamine2000 (250 μ l) suspension mix, cumulative volume 500 μ l, mix gently, room temperature leave standstill 20 minutes.
(5) DNA and Lipofectamine2000 mixed solution are added in the hole of six orifice plates, rock orifice plate all around and mix, be placed in incubator and cultivate.
(6) transfection after 4~6 hours cell change liquid, change containing the normal substratum of microbiotic the six every hole of orifice plate capacity 2ml substratum into.
(7) after transfectional cell 48~72h, collect 5/6 cell, extract for the genome of gene knockout checking.Remainder is directly separated into cell suspension, is divided in 96 orifice plates and separates mono-clonal.
three, ZFN knocks out the detection of SMN gene validity
For the special site upstream and downstream design primer that knocks out of ZFN, its sequence is:
SMN Knockout-F:5’ -GGGCGATAACCACTCGTAGA-3’(SEQ ID NO.4)
SMN Knockout-R:5’-AAGACGTAGAAAAACGCGGA-3’ (SEQ ID NO.5)
Collect the transfection Hela cell of 48~72 hours: extract full genome, step is as follows:
(1) cell is transferred in 1.5ml EP pipe, added 20 μ l Proteinase Ks and 220 μ l lysate QS.65 ℃ of temperature are bathed 10-15min.Solution should be black.Brief centrifugal to remove the globule of cap wall.
(2) add 220 μ l dehydrated alcohols, put upside down and mix back and forth up and down.Now may there is flocks, solution and cotton-shaped heavy all shallow lakes are transferred in purification column.The centrifugal 1min of 12,000 rpm, abandons filtrate.
(3) add 500 μ l protein liquid removal PS.The centrifugal 1min of 12,000 rpm, abandons filtrate.
(4) add 500 μ l rinsing liquid PE.The centrifugal 1min of 12,000 rpm, abandons filtrate.
(5) add 500 μ l rinsing liquid PE.The centrifugal 1min of 12,000 rpm, abandons filtrate.
The centrifugal 3min of (6) 12,000 rpm, thoroughly to remove liquid residual in purification column.
(7) purification column is placed in to 1.5 new ml centrifuge tubes.To purification column centre, unsettled dropping 30~100 μ l refined solution TE.Room temperature is placed 2min.
The centrifugal 2min of (8) 12,000 rpm, the pipe end, is cell genomic dna.-20 ℃ of preservations.
The transfectional cell genome extracting, uses SMN Knockout-F and SMN Knockout-R primer to carry out PCR, and its amplification condition is:
94℃ 5min;
94 ℃ of 45s, 54 ℃ of 30s, 72 ℃ of 45s, (30~35 circulations);
72℃ 5min。
After 1% agarose gel electrophoresis, detect through gel imaging system, pcr amplification ZFN target site point fragment length is 367 bp, as shown in Figure 2, reclaim test kit through sepharose DNA and reclaim object fragment connection T carrier, after transformed competence colibacillus cell E.coli.DH5 α 12 h, measure target DNA sequence with mutant proportion and mutation type in statistics PCR product fragment.As shown in Figure 3, there is cover peak in sequencing fragment, thinks that ZFN produces cutting action to target gene in this process, illustrates that pounding out region at target exists the phase shift mutation reparation after pounding out.
Carry out mono-clonal separation by limiting dilution assay again.
four, identify the mutation type of mono-clonal SMN gene knockout
When being incubated at cell clone in 96 orifice plates and growing to 80~90% degree of converging, digestion, collecting cell rear portion is frozen, extract the genomic dna of all the other cells, select altogether 17 cell strains, by pcr amplification ZFNs target site point upstream and downstream 367bp, judge mono-clonal change after ZFN effect in genomic level, and carry out identifying and knocking out insertion and the disappearance whether region has cytothesis mechanism to produce DSB double-strand break by DNA sequencing after T-A clone.
In the present embodiment, in the 17 strain monoclonal cells that adopt, detect through the order-checking of mono-clonal PCR fragment, confirming has 5 strains to have the cell of SMN transgenation.
embodiment 2 SMN gene knockout cell strain mrna expressions are identified
For identifying that SMN knocks out effective cell, extract RNA, method is referring to Trizol test kit specification sheets, after total RNA extracts, with digesting to remove trace genome DNA without the DNase of RNAase activity, measure rna content through ultraviolet spectrophotometer, electrophoresis detection is determined RNA quality; The synthetic of cDNA carries out according to the reverse transcription test kit specification sheets of Tiangen company.For (C → T) different designs primer in the 7th exon of SMN1, SMN2, carry out RT-PCR take the not identical transcript of SMN1, SMN2 as template and tentatively determine SMN1, SMN2 deletion condition according to semiquantitative situation.
But we find that the insertion of base deletion on exon and base does not affect the stability of the RNA that SMN1, SMN2 transcribe.Total length transcript transcript variant d, transcript variant b that by this conclusion, we have at SMN1 and SMN2 again design primer, and its sequence is
SMN cDNAbd-F:5’-GAAGTTACTACAAGCGGTCCTC-3’(SEQ ID NO.6)
SMN cDNAbd-R:5’-GCTCTATGCCAGCATTTCTCCT-3’(SEQ ID NO.7)
After RT-PCR, carry out T-A clone, after order-checking, utilize the point mutation of the 7th exon (C → T) with difference SMN1, SMN2 sudden change situation.According to this mode, we have obtained two strain SMN1-/-SMN2+/-genotypic HeLa cell strain.Be referred to as HS-66 and HS-1-7.
For example: the sudden change situation of HS-66 cell is as follows:
The partial sequence in the SMN1 site of wild-type is:
AGCGGCGGCAGTGGTGGCGGCGTCCCGGAGCAG[ GAGGATTCCGTGCTGTTCCGGCGCGGCACAGGCCA]GAGCGATGATTCTGACATTTGGGATGATACAGC
On the site of the SMN1 of HS-66 cell, lack the tract in above-mentioned bracket [], totally 35 bp have inserted the inserting paragraph of 103 bp simultaneously in the position of disappearance.
AGCGGCGGCAGTGGTGGCGGCGTCCCGGAGCAG::::::::::(-35)::::::::::::GAGCGATGATTCTGACATTTGGGATGATACAG;
Inserting paragraph is:
ACAACTCCAGTGAGCGGATCGACTTGATGCTGTCCCGAGGCTGCGGAAGGAGAGTTGGGCCGGAAGAAGGGTGCTGAGAGCGCTAATAGGGAGACTGCACTGG
On another allelotrope of the SMN1 of HS-66 cell, have the disappearance of 4 bp, sequence is:
GGATTCCGTGCTGTTCCGGCGCG::(-4)::AGGCCAGAGCGATGATTCTGACATTTGGGATGATACAGCA
The partial sequence in the SMN2 site of wild-type is:
AGCGGCGGCAGTGGTGGCGGCGTCCCGGAGCAG[ GAGGATTCCGTGCTGTTCCGGCGCGGCACAGGCCA]GAGCGATGATTCTGACATTTGGGATGATACAGC
On the site of the SMN2 of HS-66 cell, lack the tract in above-mentioned bracket [], totally 35 bp have inserted the inserting paragraph of 103 bp simultaneously in the position of disappearance.
AGCGGCGGCAGTGGTGGCGGCGTCCCGGAGCAG:::::::::(-35):::::::::GAGCGATGATTCTGACATTTGGGATGATACAG
Inserting paragraph is:
ACAACTCCAGTGAGCGGATCGACTTGATGCTGTCCCGAGGCTGCGGAAGGAGAGTTGGGCCGGAAGAAGGGTGCTGAGAGCGCTAATAGGGAGACTGCACTGG
On another allelotrope of SMN2, the situation that finds no sudden change occurs.
embodiment 3 Western blot detect smn protein and express
For the two strain SMN1-/-SMN2+ obtaining/-genotype Hela cell strain HS-66 and HS-1-7 in the time that it grows to 80~90% in 60mm Tissue Culture Dish, trysinization, lysing cell, extract albumen, measure protein concentration by microplate reader, get 45ug loading SDS-PAGE electrophoresis, use 10% separation gel, 5% concentrated glue, pvdf membrane is wet to be turned, transferring film time 1h, the anti-SMN Antibody of rabbit monoclonal antibody is hatched 1h, Goat-Anti-rabbit bis-is anti-hatches 1h, ECL fluoroscopic examination, and developing fixing is also taken pictures.As shown in Figure 4, obviously reduce with the amount of its SMN gene knockout cell expressing smn protein of normal HeLa cell comparison.
in embodiment 4 cells, in SMN mixture core, accumulation detects
Adopt the HS-66 and the HS-1-7 cell strain that in embodiment 2, obtain, take Gemin3 accumulation marker in smn protein mixture nucleus, as shown in Figure 5, the 1st classifies the dyeing of DAPI nucleus as, the 2nd row gemin3 dyeing, and the 3rd classifies folded figure as, i.e. two dyeing full figures that dye.Can find out, in the Hela of first row cell, SMN mixture accumulation number is normal, and knocking out in cell strain HS66 and HS-1-7, and in core, accumulation number greatly declines and even vanishes from sight, identical with pathological characters.
SEQUENCE LISTING
<110> Jiangsu Xiong Ming Pharmaceutical Technology Co., Ltd
Knockout technique and the cell model of a <120> spinal muscular atrophy gene
<130>
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 41
<212> DNA
<213> smn fragment
<400> 1
tccgtgctgt tccggcgcgg cacaggccag gtgaggtcgc a 41
<210> 2
<211> 731
<212> DNA
<213> artificial sequence
<400> 2
gaattcgcct agagatctgg cggcggagag ggcagaggaa gtcttctaac ctgcggtgac 60
gtggaggaga atcccggccc taggaccatg gactacaaag accatgacgg tgattataaa 120
gatcatgaca tcgattacaa ggatgacgat gacaagatgg cccccaagaa gaagaggaag 180
gtcggcattc atggggtacc cgccgctatg gctgagaggc ccttccagtg tcgaatctgc 240
atgcgtaact tcagtcagtc cggcgacctg acccgccaca tccgcaccca caccggcgag 300
aagccttttg cctgtgacat ttgtgggagg aaatttgccg acaccggcgc ccgcctgaag 360
cataccaaga tacacacggg cagccaaaag cccttccagt gtcgaatctg catgcgtaac 420
ttcagtcgct ccgccaacct ggcccgccac atccgcaccc acaccggcga gaagcctttt 480
gcctgtgaca tttgtgggag gaaatttgcc acctccggcc acctgtcccg ccataccaag 540
atacacacgg gatctcagaa gcccttccag tgtcgaatct gcatgcgtaa cttcagtcag 600
tccggcgacc tgacccgcca catccgcacc cacaccggcg agaagccttt ttgcctgtga 660
catttgtggg aggaaaattt gcccgccgcc agcacctgga cgcccatacc aagatacacc 720
tgcggggatc c 731
<210> 3
<211> 651
<212> DNA
<213> artificial sequence
<400> 3
gaattcgcca tggactacaa agaccatgac ggtgattata aagatcatga catcgattac 60
aaggatgacg atgacaagat ggcccccaag aagaagagga aggtcggcat ccacggggta 120
cccgccgcta tggctgagag gcccttccag tgtcgaatct gcatgcgtaa cttcagtcag 180
tccggccacc tggcccgcca catccgcacc cacaccggcg agaagccttt tgcctgtgac 240
atttgtggga ggaaatttgc cgactcctcc aaccgcgagg cccataccaa gatacacacg 300
ggcagccaaa agcccttcca gtgtcgaatc tgcatgcgta acttcagtcg ctccgacaac 360
ctgtccgtgc acatccgcac ccacaccggc gagaagcctt ttgcctgtga catttgtggg 420
aggaaatttg cccagtcctc cgacctgcgc cgccatacca agatacacac gggatctcag 480
aagcccttcc agtgtcgaat ctgcatgcgt aacttcagtc gtagtgacac cctgagccag 540
cacatccgca cccacacagg cgagaagcct tttgcctgtg acatttgtgg gaggaaattt 600
gcccacaaca gccaccgcac aaagcatacc aagatacacc tgcggggatc c 651
<210> 4
<211> 20
<212> DNA
<213> artificial sequence
<400> 4
gggcgataac cactcgtaga 20
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<400> 5
aagacgtaga aaaacgcgga 20
<210> 6
<211> 22
<212> DNA
<213> artificial sequence
<400> 6
gaagttacta caagcggtcc tc 22
<210> 7
<211> 22
<212> DNA
<213> artificial sequence
<400> 7
gctctatgcc agcatttctc ct 22

Claims (9)

1. a cell model for spinal muscular atrophy, is characterized in that: the mammalian cell that refers to SMN1 homozygous mutant gene, SMN2 genetic heterozygosis disappearance.
2. for a plasmid vector for the expression Zinc finger nuclease of SMN gene knockout, it is characterized in that: refer to the plasmid that is inserted with respectively base sequence as shown in SEQ ID NO.2 and SEQ ID NO.3.
3. the plasmid vector of the expression Zinc finger nuclease for SMN gene knockout according to claim 2, is characterized in that: described plasmid refers to pAVX.
4. the Zinc finger nuclease that the plasmid vector described in claim 2 or 3 gives expression in mammalian cell.
5. the SMN gene knockout method of the plasmid vector based on described in claim 2 or 3, it is characterized in that, comprise the steps:, by above-mentioned plamid vector transfection mammalian cell, SMN gene to be carried out to pcr amplification, then order-checking detects the cell that SMN gene knocks out to amplified production.
6. the SMN gene knockout method of plasmid vector according to claim 5, is characterized in that: described mammalian cell is any in Hela, K562, HEK293, A549, MCF-7 or LNCap.
7. the SMN gene knockout method of plasmid vector according to claim 5, is characterized in that: be to adopt liposome Lipofectamine2000 reagent to carry out transfection.
8. the SMN gene knockout method of plasmid vector according to claim 5, is characterized in that: the primer sequence using in pcr amplification is as shown in SEQ ID NO.4 and SEQ ID NO.5.
9. the SMA pathological cells model that the SMN gene that SMN gene knockout method claimed in claim 5 obtains is knocked.
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