CN105886534A - Tumor metastasis inhibition method - Google Patents
Tumor metastasis inhibition method Download PDFInfo
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- CN105886534A CN105886534A CN201610280023.1A CN201610280023A CN105886534A CN 105886534 A CN105886534 A CN 105886534A CN 201610280023 A CN201610280023 A CN 201610280023A CN 105886534 A CN105886534 A CN 105886534A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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Abstract
The invention relates to the field of biopharmacy, in particular to a tumor metastasis inhibition method. In the tumor metastasis inhibition method, DP103 genes of tumor cells are subjected to fixed-point knockout through a Crispr/cas9 technology. The tumor metastasis inhibition method has the advantage that the DP103 genes of the tumor cells are subjected to fixed-point knockout, so that tumor metastasis capability can be reduced effectively.
Description
Technical field
The present invention relates to field of biological pharmacy, particularly relate to a kind of method suppressing neoplasm metastasis.
Background technology
In in the past few decades, its toxicity to fast-growth cell is mainly investigated in the screening of antineoplastic agent
Effect.Along with the development of oncobiology research, people recognize that tumor cell not only has vigorous propagation
Ability, but also have destruction autologous tissue, infiltration adjacent tissue and infiltration blood vessel or lymphatic vessel and along with
Blood or lymph fluid transfer to the ability of its hetero-organization.The interaction of tumor and normal structure and the blood of tumor
Pipe generates, attack and the characteristic such as transfer has the biggest relation.Neoplasm metastasis has become study hotspot in recent years
One of.
Neoplasm metastasis refers to that tumor cell is diffused into the phenomenon at other position of body from original site.Statistics shows,
Tumor patient death more than 90% comes from neoplasm metastasis.Neoplasm metastasis includes: 1, tumor cell migration is passed through
Tumor tissues basement membrane；2, enter blood circulation, survive wherein and transport；3, at tumor secondary site
It is detained；4, ooze out from blood circulation；5, immerse tumor secondary site and breed processes such as forming new tumor.
Due to its complexity, although conducting extensive research it, neoplasm metastasis has remained so far and has solved
Few oncobiology process.This complex process of neoplasm metastasis is regulated by several genes.Some of which
Gene promotes the generation of neoplasm metastasis, is called neoplasm metastasis and promotes gene.For many years, various gene mistake is utilized
Expression means, people have found some neoplasm metastasis promote gene, such as, Amf, Hgf/sf, TGF-β,
MMP, UPA, β-catenin etc..
The generation of another kind of gene inhibition neoplasm metastasis, is called anti-metastasis gene.Generally, tumor turns
Move suppressor gene can suppress neoplasm metastasis and on Primary tumor growth without impact, usually through first in metastatic lesion
After base or transcription and translation, a series of mechanism such as modification show as down-regulated expression.Turn to be best understood from tumor
Move, be attempted to the effect disclosing anti-metastasis gene further in neoplasm metastasis generating process.Aobvious
And be clear to, finding anti-metastasis gene needs to be expressed by reduction anti-metastasis gene to realize.
But, owing to reducing expression means the most reliably, the most only determine very limited amount of neoplasm metastasis
The base that suppressor gene, the most only NM23, KNI1, KISS1, MKK4, BrMS1, Gas1 etc. are limited
Because meeting the definition standard of anti-metastasis gene, the most it is clear that NM23 and KNI1.Thus
Make the research to anti-metastasis gene, develop, utilize and cannot be carried out, significantly limit tumor
Transfer understanding, diagnose and treat.
The operation principle of Crispr/cas9 be crRNA (CRISPR-derived RNA) by base pairing with
TracrRNA (trans-activating RNA) combines and forms tracrRNA/crRNA complex, this complex
Guide nuclease Cas9 albumen at the sequence target site shearing double-stranded DNA matched with crRNA.And pass through people
Work design both RNA, can transform and form the sgRNA (singleguide RNA) with guiding function,
Be enough to guide Cas9 that the fixed point of DNA is cut.
The dsDNA associated proteins guided as a kind of RNA, Cas9 effector nuclease is known first
The individual unified factor (unifying factor), it is possible to position RNA, DNA and albumen altogether, thus have huge
Transformation potentiality.The Cas9 (Cas9nuclease-null) of albumen with nuclease free is merged, and expresses suitably
SgRNA, any dsDNA sequence can be targeted, and the end of sgRNA may be connected to target dna, no
Affect the combination of Cas9.Therefore, Cas9 can bring any fusion protein and RNA at any dsDNA sequence,
This research being organism and transformation bring great potential.
Summary of the invention
For solving above-mentioned technical problem, it is an object of the invention to provide a kind of based on Crispr/cas9 technology, logical
Cross fixed point cutting neoplasm metastasis and promote gene, with the method reducing the suppression neoplasm metastasis of neoplasm metastasis ability.
The present invention provides a kind of method suppressing neoplasm metastasis, and the Crispr/cas9 technology fixed point of employing knocks out tumor
The DP103 gene of cell.DP103 gene is also called DDX20 or GEMIN3, its ncbi database
(National Center for Biotechnology Information,http://www.ncbi.nlm.nih.gov/On)
The Gene ID recorded is 11218.
DP103 is one of DEAD-box family protein member, and name derives from wherein Asp-Glu-Ala-Asp
Position in unwindase, DEAD-box family protein, is the DBPA family of an ATP dependence,
Participate in the various metabolic processes such as RNA secondary structure conversion of RNA, transcription initiation, mitochondrial RNA (mt RNA) montage,
Ribosome and spliceosome assembling, mRNA degraded and the stability etc. of maintenance mRNA, participation fetal development,
Spermatogenesis and the process such as the growth of cell and division.Recent studies have found that the DP103 of one of this family member
Gene is high expressed in metastatic cancer cell.
It should be noted that, Crispr/cas9 technology, it is to become there is the gRNA of guiding function by engineer
(guide RNA) targets genes of interest, guides nuclease Cas9 albumen to DNA fixed point cutting, causes
Genes of interest can not normal expression.Need when using this technology first the genome of genes of interest to be analyzed,
This gene exon region find CAS9 enzyme effect recognition site NGG, wherein N can be A,
Any one in T, C, G, sequence according to action site front 20 bases design synthesis gRNA in experiment
Row positive-sense strand and antisense strand, i.e. gene knockout primer.Article two, strand builds to gRNA-cas9 after being annealed into double-strand
Cloning vehicle, carries out plasmid-transfected cells or is packaged into virus, subsequently infection cell, at intracellular targeting
It is cut by genes of interest so that this gene inactivation, thus reaches the purpose of gene knockout.
Further, comprise the following steps:
S1, design and synthesize the one group of gene knockout primer targeting DP103 gene；
S2, one group of gene knockout primer annealing in step S1 is become double-stranded DNA；
S3, slow virus carrier enzyme action；
S4, by step S2 obtain double-stranded DNA be connected with slow virus carrier, obtain plasmid；
S5, plasmid step S4 obtained pack the common transfectional cell of helper plasmid with slow virus, are packaged into slow
Virion, concentrates and purifies postoperative infection tumor cell.
Constitute it should be noted that, DP103 gene has 11 exons, the present invention sets in step S1
The gene knockout primer counted and synthesize targets the First Exon of DP103 gene.Outer with other parts shows
Son is compared, and First Exon is the start-up portion transcribed, and is selected as target spot, can carry to the full extent
Height knocks out efficiency to DP103 gene so that this gene complete deactivation.
Wherein, the gene order of the First Exon of DP103 gene is:
atggcggcggcatttgaagcctcgggagccttagcagcagtggcgactgctatgccggctgagcatgtggccgt
gcaggtcccggccccagagccaacacccgggcctgtgaggatcctgcggaccgctcaggatctcagcagcccgcgga cccgcacgggggatgtgctgttggcggagccggccgacttcgagtcactgctgctttcgcggccggtgctggaggggct
gcgggcggccggcttcgagaggccctcgccggtgcagctcaaggccatcccgttggggcgctgcgggctcg(SEQ
ID NO:1)
The sequence of gene knockout primer targeting DP103 gene is the part of underscore in foregoing sequences, it may be assumed that
Ggacccgcacggggg (SEQ ID NO:2)
Specifically, the gene knockout primer such as SEQ ID NO:3 employed in the method and SEQ ID NO:
Shown in 4.Particular sequence is: DP103-sgRNA-1F:CACCGCATCCCCCGTGCGGGTCCG
(SEQ ID NO:3) and DP103-sgRNA-1R:AAACCGGACCCGCACGGGGGATGC
(SEQ ID NO:4).
It should be noted that, although CAS9 ordinary plasmids the most also can reach experiment effect, but matter
Grain transfection has the shortcomings such as low, transience action time of efficiency.The appearance of virus solves these problems of plasmid,
Conventional virus mainly has slow virus and adenovirus, and slow virus commonly uses plasmid such as addgene (lentiCRISPR
V2, lentiGuide-Puro, lentiCas9-Blast), the gene expression effect lasts of lentivirus-mediated and
Stable, reason is that genes of interest is incorporated in host cell gene group, and divides with the division of cellular genome
Split.It addition, slow virus carrier can effectively infect and be incorporated in Unseparated Cell.Above characteristic makes slow virus
Carrier compared with other viral vector, the adeno-associated virus that the most unconformable adenovirus vector, integration rate are low
Traditional retroviral vector of carrier, only integration somatoblast, has the biggest advantage.Now, slow virus
The tissue of carrier mediated genes of interest long-term expression or cell include brain, liver, muscle, retina, make
Hemocytoblast, mesenchymal stem cells MSCs, macrophage etc..
Specifically, the slow virus carrier described in the method step S3 is Lenti-crispr V2.
Specifically, in step S5, plasmid transfects to 293T or 293FT jointly with slow virus packaging helper plasmid
Cell.
The present invention provides a kind of recombined lentivirus vector preparation, including the aforementioned lentiviral particle of effective dose.
Further, buffer and the saccharide maintaining the pH value range of described preparation is also included.Described saccharide
At least one in monosaccharide and non-reducing disaccharide.The most described saccharide selected from glucose, fructose,
At least one in trehalose and sucrose.On the basis of recombined lentivirus vector preparation, described contents of saccharide
Quality volume percent range is 2-10% (w/v).
By such scheme, the present invention at least has the advantage that and promotes base by fixed point cutting neoplasm metastasis
Because of DP103, can effectively reduce neoplasm metastasis ability.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technology of the present invention
Means, and can being practiced according to the content of description, below with presently preferred embodiments of the present invention and coordinate attached
After figure describes in detail such as.
Accompanying drawing explanation
Fig. 1 is DP103 gene expression testing result figure in different cells in the present invention；
Fig. 2 is different cell scratch experiments migration area result figure of cell after 48 hours in the present invention.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment, the detailed description of the invention of the present invention is described in further detail.With
Lower embodiment is used for illustrating the present invention, but is not limited to the scope of the present invention.
Embodiment 1
A preferred embodiment of the present invention provides a kind of method suppressing neoplasm metastasis, specifically includes following steps:
S1, the expression of detection DP103 gene in tumor cell
Extract kinds of tumor cell and the total serum IgE of non-tumor cell, and reverse transcription becomes cDNA, uses QPCR
Method detection DP103 gene expression in each cell line.Do with non-tumor cell HEK293 cell
For compared with control cells.Testing result is as shown in Figure 1, it is seen that in each tumor cell, DP103 gene exists different journey
The high expressed of degree is the most especially the highest with the expression values of DP103 gene in malignant melanoma cell system A375.
Wherein QPCR primer is as follows: F:GCTGCGGGCTCGATTTAATTG (SEQ ID NO:5),
R:GTCCAAAGCTATGGTGGAGAAC (SEQ ID NO:6).
S2, design and synthesize the targeting sequence for DP103 gene
According to crispr/cas9 shot design principle, design and synthesize the single stranded nucleotide targeting DP103 gene
Acid (oligo):
DP103-sgRNA-1F:CACCGCATCCCCCGTGCGGGTCCG (SEQ ID NO:3)
DP103-sgRNA-1R:AAACCGGACCCGCACGGGGGATGC (SEQ ID NO:4)
The nucleotides sequence of DP103 gene is classified as:
atggcggcggcatttgaagcctcgggagccttagcagcagtggcgactgctatgccggctgagcatgtggccgt
gcaggtcccggccccagagccaacacccgggcctgtgaggatcctgcggaccgctcaggatctcagcagcccgcgga cccgcacgggggatgtgctgttggcggagccggccgacttcgagtcactgctgctttcgcggccggtgctggaggggct
gcgggcggccggcttcgagaggccctcgccggtgcagctcaaggccatcccgttggggcgctgcgggctcgatttaatt
gttcaagctaaatctggcaccgggaaaacctgtgtgttctccaccatagctttggactctcttgttcttgaaaacttaagtaccc
agattttgatcttggctcctacaagagaaattgctgtacagatacattctgttattacagccattggaataaaaatggaaggctt
agagtgtcatgtctttattggagggaccccattatcacaagacaaaaccagacttaaaaagtgtcatattgctgttggatctcc
tggcagaattaagcaactcatagaacttgactacttgaacccaggcagtatacgcctctttattcttgatgaagcagataagct
tttagaagaaggcagcttccaggagcaaataaattggatttattcttccttgcctgccagtaaacagatgctggcagtatcag
ctacttatcccgaatttttggctaatgctttgacaaagtacatgagagatcccacttttgtaagactgaattccagtgatccaagt
ctcataggtttgaagcagtattacaaagttgtcaattcataccctttggcacataaggtttttgaggaaaagactcagcatttac
aggaactgttcagcagaattccatttaatcaagctttagtcttttctaatttgcacagcagagcacaacatttggctgatatcctt
tcttctaaaggctttcctgctgagtgcatttcaggcaatatgaatcagaatcagcgtcttgatgctatggctaaactgaagcac
tttcattgcagagtcctcatttccacagatttgacttctcgtgggattgatgctgagaaggtgaatctggttgtaaatctggatgt
accattggattgggagacatacatgcatcggattgggagagctggccgttttggtacattggggctgacagtgacctactgt
tgccggggagaggaagaaaatatgatgatgagaattgcccagaaatgtaatatcaaccttctccctttaccagatcccattc
cttctggtctgatggaagaatgtgtggattgggatgtggaagttaaagctgctgtgcatacatatggtatagcaagtgtacct
aaccaacccttaaaaaagcaaattcagaaaatagagagaacccttcaaattcagaaagctcatggtgaccacatggcttcct
ctagaaataattctgtatctggactatcagtcaaatcaaaaaataataccaaacaaaagcttcctgtgaaaagccactcagaa
tgtggaatcatagaaaaagcaacgtcaccaaaagaactgggctgtgacaggcaatccgaagagcaaatgaagaattctgt
tcagactcccgttgaaaactccaccaacagtcagcaccaggtcaaagaagctttacctgtgtcactcccccagattccttgtc
tgtcttcctttaaaatccatcagccatacacgttgacttttgctgaattggtagaggattatgaacattatattaaagaggggtta
gagaaacctgtggaaatcatcaggcactacacaggccctggggatcagactgtgaatcctcaaaatggttttgtgagaaat
aaagttattgaacagagagtccctgtgttggcaagtagtagccaatctggagactctgagagtgacagtgattcttacagct
caagaacctcttcccagagcaaaggaaataagtcatacttggaaggctcttctgataatcagctgaaagactctgaatctac
gcctgtggatgatcgtatttctttggaacaaccaccaaatggaagtgacacccccaatccagagaaatatcaagaatcacct
ggaatccagatgaagacaagacttaaagagggggctagccagagagctaagcagagccggagaaacctacccaggc
ggtcttccttcagattgcagactgaagcccaggaagatgattggtatgactgtcatagggaaatacgtctgagtttttctgata
cctatcaggattatgaggagtactggagagcttactacagggcatggcaagaatattatgctgccgcttctcattcatattatt
ggaatgctcagagacatccaagttggatggcagcttatcacatgaataccatttatctacaagaaatgatgcatagtaacca
Gtga (SEQ ID NO:7)
Wherein, in foregoing sequences, the position of underscore is the target area of single-stranded nucleotide.
The aminoacid sequence of DP103 gene is:
MAAAFEASGALAAVATAMPAEHVAVQVPAPEPTPGPVRILRTAQDLSSPR
TRTGDVLLAEPADFESLLLSRPVLEGLRAAGFERPSPVQLKAIPLGRCGLDLIV
QAKSGTGKTCVFSTIALDSLVLENLSTQILILAPTREIAVQIHSVITAIGIKMEGL
ECHVFIGGTPLSQDKTRLKKCHIAVGSPGRIKQLIELDYLNPGSIRLFILDEADK
LLEEGSFQEQINWIYSSLPASKQMLAVSATYPEFLANALTKYMRDPTFVRLNS
SDPSLIGLKQYYKVVNSYPLAHKVFEEKTQHLQELFSRIPFNQALVFSNLHSR
AQHLADILSSKGFPAECISGNMNQNQRLDAMAKLKHFHCRVLISTDLTSRGID
AEKVNLVVNLDVPLDWETYMHRIGRAGRFGTLGLTVTYCCRGEEENMMMR
IAQKCNINLLPLPDPIPSGLMEECVDWDVEVKAAVHTYGIASVPNQPLKKQIQ
KIERTLQIQKAHGDHMASSRNNSVSGLSVKSKNNTKQKLPVKSHSECGIIEKA
TSPKELGCDRQSEEQMKNSVQTPVENSTNSQHQVKEALPVSLPQIPCLSSFKI
HQPYTLTFAELVEDYEHYIKEGLEKPVEIIRHYTGPGDQTVNPQNGFVRNKVI
EQRVPVLASSSQSGDSESDSDSYSSRTSSQSKGNKSYLEGSSDNQLKDSESTP
VDDRISLEQPPNGSDTPNPEKYQESPGIQMKTRLKEGASQRAKQSRRNLPRRS
SFRLQTEAQEDDWYDCHREIRLSFSDTYQDYEEYWRAYYRAWQEYYAAASH
SYYWNAQRHPSWMAAYHMNTIYLQEMMHSNQ (SEQ ID NO:8)
S3, single-stranded nucleotide are annealed into double-stranded DNA
Will synthesis two single-stranded nucleotide, be dissolved into the solution of 20uM/ml with ultra-pure water, respectively take 3ul with
14ul ultra-pure water is configured to 20ul reaction system in 200ul PCR pipe, is placed in PCR instrument according to 95 DEG C:
10 minutes, 90 DEG C: 5 minutes, 85 DEG C: 5 minutes, 80 DEG C: 5 minutes, 75 DEG C: 5 minutes, 70 DEG C: 5
Minute, 65 DEG C: 5 minutes, 60 DEG C: 5 minutes, 55 DEG C: 5 minutes, 50 DEG C: 5 minutes, 45 DEG C: 5 points
Clock, 40 DEG C: 5 minutes, 35 DEG C: 5 minutes, 30 DEG C: 5 minutes, 25 DEG C: 5 minutes, 20 DEG C: 5 minutes.
Two strands are annealed into double-stranded DNA.
S4, slow virus carrier enzyme action
By Lenti-crispr V2 carrier BsmBI (NEB, R0580S) enzyme action, system is as follows:
BsmBI (NEB, R0580S) 1ul；
Lenti-crispr V2 vector plasmid 3ug；
NEBuffer3 2ul；
Moisturizing is to 20ul.
55 DEG C of water-bath 1h, reclaim purpose carrier about 12Kb with concentration 1% agarose gel electrophoresis.
S5, double-stranded DNA containing target sequence and slow virus carrier connect
Annealing double-stranded DNA 8ul, Lenti-crispr V2 BsmBI enzyme action glue is returned product 6ul, 5X T4
DNA Ligase buffer (invitrogen) 4ul, T4 DNA Ligase (invitrogen) 2ul, is formulated as
The reaction system of cumulative volume 20ul, is placed in 1.5mlEP pipe, 25 DEG C of water-baths 1 hour.
Taking 10ul and convert 100ul DH5a competent cell, coated plate (AMP+), 37 DEG C of constant temperature culture are overnight.
S6, slow virus packaging and infected tumor's cell
DH5a cell step S5 obtained, after extracting plasmid, packs helper plasmid helper1 with slow virus
Jointly transfect 293FT cell with plasmid helper2, be packaged into lentiviral particle, and concentrate and purify.
Tumor cell (MB231, A375 and BT549) and three strains with postoperative infection three Expression of Plant Height DP103 gene
The tumor cell (SGC7901, MCF7, A549) of low expression DP103 gene.
S7, cellular genome are extracted
Tumor cell gene group after slow virus infection in extraction step S6, carries out Genomic PCR.Its primer
For:
DP103-PCR-F5:CCCTCTTGGGGCTTTCCT (SEQ ID NO:9)
DP103-PCR-R5:AACGGGATGGCCTTGAGC (SEQ ID NO:10)
Amplified production is 485bp, and pcr amplification product purification reclaims.
S8, mispairing method detection gene knockout result
The PCR purified product that will obtain in step S7, carries out PCR order-checking with the primer in step S2, moves back
Carrying out enzyme action with T7E1 enzyme (NEB) after fire, 2% agarose gel electrophoresis detects gene knockout result, can
To detect tri-DNA bands of 484bp, 137bp and 344bp.
On transcellular impact after S9, scratch experiment detection DP103 gene knockout
By the tumor cell of isopycnic DP103 gene knockout and do not carry out the tumor cell of gene knockout pass in
In Tissue Culture Dish, cell density about 90% after passing on latter 24 hours, carry out scratch experiment.Count after 48 hours
Calculate the area of cell migration, and contrast.
As in figure 2 it is shown, the tumor cell of high expressed DP103 gene transfer velocity after by this gene knockout is bright
Aobvious less than the tumor cell not knocking out this gene.Rather than the tumor cell of high expressed DP103 gene is by this base
After knocking out, cell transfer velocity also has reduction in various degree.
Embodiment 2
A preferred embodiment of the present invention provides a kind of recombined lentivirus vector preparation, aforementioned including effective dose
Lentiviral particle.
Also include maintaining the buffer of the pH value range of preparation and as the saccharide of stabilizer.Saccharide is selected from single
At least one in sugar and non-reducing disaccharide.More preferably saccharide is selected from glucose, fructose, trehalose and sugarcane
At least one in sugar.On the basis of recombined lentivirus vector preparation, the quality percent by volume of contents of saccharide
Scope is 2-10% (w/v).
The above is only the preferred embodiment of the present invention, is not limited to the present invention, it is noted that
For those skilled in the art, on the premise of without departing from the technology of the present invention principle, also
Can make some improvement and modification, these improve and modification also should be regarded as protection scope of the present invention.
Claims (9)
1. the method suppressing neoplasm metastasis, it is characterised in that: the Crispr/cas9 technology fixed point of employing knocks out
The DP103 gene of tumor cell.
The method of suppression neoplasm metastasis the most according to claim 1, it is characterised in that: include following step
Rapid:
S1, design and synthesize the one group of gene knockout primer targeting DP103 gene；
S2, one group of gene knockout primer annealing in step S1 is become double-stranded DNA；
S3, slow virus carrier enzyme action；
S4, by step S2 obtain double-stranded DNA be connected with slow virus carrier, obtain plasmid；
S5, plasmid step S4 obtained pack the common transfectional cell of helper plasmid with slow virus, are packaged into slow
Virion, concentrates and purifies postoperative infection tumor cell.
The method of suppression neoplasm metastasis the most according to claim 2, it is characterised in that: in step S1
The gene knockout primer designed and synthesized targets the First Exon of DP103 gene.
The method of suppression neoplasm metastasis the most according to claim 3, it is characterised in that: in step S1
The gene knockout primer designed and synthesized targets the sequence as shown in SEQ ID NO:2.
The method of suppression neoplasm metastasis the most according to claim 4, it is characterised in that: the base used
Because knocking out primer as shown in SEQ ID NO:3 and SEQ ID NO:4.
The method of suppression neoplasm metastasis the most according to claim 2, it is characterised in that: in step S3
Described slow virus carrier is Lenti-crispr V2.
The method of suppression neoplasm metastasis the most according to claim 2, it is characterised in that: in step S5
Plasmid transfects to 293T or 293FT cell jointly with slow virus packaging helper plasmid.
8. a recombined lentivirus vector preparation, it is characterised in that: include effective dose according to claim
The lentiviral particle that the method for the suppression neoplasm metastasis described in 2 obtains.
Recombined lentivirus vector preparation the most according to claim 8, it is characterised in that: also include maintaining
The buffer of the pH value range of described preparation and saccharide.
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| CN201610280023.1A CN105886534A (en) | 2016-04-29 | 2016-04-29 | Tumor metastasis inhibition method |
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| CN201610280023.1A CN105886534A (en) | 2016-04-29 | 2016-04-29 | Tumor metastasis inhibition method |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103316356A (en) * | 2012-03-22 | 2013-09-25 | 北京三诺佳邑生物技术有限责任公司 | Recombinant lentivirus vector preparation |
| CN105492608A (en) * | 2015-06-11 | 2016-04-13 | 深圳市第二人民医院 | Method using CRISPR-Cas9 to specifically knock off pig PDX1 gene and sgRNA of PDX1 gene for specific targeting |
-
2016
- 2016-04-29 CN CN201610280023.1A patent/CN105886534A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103316356A (en) * | 2012-03-22 | 2013-09-25 | 北京三诺佳邑生物技术有限责任公司 | Recombinant lentivirus vector preparation |
| CN105492608A (en) * | 2015-06-11 | 2016-04-13 | 深圳市第二人民医院 | Method using CRISPR-Cas9 to specifically knock off pig PDX1 gene and sgRNA of PDX1 gene for specific targeting |
Non-Patent Citations (4)
| Title |
|---|
| EUN MYOUNG SHIN等: "DEAD-box helicase DP103 defines metastatic potential of human breast cancers", 《THE JOURNAL OF CLINICAL INVESTIGATION》 * |
| 吴梧桐: "《生物制药工艺学》", 31 August 2015, 中国医药科技出版社 * |
| 苏甲林: "建立CRISPR/Cas9慢病毒系统高效敲除人源AIP1基因", 《生物技术通报》 * |
| 赵广荣: "《现代制药工艺学》", 31 January 2015, 清华大学出版社 * |
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