Background technology
SLC30A1 (zinc operating body) member 1 be a kind of protein in the mankind by SLC30A1 encoding genes.Required micro memberPlain zinc participates in a variety of enzymes of body and protein function by catalysis and structure function, with body development, brain function, bone growth, lifeIt is closely related to grow health and immune function etc..Supplement zinc can prevent to a certain degree children's diarrhae, chronic hepatitis C, it is acute underThe diseases such as respiratory tract infection and flu, however excessive zinc has toxicity.Therefore, there is complicated zinc ion stable state body in bodyIt is the equilibrium process of the absorption for maintaining zinc ion, storage and loss.SLC30A families are also known as ZnT families, share 10 members, moreA family member can assist zinc ion to flow out to extracellular or flow into organelle out of cytoplasm.Research prompting ZnT1,ZIP4 and ZIP5 participation small intestine zinc ion absorption processes, ZIP10 and ZnT1 participation kidney zinc ion resorption process, ZIP5,ZnT2 and ZnT1 participates in pancreas zinc ion secretion loss process.Separately prove that the albumen of SLC30A families may also participate in perhaps on evidenceMore diseases include the occurrence and development of tumour and diabetes.
Breast cancer is to threaten a big disease of women life and health, inquires into its pathogenesis and finds the side of being effectively prevented and treatedMethod is particularly important.Zinc horizontal abnormality in breast cancer increases, specific transmembrane transport protein zinc transporter and and metal matrixThis known tumor correlated albumen of protease has similar structure, in addition two zinc transhipment in LIV-1 estrogen-regulated genesBody member ZIP6 and ZIP10 and the transfer of breast cancer have it is very close be associated with, therefore, inquire into the different breast of estrogen receptor characterThe influence that the expression of zinc transporter and zinc express zinc transporter in adenocarcinoma cell MDA-MB-231 is necessary.
CRISPR/Cas9 is that the Cas9 nucleases by guide RNA found in recent years target target gene into edlinEmerging technology.CRISPR/Cas9 has found in archeobacteria at first, is that researcher has found archeobacteria to adventive information notBreak and attack and develop a kind of acquired defense mechanism come in CRISPR/Cas9 systems, crRNA (CRISPR-DerivedRNA it) is combined by DNA base pairing mechanism and tracrRNA (trans-activating RNA) and forms double-strandRNA instructs Cas9 albumen to target purpose site cut-out double-stranded DNA in the DNA sequence dna of guide crRNA.Compared to traditional zinc fingerNuclease (ZFNs) technology, transcriptional activation sample effector nuclease（TALENs）Technology etc., CRISPR-Cas technologies have onlySpecial advantage：Simpler, specific higher is designed, speed is fast, the system genitale transfer ability is strong and the characteristics of simple economy.
Bibliography：
1)Moonhuhn Ryu, Louis A. Lichten, Juan P. Liuzzi, et al. ZincTransporters ZnT1 (Slc30a1), Zip8 (Slc39a8), and Zip10 (Slc39a10) in MouseRed Blood Cells Are Differentially Regulated during Erythroid Development andby Dietary Zinc Deficiency[J]. Journal of Nutrition, 2008, 138(11):2076-2083.
2)Fernandez E L, Dencker L, Tallkvist J. Expression of ZnT-1 (Slc30a1)and MT-1 (Mt1) in the conceptus of cadmium treated mice[J]. ReproductiveToxicology, 2007, 24(3–4):353-358.
3)Muraina, Issa. Reverse genetics analysis of biological functions ofzinc transporters Znt1 (Slc30a1) & Zip10 (Slc39a10) in zebrafish[J]. KingsCollege London, 2013。
Invention content
The shortcomings that primary and foremost purpose of the present invention is to overcome the prior art and deficiency, provide a kind of targeting knock out SLC30A1The sgRNA of gene.
It is sick slowly another object of the present invention is to provide a kind of CRISPR/Cas9 of targeting knock out SLC30A1 genesMalicious systematic difference.
Another object of the present invention is to provide people's colon-cancer cell MDA-MB-231 cells of targeting knock out SLC30A1 genesApplication.
The purpose of the present invention is achieved through the following technical solutions：A kind of sgRNA of targeting knock out SLC30A1 genes, is selected fromThe following SLC30A1sgRNA of DNA sequence dna：
The sequence of SLC30A1sgRNA is as follows：
SLC30A1sgRNAoligo1：5’-cacc GCTCTTAACGCGAGGCCCCT-3’；
SLC30A1sgRNAoligo2：5’-aaac AGGGGCCTCGCGTTAAGAGC-3’；
A kind of CRISPR/Cas9 slow virus systems of targeting knock out SLC30A1 genes, contain above-mentioned targeting knock out SLC30A1 basesThe DNA sequence dna of the sgRNA of cause.
The structure of the CRISPR/Cas9 slow virus systems of the targeting knock out SLC30A1 genes, includes the following steps：
（1）Using BsmBI digestion CRISPR/Cas9 slow virus carrier LentiCRISPRV2, the CRISPR/ after digestion is obtainedCas9 slow virus carriers；
（2）By after the DNA sequence dna phosphorylation of the sgRNA of above-mentioned targeting knock out SLC30A1 genes with the CRISPR/Cas9 after digestionSlow virus carrier connects, and obtains the CRISPR/Cas9 slow virus systems of targeting knock out SLC30A1 genes.
DNA sequence dna described in step (2) be by oligonucleotide chain 1 (oligo1) and oligonucleotide chain 2 (Oligo2) annealing obtains double-stranded sequence.
The CRISPR/Cas9 slow virus system of the targeting knock out SLC30A1 genes is preparing knockout RITA genesApplication in cell strain.
A kind of cell strain for knocking out SLC30A1 genes, is by the CRISPR/ of the targeting knock out SLC30A1 genesCas9 slow virus system transfections aim cell strains obtain.
The cell strain of the knockout SLC30A1 genes, builds to obtain particular by following steps：
1）The CRISPR/Cas9 slow virus systems of the targeting knock out SLC30A1 genes are packed by incasing cells,Obtain lentiviral particle；
2）Lentiviral particle is infected into aim cell strain, obtains knocking out the cell strain of SLC30A1 genes.
The aim cell strain is preferably tumor cell line.
The tumor cell line is preferably breast carcinoma cell strain.
The breast carcinoma cell strain is preferably human breast carcinoma cancer immortalized cells MDA-MB-231.
The cell strain of the knockout SLC30A1 genes is the SLC30A1 gene delections or slotting in MDA-MB-231 cellsEnter the cell strain that nucleotide obtains.
The present invention is had the following advantages relative to the prior art and effect：
The present invention provides the sgRNA energy efficient targeting SLC30A1 genes of SLC30A1 genes, is built into CRISPR/Cas9Slow virus system, the system can knock out SLC30A1 genes, obtain knocking out the cell strain of SLC30A1 genes, so as to be conducive to studyThe mechanism of action of SLC30A1 in cell strain.
Embodiment 1
1, knocks out SLC30A1 plasmids using CRISPR/Cas9 technologies structure
1 .1sgRNA oligonucleotide chains synthesize
Use CRISPR Photographing On-line tools (http://crispr .mit .edu/) according to points-scoring system, in SLC30A1Exon 2 on design 1 20bp sgRNA, and by BLAST verify without non-specific gene.The end of coding strand template 5 ' addsAdd CACC, the cohesive end formed after the addition of the end of noncoding strand template 3 ' AAAC, with BsmBI digestions is complementary, designs 1 couple of CRISPROligonucleotide chain is shown in Table 1 SLC30A1 target sites and sgRNA oligonucleotide sequences
1 .2 vector constructions
1.2.1 using 2 μ g lentiCRISPRv2 plasmids (being purchased from Addgene companies) of BsmBI digestions, 2h, 37 DEG C of
1.2.2 digested plasmid product is purified using GENRY plastic recovery kits, by specification is operated
1.2.3 phosphorylation and the sgRNAoligos that anneals：
PCR instrument cycle of annealing：37 DEG C of 30min, 95 DEG C of maintenance 5min, per minute to reduce by 5 DEG C to 25 DEG C, 4 DEG C of maintenances.
1.2.4 the lentiCRISPRv2 carriers after anneal the oligo double-strands formed and digestion are directly connected to, at room temperature,10min。
1.2.5 the plasmid after connection is converted into competent cell DH5 α, is uniformly applied in LB solid medium tablets,It is placed in 37 DEG C of incubators and cultivates 12-16 hours, single bacterium colony may occur in which.
The expansion of 1 .3 pickings single bacterium colony, which is cultivated, and plasmid is small carries.
1.4 sequencing identification plasmid construction successes, and it is named as lentiCRISPRv2-hSLC30A1sg.
1.5 knock out efficiency verification
5%CO2 is based on the DMEM in high glucose culture containing 10% fetal calf serum, 37 DEG C of constant temperature incubation 293T cells (are purchased from the U.S.ATCC cell banks).Phase cell take the logarithm with 1 × 105/ hole is inoculated into 24 orifice plate cultures.Treat that cell fusion degree reaches 70%~8 0Opti-MEM culture mediums are replaced with during %, corresponding CRISPR/Cas9 is knocked out into 0.8 μ g of plasmid through Lipo2000 after 1 hourReagent is transfected into 293T cells, and as a child, cell is collected in fluorescence microscopy Microscopic observation transfection, digestion for transfection 48, is usedGenome DNA extracting reagent kit extracts cell genomic dna;Using each group cell genomic dna as template, the primer of table 2 is utilizedPCR amplification targets sequence.PCR is carried out with SLC30A1-4-1 and SLC30A1-4-2, the segment a (high GC) of 904bp is obtained, with aFor template, PCR is carried out with SLC30A1-4-3 and SLC30A1-4-4, obtains the segment b (high GC) of 400bp, after the fragment electrophoreticGlue recycling is for sequencing primer SLC30A1-4-3, table 2
2, pack slow virus
100mm ware kinds enter 293TN cells 4X106, for 24 hours after (37 DEG C, 5%CO2), replacement fresh culture transfects for cultureIncasing cells：
3 plasmids below a mixings：
12 μ g of packaging plasmid psPAX2
10 μ g of helper plasmid pLP-VSVG
22 μ g of expression plasmid lentiCRISPRv2- hSLC30A1sg
Add in CaCl2 250μl
Add in ddH2O is 500 μ l to total volume, and 37 DEG C are placed 20min；
B .2 X BES 500 μ l, 37 DEG C of placement 20min；
C 500 μ l in a, tri- plasmid mixed liquors are added dropwise in b, and gently mixing；
D. it is careful that mixed liquor addition kind in c has in the 100mm wares of 293TN；Infection liquid is removed after cultivating 12h, replaces 10mlFresh culture produces virus liquid；After cultivating 48h, harvest in virus liquid to 50ml centrifuge tubes, and mended again into 100mm wares10ml culture mediums is added to produce virus again；After culture for 24 hours, virus liquid, room temperature centrifugation, 1000rpm, 10min are harvested again；It uses0.22 μm of filter filter virus supernatant；Vial supernatant is fitted into and is surpassed from pipe, 4 DEG C, 100000g, 2h；Discard supernatant liquidIt is resuspended overnight with Opti-MEM afterwards；Reuse the virus that 0.22 μm of filter filtering is resuspended；After packing, -80 DEG C of preservations.
3, viruses infect
1) it in the MDA-MB-231 cell inoculations to six orifice plates that will be infected, overnight, when cell fusion degree is about 50%, movesCulture medium is removed, replaces 2ml fresh cultures；
2) 50 μ l virus liquids are added in per hole, 12 μ l polybrene polybrene are added, until final concentration of 6 μ g/ml；
3) after 37 DEG C of culture 12h, culture solution is removed, continues to cultivate 48h after replacing fresh medium.
4, screen stable cell line
After infection terminates 48h, puromycin (2 .0 μ g/ml) is added in every hole, changes liquid every other day, and keep the purine of culture mediumMycin constant concentration, screening positive clone cell, gained cell strain are named as MDA-MB-231-SLC30A1, and using limited diluteInterpretation of the law selects MDA-MB-231-SLC30A1 monoclonals and knocks out cell strain.
5, stable cell lines are identified
The monoclonal cell strain genomic DNA sequencing that each group knocks out SLC30A1 is extracted, it is active to identify sgRNA（Figure5）, and find the stable cell line (Fig. 6-9) with SLC30A1 gene delections or insertion mutation of 4 types.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodimentLimitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,Equivalent substitute mode is should be, is included within protection scope of the present invention.
Sequence table
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