CN107828738A - A kind of dnmt rna deficiency Chinese hamster ovary celI system and preparation method and application - Google Patents
A kind of dnmt rna deficiency Chinese hamster ovary celI system and preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of dnmt rna deficiency Chinese hamster ovary celI system and its preparation method and application, belong to gene engineering technology field.The present invention knocks out Chinese hamster ovary celI dnmt rna Dnmt3a genes using CRISPR/Cas9 gene editings technology, screen and identify and obtain dnmt rna Dnmt3a deficiency Chinese hamster ovary celIs, the novel C HO cell expression systems based on dnmt rna deficiency are established by transfecting carrier for expression of eukaryon and screening stable expression Recombinant CHO cell line.The present invention establishes recombination expressing cho cell system by host CHO cell genetic modification, the problem of can significantly improving the expression of recombinant protein while overcome expression of recombinant proteins unstable, improves expression of recombinant proteins stability.
Description
Technical field
The present invention relates to a kind of preparation method of dnmt rna deficiency Chinese hamster ovary (CHO) cell line and
Using belonging to gene engineering technology field.
Background technology
The 1980s tissue plasminogen activator (tissue type plasminogen activator,
T-PA) successful expression and U.S. is obtained first in Chinese hamster ovary (Chinese hamster ovary, CHO) cell is recombinated
State FDA ratifies to be used for clinic, indicates the arrival in the bio-pharmaceuticals epoch based on Chinese hamster ovary celI.Chinese hamster ovary celI source produces at present
Recombinant pharmaceutical proteins accounted for mammalian cell production nearly 70%.But at this stage there is expression quantity in expressing cho cell system
It is low, especially unstable expression the problems such as, can not meet that being used for vaccine development, clinical treatment and gene therapy etc. increasingly increases
Long demand.Therefore, Chinese hamster ovary celI recombination expression research is carried out using the method for gene and cell engineering, turns base for improving it
Because expression and expression stability, the recombinant protein c HO cell expression systems for establishing stable high yield have highly important meaning
Justice.Research and render transgenic by reducing promoter Zhong CpG islands (CpG islands) to methylating it has proven convenient that insensitive have
Beneficial to the efficient and continuous expression of recombinant protein.For example, the C-179 of hCMV-MIE promoter transcription initiation sites upstream is sported
G can significantly improve the continuous expression of recombinant protein in stable transfection Chinese hamster ovary celI.Therefore, DNA methylation is furtherd investigate to turning base
Influence and its mechanism because of expression will be helpful to the recombinaant CHO cell system that screening obtains stable high yield.
DNA methylation (DNA methylation) is a kind of common apparent something lost in protokaryon and eukaryotic gene group
Pass modification, and in mammal gene expression important regulating and controlling mode, played a crucial role in terms of genetic transcription suppression：Gene
The hyper-methylation state on promoter region (including Binding site for transcription factor) CpG islands can directly prevent the combination of transcription factor
And cause the change of chromatin Structure, combined so as to limit transcription factor with gene promoter, so to genetic transcription, montage,
Translation and expression quantity etc. impact.The Effect study of expression of recombinant proteins is mainly collected in Chinese hamster ovary celI about DNA methylation
In in the transformation of promoter, including the point mutation of promoter methylation site, the application without CpG islands promoter, synthetic promoter
And insertion of core CpG islands element etc..DNA methylation need dnmt rna (DNA methyltransferase,
Dnmt) include from the beginning transmethylase Dnmt3a and Dnmt3b and DNA methylation maintains enzyme Dnmt1 catalysis, particularly
The promoter methylation of Dnmt3a mediations and the unstable expression of transgenosis are closely related.
Currently, carry out transformation to cell using cell engineering to have been widely used, especially by gene knockout or base
Because of the expression of the influences such as silence and special biological related gene, changed with to obtain special biological
Cell.To be current and following transformation CHO because it has easy to operate, stably, flexibly and the technical advantage such as efficiency height
Cell constantly lifts the major technique strategy of its recombinant protein production capacity.
The content of the invention
In order to solve the above technical problems, the present invention knocks out Chinese hamster ovary celI DNA first using CRISPR/Cas9 gene editings technology
Based transferase Dnmt3a genes, screen and identify acquisition dnmt rna Dnmt3a deficiency Chinese hamster ovary celIs, it is true by transfecting
Nuclear expression carrier simultaneously screens stable expression Recombinant CHO cell line and established based on the new of dnmt rna Dnmt3a deficiencies
Type is without the expressing cho cell system to methylate.
In order to realize the above object the technical solution adopted in the present invention is as follows：
A kind of Chinese hamster ovary celI system of dnmt rna Dnmt3a gene defection types.
The dnmt rna Dnmt3a genomic dna sequences are GenBank：NW_003613640.1, Dnmt3a are lacked
It is 41138-41336 bit bases that swaged Chinese hamster ovary celI genomic dna sequence, which knocks out 199 bases of missing,.
Further, any one of the Chinese hamster ovary celI system in CHO-K1, CHO-S, CHO-DG44.
The present invention also provides the preparation method of the Chinese hamster ovary celI system of above-mentioned dnmt rna Dnmt3a gene defection types, can
Chinese hamster ovary celI dnmt rna Dnmt3a genes are knocked out using CRISPR/Cas9 gene editings technology and are obtained；Using
CRISPR/Cas9 gene editing technology knockout dna transmethylase Dnmt3a genes, screen and identify acquisition Dnmt3a deficiencies
Chinese hamster ovary celI, bred by cell, can the characteristics of cell biology such as Apoptosis detection checking deficient cell system be carried out normally
Propagation and Secondary Culture.
Specifically, the above method comprises the following steps：
1) target practice site is determined for candidate gene：According to Chinese hamster dnmt rna base in NCBI GenBank
Because Dnmt3a genome sequences (No.NW_003613640.1) and mRNA sequence (No.XM_016964036.1) design primer enter
Row corresponding gene group DNA genetic fragments PCR is expanded, and PCR amplification genes fragment is carried out to the accuracy of cloning and sequencing checking sequence；
Using online tool (http://crispr.mit.edu/) Computer Aided Design Dnmt3a genes chimeric single-chain guide RNA
(chimeric single-guide RNA, chimeric sgRNA) target practice site, separately design two pairs according to targeting sequence and draw
Thing is with construction expression CRISPR/Cas9 target practice sgRNA carriers；
2) structure of sgRNA carriers：Use Bbs I restriction enzymes linearisation px330 vector plasmids and glue reclaim line
Property fragment；The single-stranded annealing of sgRNA oligonucleotides forms double-strand；Linearisation px330 carriers were attached with double-strand sgRNA
Night；Connection product converts DH5 α competent cells, the screening of ammonia benzyl resistance coated plate, and picking single bacterium colony, 37 DEG C of shaking tables shake bacterium and carried overnight
Take plasmid and carry out sequence verification；Choose and correct positive colony plasmid progress cell transfecting is sequenced；
3) Chinese hamster ovary celI is transfected, monoclonal is selected and cell knocks out identification
By 2.0 × 10 before transfection5Individual Chinese hamster ovary celI is inoculated in 24 well culture plates, when cell fusion degree reaches 80%-90%
When add the above-mentioned recombinant plasmid corotation transfected cho cell containing sgRNA；Transfectional cell is subjected to gradient dilution and Method of Limited Dilution,
The monoclonal mutant cell with independently knocking out is picked out to reach；To obtain the defects of type cell line extract its genome
DNA, expand Dnmt3a genetic fragments using PCR and be sequenced to determine gene knockout success.
The eucaryon that the present invention also provides the Chinese hamster ovary celI system comprising above-mentioned dnmt rna Dnmt3a gene defection types is thin
Cellular expression system.
The present invention also provides the preparation method of above-mentioned eukaryotic cell expression system, including：Target gene is inserted into expression to carry
In body, structure obtains recombinant expression carrier；The recombinant expression carrier is transfected to the Chinese hamster ovary celI system for entering the deficiency, through sieve
Choosing obtains expression system.
Further, constructed expression vector can be transfected to Dnmt3a deficiencies and normal control Chinese hamster ovary celI, screening is more
Clone CHO recombinant cell strains and Secondary Culture, detection and analysis target gene transient expression and steady in a long-term in recombinaant CHO cell
Expression.
The present invention also provides the Chinese hamster ovary celI system of above-mentioned dnmt rna Dnmt3a gene defection types, above-mentioned eukaryotic
Expression system is preparing the application of destination protein etc..
Target gene, which can not continue high efficient expression or even expression decline occurs, in recombinant cell strain is carried out using Chinese hamster ovary celI
Recombinant protein production is universal and protrudes the problem of existing.It is now recognized that DNA methylation is the master for causing this unstable expression
Want reason, the promoter methylation and the close phase of unstable expression of transgenosis of particularly dnmt rna DNMT3a mediations
Close.In recent years, cell engineering Reconstruc-tion policy is sunk using cell engineering particularly gene knockout, gene overexpression or gene
The technology such as silent, change the biological function gene expression dose related to expression of recombinant proteins amount or activity, show to improve weight
The great potential of histone yield and (or) activity.Therefore, host cell genetic modification and expression vector are optimized phase by the present invention
With reference to, utilize CRISPR/Cas9 gene editings technique construction based on dnmt rna deficiency novel C HO cells expression
System, the expression of recombinant protein can be significantly improved, overcome expression existing for the system of being presently expressed by it is relatively low, expression
The problem of unstable.
Brief description of the drawings
Fig. 1：Chinese hamster ovary celI Dnmt3a gene knockout monoclonal Dnmt3a-30 sequencing identification results；Wherein, strikethrough is drawn
For 199 base sequences of gene knockout missing, arrow meaning is deletion mutation site, and underscore expands for Dnmt3a genetic fragments
Increase primer, wave is sgRNA target practices site.
Fig. 2：CCK-8 methods detect Dnmt3a deficiency Chinese hamster ovary celI monoclonal cell Dnmt3a-30 and Dnmt3a-40 and just
Normal CHO-K1 Cell proliferation results figures.
Fig. 3：Flow cytometry Dnmt3a deficiencies Chinese hamster ovary celI and normal Chinese hamster ovary celI apoptosis result figure.
Fig. 4：The carrier for expression of eukaryon pWTY-02 schematic diagrames of CMV promoter driving.
Fig. 5：The eGFP genes driven by CMV promoter are expressed in Dnmt3a deficiencies Chinese hamster ovary celI and normal Chinese hamster ovary celI
Positive cell rate.
Fig. 6：The eGFP of CMV promoter driving expression is expressed surely in Dnmt3a deficiencies Chinese hamster ovary celI and normal Chinese hamster ovary celI
Qualitative experiment result.
Fig. 7：Expression quantity detection and analysis knot of the recombinant epo albumen in Dnmt3a deficiencies Chinese hamster ovary celI and normal Chinese hamster ovary celI
Fruit.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.It is unreceipted specific in embodiment
Technology or condition person, according to the technology or condition described by document in the art, such as Sambrook equimolecular cloning experimentation hands
Volume (Sambrook J＆Russell DW, Molecular Cloning:A Laboratory Manual, 2001), or according to
Product description is carried out.Agents useful for same or the unreceipted production firm person of instrument, it is that can be commercially available by regular distributor
Conventional products.
The knockout of embodiment 1CHO cell Dnmt3a genes
1.1 determine target practice site for candidate gene
According to Chinese hamster dnmt rna gene Dnmt3a genome sequences (No.NW_ in NCBI GenBank
003613640.1) and mRNA sequence (No.XM_016964036.1) designs primer (D3a-Ex1seq-L:5′-
GACCACAAGAATTCCGGCTC-3′,D3a-Ex1seq-R:5 '-CGTGTGTGAATCTGTGTGGG-3 ') carry out corresponding gene
Fragment PCR is expanded, and pcr amplified fragment is carried out to the accuracy of cloning and sequencing checking sequence.Using online tool (http://
Crispr.mit.edu/) the chimeric single-chain guide RNA target practices site of the Dnmt3a genes of Computer Aided Design is as follows：
D3a-Ex1-98rev:5′-ATCATCCTCCCGCTCCAAAGTGG-3′；
D3a-Ex1-308fw:5′-TTTGAGGGGTCATCCTTGCAGGG3′
Two pairs of primers are separately designed with construction expression CRISPR/Cas9 target practice sgRNA carriers according to targeting sequence.
The structure of 1.2sgRNA carriers
Use Bbs I restriction enzymes linearisation px330 vector plasmids and glue reclaim linear fragment.SgRNA few nucleosides
The single-stranded annealing of acid forms double-strand.Linearisation px330 carriers and double-strand sgRNA are attached overnight.Connection product conversion DH5 α
Competent cell, the screening of ammonia benzyl resistance coated plate, picking single bacterium colony, 37 DEG C of shaking tables, which shake bacterium and extract plasmid overnight and carry out sequencing, to be tested
Card.Choose and correct positive colony plasmid progress cell transfecting is sequenced.
1.3CHO cell transfectings, monoclonal are selected and cell knocks out identification
By 2.0 × 10 before transfection5Individual CHO-K1 cells are inoculated in 24 well culture plates, when cell fusion degree reaches 80%-
The above-mentioned recombinant plasmid cotransfection CHO-K1 cells containing sgRNA are added when 90%.Transfectional cell is subjected to gradient dilution and pole
Limit dilution, the monoclonal mutant cell with independently knocking out is picked out to reach.Utilize CRISPR/Cas9 gene knockout methods
Expand the result with PCR, our final screenings obtain 6 plants of Dnmt3a deficiency Chinese hamster ovary celIs monoclonals (clone 30,31,
32,33,40,41).The sequencing result of the wherein pcr amplification product of Dnmt3a deficient cells strain clone 30 is shown, clpp gene
Except the deletion mutation for causing 199 bases of Dnmt3a genes, gene knockout success (result is shown in Fig. 1).
The biological characteristics checking of embodiment 2Dnmt3a deficiency Chinese hamster ovary celIs
Using wild type CHO-K1 cells as control, cell is carried out to the Dnmt3a deficiency Chinese hamster ovary celIs monoclonal of acquisition
Growth characteristics are verified, including cellular morphology and growth conditions observation, CCK-8 methods detection cell proliferative conditions, flow cytometry
(FCM) Apoptosis situation is detected, can checking Dnmt3a deficiency Chinese hamster ovary celIs system normally be grown and passed on.CCK-8
(result is shown in figure to kit (Cell Counting Kit-8 kits, green skies Beyotime biotech firms) detection cell propagation
2) prompted with Apoptosis by Flow Cytometry result (result is shown in Fig. 3), the Dnmt3a deficiency Chinese hamster ovary celI strains of acquisition can
It is normally carried out growing and passes on, the biological characteristics such as cell growth state, form, cell propagation, Apoptosis and normal CHO is thin
Born of the same parents are without difference.
The structure of the recombinant expression carrier of the different promoters of embodiment 3 driving
The present invention for maternal carrier, is built and driven by CMV promoter with eukaryotic expression vector pIRES neo2 (Clontech companies)
The carrier for expression of eukaryon pWTY-02 of dynamic expressing green fluorescent protein (eGFP) (see Fig. 4).
Expression analysis of the target gene eGFP of embodiment 4 in recombinaant CHO cell
4.1CHO cell transfecting
CHO-K1 cells are cultivated, 1 day before cell transfecting, with 1 × 105Fresh free serum culture is arrived in cell density passage
Base, liposome 3000 (Invitrogen, USA) can use to be transfected when cell fusion degree reaches 80%-90%.Transfect respectively
Two groups of Chinese hamster ovary celIs：Dnmt3a deficiencies and normal control CHO-K1 cells.3 multiple holes of each parallel transfection of plasmid.
4.2 transfection cell strain transient expressions are observed
24h is transfected, inverted fluorescence microscope observation transiently transfects the expression of eGFP in cell.As a result show, eucaryon
Expression vector is suitable in Dnmt 3a deficiencies and normal CHO-K1 cell transfectings efficiency, both eGFP express positive cell rate without
Significant difference (see Fig. 5).This explanation Dnmt 3a gene knockout does not influence on cell transfecting efficiency.
The 4.3 stable polyclonal Chinese hamster ovary celI strains of expression are screened and eGFP expression analysis steady in a long-term
G418 is added to screen, screening and culturing obtains the polyclonal cells strain of stable conversion and having screening pressure (to add after two weeks
G418) and without the generation of Secondary Culture 50 under screening pressure (being not added with G418), in every 10 generation, is respectively by each experimental group Chinese hamster ovary celI according to each
Sample 106Individual cell carries out flow cytomery analysis, measure eGFP average fluorescent strengths (Mean fluorescence
intensity,MFI).Flow cytometer detection result shows, regardless of whether G418 screening pressures be present, is driven and expressed by CMV promoter
EGFP can be expressed steadily in the long term (see Fig. 6) in Dnmt3a deficiencies Chinese hamster ovary celI.
Expression analysis of the hematopoietin of embodiment 5 (EPO) destination protein in recombinaant CHO cell
For further effect of the test Dnmt3a deficiencies Chinese hamster ovary celI to recombinant protein expression stability, we are with plasmid
Based on pWTY-02, construct and hematopoietin (Erythropoietin, EPO) gene expression is driven by CMV promoter
Carrier for expression of eukaryon.The plasmid of expression vector containing EPO of structure is transfected into Dnmt3a deficiencies and normal CHO-K1 respectively
Cell.The cell of transfection cultivates 15 days to screen the recombinant cell of stable transfection in the culture medium containing G418 (800 μ g/mL)
Pond (cell pool), recombinaant CHO cell is subjected to Secondary Culture to 50 generations every 3 days.Then by recombinaant CHO cell in 125mL
Cultivate CD CHO culture mediums (the Life Technologies public affairs determined in shaking flask with 25mL without albumen, serum-free, chemical composition
Department, the Glu containing 8mM in culture medium) culture 5 days to cell number reaches 8 × 106, be collected by centrifugation supernatant be used for weight
The detection of expression analysis of group epo protein.
Recombinant epo albumen is tested and analyzed in Dnmt3a deficiencies and normal CHO-K1 cell pools using Western blot
Expression.Specifically, the supernatant containing recombinant epo is added the bath to boil water of 5 × SDS sample buffers and boiled 10 minutes and fully become
Property.Taking 10 μ L protein samples respectively, wet robin is transferred to cellulose nitrate after the separation of 10%SDS- polyacrylamide gel electrophoresises
Plain film.5%BSA close membranes 2 hours, 1:4 DEG C of overnight incubations of anti-EPO primary antibodies (Santa Cruz, CA) of 1000 dilutions, TBST are washed
Add 1 after film:37 DEG C of the anti-mouse IgG secondary antibodies (Santa Cruz, CA) of the HRP marks of 5000 dilutions are incubated 1 hour, and TBST washes film
Afterwards plus chemiluminescence nitrite ion develops, and is observed in gel imager and gathers image result, the gray value of analysis purpose albumen
Represent the expression of testing protein.Western blot testing results show that recombinant epo albumen is in Dnmt3a deficiencies CHO
Average expression amount (860.5 ± 42.9mg/L) in cell pool is significantly higher than the average expression in non-deficiency Chinese hamster ovary celI pond
Measure (373.7 ± 29.6mg/L).Recombinant epo albumen is in the restructuring Dnmt3a deficiency Chinese hamster ovary celI ponds in 50 generations of stable culture and not
Expression quantity in deficiency Chinese hamster ovary celI pond is respectively：672.7 ± 35.1mg/L and 157.9 ± 16.2mg/mL.Expression difference
Statistical significance is respectively provided with (see Fig. 7, P<0.05).These results illustrate that Dnmt3a deficiencies Chinese hamster ovary celI can significantly improve restructuring
The expression and long-term expression stability of epo protein.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
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Claims (10)
- A kind of 1. Chinese hamster ovary celI system of dnmt rna Dnmt3a gene defection types.
- 2. Chinese hamster ovary celI system according to claim 1, it is characterised in that the dnmt rna Dnmt3a genomes DNA sequence dna is GenBank：NW_003613640.1, Dnmt3a deficiency Chinese hamster ovary celI genomic dna sequence knock out missing 199 Base is 41138-41336 bit bases；And/orPreferably, any one of the Chinese hamster ovary celI system in CHO-K1, CHO-S, CHO-DG44.
- 3. the preparation method of the Chinese hamster ovary celI system of claim 1 or 2, it is characterised in that utilize CRISPR/Cas9 gene editings Technology knocks out Chinese hamster ovary celI dnmt rna Dnmt3a genes and obtained.
- 4. preparation method according to claim 3, it is characterised in that comprise the following steps：1) target practice site is determined for candidate gene：According to Chinese hamster dnmt rna gene in NCBI GenBank Dnmt3a genome sequences No.NW_003613640.1 and mRNA sequence No.XM_016964036.1 design primers carry out corresponding Genomic DNA genetic fragment PCR is expanded, and PCR amplification genes fragment is carried out to the accuracy of cloning and sequencing checking sequence；Apply The chimeric single-chain guide RNA target practices site of the Line tool Computer Aided Design Dnmt3a genes, separately design two pairs according to targeting sequence and draw Thing is with construction expression CRISPR/Cas9 target practice sgRNA carriers；2) structure of sgRNA carriers：Use Bbs I restriction enzymes linearisation px330 vector plasmids and the linear piece of glue reclaim Section；The single-stranded annealing of sgRNA oligonucleotides forms double-strand；Linearisation px330 carriers and double-strand sgRNA are attached overnight；Even Thing of practicing midwifery converts DH5 α competent cells, the screening of ammonia benzyl resistance coated plate, and picking single bacterium colony, 37 DEG C of shaking tables shake bacterium and extract plasmid overnight And carry out sequence verification；Choose and correct positive colony plasmid progress cell transfecting is sequenced；3) Chinese hamster ovary celI is transfected, monoclonal is selected and cell knocks out identification；Preferably, by 2.0 × 10 before transfection5Individual Chinese hamster ovary celI is inoculated in 24 well culture plates, when cell fusion degree reaches 80%- The above-mentioned recombinant plasmid corotation transfected cho cell containing sgRNA is added when 90%；Transfectional cell is subjected to gradient dilution and the limit is dilute Release, the monoclonal mutant cell with independently knocking out is picked out to reach；To obtain the defects of type cell line extract its gene Group DNA, expand Dnmt3a genetic fragments using PCR and be sequenced to determine gene knockout success.
- 5. preparation method according to claim 4, it is characterised in that for Dnmt3a genomic DNA fragments PCR amplifications Primer is as follows：D3a-Ex1seq-L:5′-GACCACAAGAATTCCGGCTC-3′；D3a-Ex1seq-R:5′-CGTGTGTGAATCTGTGTGGG-3′；And/orThe chimeric single-chain guide RNA target practices site is as follows：D3a-Ex1-98rev:5′-ATCATCCTCCCGCTCCAAAGTGG-3′；D3a-Ex1-308fw:5′-TTTGAGGGGTCATCCTTGCAGGG3′。
- 6. the Chinese hamster ovary celI system prepared comprising any one of the Chinese hamster ovary celI system of claim 1 or 2 or claim 3-5 methods described Eukaryotic cell expression system.
- 7. the preparation method of eukaryotic cell expression system described in claim 6, it is characterised in that including：Target gene is inserted In expression vector, structure obtains recombinant expression carrier；The recombinant expression carrier is transfected to the Chinese hamster ovary celI for entering the deficiency System, expression system is obtained through screening.
- 8. eukaryotic cell expression system described in the Chinese hamster ovary celI system or claim 6 described in claim 1 or 2 is preparing purpose egg The application of white aspect.
- 9. the primer for Chinese hamster ovary celI dnmt rna gene Dnmt3a genomic DNA fragments PCR amplifications：D3a-Ex1seq-L:5′-GACCACAAGAATTCCGGCTC-3′；D3a-Ex1seq-R:5′-CGTGTGTGAATCTGTGTGGG-3′。
- 10. for selectively targeted Chinese hamster ovary celI dnmt rna gene Dnmt3a chimeric single-chain guide RNA, its target practice position Point is as follows：D3a-Ex1-98rev:5′-ATCATCCTCCCGCTCCAAAGTGG-3′；D3a-Ex1-308fw:5′-TTTGAGGGGTCATCCTTGCAGGG3′。
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| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
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| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015092735A1 (en) * | 2013-12-20 | 2015-06-25 | Novartis Ag | Novel eukaryotic cells and methods for recombinantly expressing a product of interest |
| CN107090441A (en) * | 2010-12-09 | 2017-08-25 | 巴斯德研究所 | The method based on MGMT for obtaining high yield expression of recombinant proteins |
-
2017
- 2017-11-28 CN CN201711211535.3A patent/CN107828738A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107090441A (en) * | 2010-12-09 | 2017-08-25 | 巴斯德研究所 | The method based on MGMT for obtaining high yield expression of recombinant proteins |
| WO2015092735A1 (en) * | 2013-12-20 | 2015-06-25 | Novartis Ag | Novel eukaryotic cells and methods for recombinantly expressing a product of interest |
| CN106029692A (en) * | 2013-12-20 | 2016-10-12 | 诺华股份有限公司 | Novel eukaryotic cells and methods for recombinantly expressing a product of interest |
Non-Patent Citations (4)
| Title |
|---|
| LOWE,T.M.等: "Accession号：NW_003613640.1，Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold399, whole genome shotgun sequence", 《GENBANK》 * |
| MARINA AGA等: "Construction of a gene knockout CHO cell line using a simple gene targeting method", 《BMC PROCEEDINGS》 * |
| 孙涛等: "Crispr/Cas9 技术在CHO 细胞中基因敲除的应用", 《中国医药工业杂志》 * |
| 无作者: "Genbank：XM_016964036.1，PREDICTED: Cricetulus griseus DNA (cytosine-5-)-methyltransferase 3 alpha (Dnmt3a),transcript variant X1, mRNA", 《GENBANK》 * |
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