CN107034234B - A kind of kit for being used to knock out two kinds of genes of FUT8 and DHFR in Chinese hamster ovary celI - Google Patents
A kind of kit for being used to knock out two kinds of genes of FUT8 and DHFR in Chinese hamster ovary celI Download PDFInfo
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- CN107034234B CN107034234B CN201710357070.6A CN201710357070A CN107034234B CN 107034234 B CN107034234 B CN 107034234B CN 201710357070 A CN201710357070 A CN 201710357070A CN 107034234 B CN107034234 B CN 107034234B
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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
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
- C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
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Abstract
It is used to knock out the kit of FUT8 genes and DHFR genes in Chinese hamster ovary celI the invention provides a kind of, it includes SEQ ID NO：1 and/or SEQ ID NO：SgRNA sequences described in 3, and SEQ ID NO：SgRNA sequences described in 23.Present invention also offers a kind of while knock out the method and its engineering cell strain of FUT8 and DHFR genes in Chinese hamster ovary celI.Kit of the present invention can be used for FUT8 the and DHFR genes for fast and efficiently knocking out Chinese hamster ovary celI, the antibody class medicine modified available for high efficient expression without fucose, and antibody production height, application prospect are good.
Description
Technical field
The invention belongs to genetic engineering field, and in particular to one kind is used to knock out FUT8 genes and DHFR bases in Chinese hamster ovary celI
The kit of cause.
Background technology
Chinese hamster ovary cell (Chinese hamster ovary cell, Chinese hamster ovary celI) is current production medicine egg
White the most frequently used host cell, is widely used in the research and development and production of the products such as antibody, recombinant protein medicine and vaccine.Antibody
Medicine is current most important biotechnology class medicine, how further to improve the efficiency and yield of Chinese hamster ovary celI antibody drug, is
The focus studied at present.
α -1,6 fucosyltransferases (α -1,6fucosyltrasnferase, FUT8) are fucosyl transferase genes
A member of superfamily, the core fucosylation of FUT8 catalysis is the important posttranslational modification of glycoprotein and function controlling mode.
During using host cell expression antibody drug, its function and stability in vivo of the glycosylation modified influence of antibody.Study table
Bright, compared with the antibody of fucosylation, removing or reduce the antibody of fucosylation, to show stronger ADCC effects (anti-
Body dependent cellular cytotoxicity), antibody performance is good.Therefore, the FUT8 of engineering cell strain is knocked out, makes the antibody of expression without fucosido
Change modification, be the effective means for improving antibody performance.
Dihyrofolate reductase (dihydrofolate reductase, DHFR) is that one kind is catalyzed 5,6- dihydrofoilic acid also
Originally it was the protease of 5,6,7,8- tetrahydrofolic acids, and participated in intracellular purine metabolism process.When cell line has lacked DHFR genes,
The expression vector transfectional cell of DHFR genes will be carried, positive cell also just obtains DHFR genes, now adds in the medium
Entering methopterin (Methotrexate, MTX), DHFR gene magnifications can be caused, its related both sides fragment is also accordingly expanded,
The copy number of target gene can be made increase by hundreds of Dao thousands of times, so as to the high efficient expression for the gene that achieves the goal.Therefore, urgent need will strike
Except the DHFR genes of Chinese hamster ovary celI strain, to improve exogenous gene albumen expression quantity.
Crispr/Cas9 systems, be bacterium and one kind that archeobacteria develops out during evolution be used to resisting it is external
The immune intrusion system of infringement, in modern genetic engineering application field and TALEN (transcription activator-like
Effector nuclease) and ZFN (zinc-finger nuclease) technology turn into three big genome editor works side by side
Tool.CRISPR-Cas9 technologies have specific DNA recognition capability, and Cas9 endonucleases are in guide ribonucleic acid (guide
RNA, sgRNA) the lower cutting double-stranded DNA of guiding, is caused genome double-strand break, is produced using the unstability of cellular genome reparation
Raw non-specific restructuring repairs mistake (insertion or missing) to produce, and gene function is caused so as to produce frameshift mutation
Lose, realize the purpose of gene knockout.TALEN and ZFN technologies are compared to, its sgRNA design and synthetic work amount are much
It is easily operated less than the building process of TALEN and ZFN identification modules and more efficient, be easier to obtain homozygous mutation
Body.
At present in research, have no by CRISPR-Cas9 systems, knock out two kinds of genes of FUT8 and DHFR in Chinese hamster ovary celI
Report.
The content of the invention
It is used to knock out the kit of FUT8 genes and DHFR genes in Chinese hamster ovary celI it is an object of the invention to provide a kind of
And application thereof.
FUT8 genes：For α-(1,6)-fucosyl transferase gene；
DHFR genes：For dihydrofolate reductase gene.
It is used to knock out the kit of FUT8 genes and DHFR genes in Chinese hamster ovary celI the invention provides a kind of, it includes SEQ
ID NO：1 and/or SEQ ID NO：SgRNA sequences described in 3, and SEQ ID NO：SgRNA sequences described in 23.
It is used to knock out the kit of FUT8 genes and DHFR genes in Chinese hamster ovary celI present invention also offers a kind of, it includes
SEQ ID NO：13、SEQ ID NO：17 and SEQ ID NO：SgRNA sequences described in 31
Present invention also offers mentioned reagent box in the purposes for knocking out two kinds of genes of FUT8 and DHFR in Chinese hamster ovary celI.
Present invention also offers purposes of the mentioned reagent box in the cell line for preparing FUT8 and DHFR genes while missing.
Present invention also offers method that is a kind of while knocking out FUT8 and DHFR genes in Chinese hamster ovary celI, and it is to utilize
CRISPR/Cas9 systems are transformed two kinds of genes in Chinese hamster ovary celI, and step is as follows：
A, SEQ ID NO are synthesized：Sequence and its complementary strand described in 1 and/or 3, denaturation, annealing, form double-strand, are inserted into
The multiple cloning sites of Cas9 expression vectors, obtain recombinant expression plasmid；
B, by step a recombinant expression carrier transfection CHO cell, picking individual cells, inoculated and cultured；
C, after individual cells propagation is monoclonal, identify and confirm that FUT8 genes are knocked, and obtain FUT8 genes and lack
The cell line FUT8- of mistake/-, it is standby；
D, SEQ ID NO are synthesized：Sequence and its complementary strand described in 23, denaturation, annealing, form double-strand, are inserted into Cas9
The multiple cloning sites of expression vector, obtain recombinant expression plasmid；
E, step c FUT8-/- cell line is taken, is transfected with step d recombinant expression carrier, picking individual cells, inoculation
Culture；
F, after individual cells propagation be monoclonal after, identify and confirm that DHFR genes are knocked, and obtain FUT8 genes and
The cell line that DHFR genes lack simultaneously.
Wherein, the Chinese hamster ovary celI is CHO-S sub-types of cells.
Wherein, the Cas9 expression vectors are CRISPR/Cas9 carriers.
Present invention also offers the FUT8 deletion cells strains that the above method is prepared.
FUT8 the and DHFR genes while the cell line of missing being prepared present invention also offers the above method.
The present invention include the kits of sgRNA sequences, can be used for FUT8 that is quick, efficient while knocking out Chinese hamster ovary celI and
DHFR genes, the silence to FUT8 and DHFR genes in genomic level is realized, it is easy to operation；The FUT8 of acquisition and
The cell line that DHFR genes lack simultaneously, there are identical cellular morphology and growth characteristics with wild type CHO-S cells, but expand
Increasing Efficiency is higher, the antibody class medicine modified available for high efficient expression without fucose, and antibody production is high, is carried for antibody expression
Selected for a kind of good engineering cell strain.
Obviously, according to the above of the present invention, according to the ordinary technical knowledge and customary means of this area, do not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The embodiment of form by the following examples, the above of the present invention is remake further specifically
It is bright.But the scope that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following example.It is all to be based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description of the drawings
Fig. 1 is CHO-S/FUT8-/-/SD8 cell growth characteristics figures.
Fig. 2 is CHO-S/FUT8-/-/SD8 cell detection of mycoplasma figures (PC:Positive control；NC:Negative control；SE2/
SD8/SE7:Monoclonal cell strain is numbered).
Fig. 3 is that cell surface fucosido detects and cellular morphology observation is schemed,
Upper left and lower-left：WtCHO-S cells；Upper right and bottom right：CHO-S/FUT8-/-/SD8 cells.
Fig. 4 is TNFR2-Fc Identification of Fusion Protein figures.
Fig. 5 is capillary electrophoresis analysis TNFR2-Fc glycans size figures.
Fig. 6 is that CHO-S cell DHFR gene Exon1 partial sequences and sgRNA target sequence chart.
Fig. 7 is that fluorescein enzyme process identifies the efficiency chart that each sgRNA guides Cas9 to cut target dna.
Fig. 8 is CHO-S/DHFR-/-/G3 monoclonal cell speed of growth figures.
Fig. 9 is cellular morphology observation figure,
Left figure：CHO-S/DHFR-/-/G3 monoclonal cells；Right figure：Wild type CHO-S cells (control)；
Figure 10 is CHO-S/DHFR-/-/G3 cell detection of mycoplasma figures (PC：Positive control；NC：Negative control；A2, G3：
Monoclonal cell strain is numbered).
Figure 11 is that the lower TNFR2-Fc of different MTX dosage processing expresses spirogram.
Figure 12 is CHO-S/FUT8-/-/DHFR-/-/G3 cell growth characteristics figures.
Figure 13 is CHO-S/FUT8-/-/DHFR-/-/G3 cell detection of mycoplasma figures (PC:Positive control；NC:It is negative right
According to；B8/D11:Monoclonal cell strain is numbered).
Embodiment
It is described further below with embodiment, but the present invention is not limited to these embodiments.
Experiment reagent used in the present invention is as follows：
Lipofectamine 2000, Invitrogen company, REF.11668-030, lot.1828123；
Trypsin0.25%, Hyclone company, SH42605.01, Cas.9002-07-7；
DEME/F-12, Gibco company, REF.C11330500CP, Lot.8115411；
Foetal Bovine Serum, Hyclone companies, Cat.SH30084.03, Lot.YK0119；
Opti-MEM, Gibco company, REF.31985-062, Lot.1800574；
Phosphate Buffered Saline, Hyclone companies, Cat.SH30256.01B, Lot.NAG1428
DEME, Gibco company, REF.C11995500CP, Lot.8116413；
Renilla Luciferase Assay Substrate, Promega companies, REF.E289A,
Lot.0000119245；
Renilla Luciferase Assay Buffer, Promega companies, REF.E290A, Lot.0000141517；
Stop＆Glo Substrate, Promega companies, REF.E640A, Lot.0000133610；
HT Supplement, Gibco companies, REF.11067-030, Lot.1797757；
UCA CRISPR/Cas9 rapid builds and activity detection kit, hundred Ao Saitu companies, Cat.BCG-DX001,
Lot.BBCTG201409。
CHO-S cells：Purchased from invitrogen companies, article No.：A1155701.
Embodiment 1 knocks out FUT8 the and DHFR genes of Chinese hamster ovary celI using kit of the present invention
First, the intracellular FUT8 genes of CHO-S are knocked out
1st, the sgRNA of design targeting CHO-S cell FUT8 genes
According to the FUT8 gene orders of the Genbank Chinese hamster ovary celIs announced, design is for different extrons (Exon)
SgRNA sequences, the sgRNA sequences such as table 1 of design：
The sgRNAs sequences of table 1
| Sequence number | SgRNA sequences (5---3) | Direction | PAM sequences | SgRNA sequence locations |
| SgRNA2‐f(SEQ ID NO：1) | UGGGAUACCCACCACACUGC | Reversely | AGG | Exon8 |
| SgRNA4‐f(SEQ ID NO：2) | ACAUGGACUCUGGGGAGAAG | Reversely | TGG | Exon9 |
| SgRNA5‐f(SEQ ID NO：3) | GUCCAGCUUUGCAAGAAUCU | Reversely | TGG | Exon1 |
CHO-S cell genomic dnas are extracted, use the primer amplifying genom DNA of table 2, gel recovery amplified production, connection
To precut pUCA (LUC) carrier, apply Amp resistant panels, 4 clones of picking (mark respectively precut pUCA (LUC)-
FUT8-2/precut pUCA (LUC)-FUT8-4/precut pUCA (LUC)-FUT8-5, corresponding sgRNA2,4 and 5) surveyed
Sequence, and the FUT8 gene orders announced with Genbank compare, detection sgRNA target position sequences whether there is mutation, as a result show
SgRNA target sequences are without mutation.
Table 2 expands FUT8 gene sgRNA target spot the primers
SEQ ID NO：10 FUT8 gene sgRNA2-f target spot surrounding DNA sequences
SEQ ID NO：11 FUT8 gene sgRNA4-f target spot surrounding DNA sequences
SEQ ID NO：12 FUT8 gene sgRNA5-f target spot surrounding DNA sequences
2nd, the CRISPR/Cas9/sgRNA carriers of structure targeting FUT8 genes
SgRNA forward and reverse complementary series is respectively synthesized, and CACC joint sequences are added at the positive end of sequence 5, anti-
AAAC joint sequences are added to the end of sequence 5, composition sequence such as table 3, double-strand sgRNA fragments are set up by being denatured method for annealing.
Table 3 sets up each sgRNA forward and reverses sequence of CRISPR/Cas9/sgRNA carriers
| Sequence number | Direction | SgRNA sequences (5---3) |
| SgRNA2-f(SEQ ID NO：13) | It is positive | CACCGTGGGATACCCACCACACTGC |
| SgRNA2-f(SEQ ID NO：14) | Reversely | AAACGCAGTGTGGTGGGTATCCCAC |
| SgRNA4-f(SEQ ID NO：15) | It is positive | CACCGACATGGACTCTGGGGAGAAG |
| SgRNA4-f(SEQ ID NO：16) | Reversely | AAACCTTCTCCCCAGAGTCCATGTC |
| SgRNA5-f(SEQ ID NO：17) | It is positive | CACCGTCCAGCTTTGCAAGAATCT |
| SgRNA5-f(SEQ ID NO：18) | Reversely | AAACAGATTCTTGCAAAGCTGGAC |
With BsmB I digestion CRISPR/Cas9 carrier (the UCA CRISPR/Cas9 rapid builds of hundred Olympic Competition figure companies and work
Property detection kit in reagent), 1% Ago-Gel recovery purpose fragment, T4DNA ligases connect each sgRNA fragments and enzyme
Carrier is cut, and converts DH5a competent cells, applies Amp resistant panels, each flat board selects 5 positive colonies and carries out sequencing core
It is real.Sequencing correct plasmid is purified and preserved with high-purity kit.
4th, for the sgRNA activity identifications of FUT8 genes
PCR sequencing PCR is taken to identify the activity of each sgRNA guiding Cas9 cutting target genes.Concretely comprise the following steps：It is thin to pass on CHO-S
(cell culture medium is born of the same parents：DEME/F12+10%FBS) to 24 orifice plates, secondary daily lipofection transfection CRISPR-Cas9-
SgRNA-f and precut pUCA (LUC)-FUT8 carriers (wherein, Lipofectamine 2000 transfection reagent 2ul, CRISPR-
Cas9-sgRNA-f carriers 0.8ug, precut pUCA (LUC)-FUT8 0.2ug), transfection extracts cellular genome after 72 hours
DNA, PCR expand each sgRNA target areas domain gene, convert DH5a competent cells after being connected to carrier T, then apply Amp flat boards, often
Individual sgRNA corresponds to each 30 bacterial clones of picking on flat board and is sequenced, and analyzes sgRNA target gene site sequence catastrophes.
Sequencing situation is shown in Table 4.
The efficiency of each sgRNA guiding Cas9 cutting target dnas of the sequencing analysis of table 4
| Total sequencing number | Mutant target gene number | Mutation rate | |
| SgRNA2-f | 30 | 10 | 33% |
| SgRNA4-f | 30 | 4 | 13% |
| SgRNA5-f | 30 | 8 | 26% |
It can be seen that after carrying SgRNA2-f, SgRNA5-f plasmid-transfected cells, to target gene FUT8 mutation effect compared with
Good, mutation rate reaches as high as 33%, and after carrying SgRNA4-f plasmid-transfected cells, it is bad to the mutation effect of target gene.
To improve the efficiency of the inactivation intracellular FUT8 gene functions of CHO-S, using mixing transfection CRISPR-Cas9- in follow-up test
SgRNA2-f and CRISPR-Cas9-sgRNA5-f.
5th, the intracellular FUT8 genes of CHO-S are knocked out
Recovery CHO-S cells, reach 6 orifice plates, using lipofection, select CRISPR-Cas9-sgRNA2-f and
Two plasmid co-transfection CHO-S cells of CRISPR-Cas9-sgRNA5-f are (during transfection：The transfection reagents of Lipofectamine 2000
4ul, CRISPR-Cas9-sgRNA2-f carrier 1ug, CRISPR-Cas9-sgRNA5-f carrier 1ug), next day reaches 25cm2Side
In bottle, pressurization screening is carried out from the serum free growth medium containing 75ug/ml LcAs, it is every more to renew within 2-3 days
Fresh culture medium, after pressurizeing 2 weeks, limiting dilution assay monoclonal is carried out to survivaling cell and (coagulated in culture medium containing 50ug/ml lens
Collection element), cultivate into 4 piece of 96 orifice plate, continue culture 2 weeks.
6th, CHO-S/FUT8-/- monoclonal cell screening
Cell culture about 2 weeks in 96 orifice plates, select 30 monoclonals that cellular morphology is good, the speed of growth is fast and digested,
1/3 cell reaches one piece of 48 orifice plate (A plates), and another 2/3 cell reaches another piece of 48 orifice plates (B plates), and next day extracts cell base on B plates
Because of a group DNA (30 DNA samples), sequence around sgRNA2-f and sgRNA5-f target spots, agar are expanded respectively using the primer of table 2
Sugared gel reclaims purpose fragment, is then connected to carrier T, converts DH5a competent cells, applies Amp resistant panels and (puts down for totally 60 pieces
Plate), next day is from 5 cloning and sequencings of picking on each flat board.
It is compared with former sequence, selects the list that all missense mutations, cell growth state are good, the speed of growth is fast
Clone carries out secondary Colony Culture.5 monoclonals are selected from secondary monoclonal to be identified, it is prominent to select all wrong meanings
Become, the monoclonal that cell growth state is good, the speed of growth is fast and detection of mycoplasma is negative is amplified culture and freezes.
The speed of growth, cellular morphology observation point are carried out to monoclonal (CHO-S/FUT8-/-/SD8) cell finally chosen
Analysis and detection of mycoplasma, as a result show the CHO-S/FUT8-/-/SD8 monoclonal cell speeds of growth for knocking out FUT8 genes (see figure
1), form (see Fig. 3) and wild type CHO-S cell no significant differences, and without mycoplasma infection (see Fig. 2).
Wherein, the catastrophe of CHO-S/FUT8-/-/SD8 monoclonal cells is as follows：Double-stranded DNA breaks are different, and
Insertion/deletion bp numbers are not 3 integral multiples, wherein a chain (mutant A-) lacks 4bp, another chain (mutant B-) is more
1bp。
SEQ ID NO：19 CHO-S/FUT8-/-/SD8 monoclonal cells sequencing (mutant A- lacks 4bp)
SEQ ID NO：20 CHO-S/FUT8-/-/SD8 monoclonal cells sequencing (the more 1bp of mutant B-)
7th, the performance verification of CHO-S/FUT8-/- cell
7.1 cell surface fucoses detect
CHO-S/FUT8-/-/SD8 cells and wtCHO-S cells are spread to 48 orifice plates, next day removes culture medium, addition 10ug/
The LcA of ml luciferase mark, 37 degree are incubated 20 minutes, PBS 2 times, add 100ul PBS, use fluorescence
Micro- sem observation cell situation.As a result CHO-S/FUT8-/-/SD8 cell redgreen fluorescence is shown, and on wtCHO-S cell membranes
Green fluorescence is presented (see Fig. 3).
It can be seen that CHO-S/FUT8-/-/SD8 the cell lines for knocking out FUT8 do not express fucose.
The glycosylation modified detection of 7.2 expression products
1) CHO-S/FUT8-/-/SD8 cells and CHO-S cells expression TNFR2-Fc
Expression vector of the structure containing TNFR2-Fc fusions (i.e. Tumor Necrosis Factor Receptors-Fc fusion proteins)
(construction method is pcDNA3.1-TNFR2-Fc：Synthesize TNFR2-Fc genes, using NheI and XhoI double digestions TFNR2-Fc and
PcDNA3.1,1% Ago-Gel recovery purpose fragment, converts DH5a competent cells after the connection of T4DNA ligases, applies Amp
Resistant panel, cloning and sequencing is chosen, the correct clone of selection sequencing, plasmid is extracted using high-purity plasmid extraction kit), turn
Contaminate CHO-S/FUT8-/-/SD8 cell lines and wtCHO-S cell lines, and carry out cell pressurization culture (G418,800ug/ml) and
Monoclonal screens, and selects and expresses higher cell line, in 225cm2Cell culture and protein expression are carried out in square vase.
TNFR2-Fc Identification of Fusion Protein：Purify recovery Fc fusion proteins (in detail with the Protein A affinity columns of GE companies
Method is shown in company's specification), protein concentration, PAGE gel analyzing proteins purity are determined by BCA determination of protein concentration method
And size.As a result show that recovery protein band is single, purity is up to 97%, and molecular weight is with being expected unanimously.At deglycosylating enzyme
Manage TNFR2-Fc fusion proteins, PAGE gel electrophoresis showed glycans size about 3kda.As a result Fig. 4 is seen.
2) the TNFR2-Fc protein glycosylations analysis of CHO-S/FUT8-/-/SD8 cells expression
With the glycosyl of glycosylase cutting TNFR2-Fc fusion proteins, capillary electrophoresis analysis glycosyl size, molecule
Ladder is mannose Marker.As a result Fig. 5 is seen, the antibody glycosyl of CHO-S/FUT8-/-/SD8 cell lines expression compares wtCHO-S
The small mannose molecules size of antibody glycosyl of cell expression, molecular weight about 160, and fucosido is also about 160, from figure
As a result it is consistent with being contemplated to be from the point of view of, illustrate that CHO-S/FUT8-/-/SD8 fucose generation key gene FUT8 is destroyed
Afterwards, the recombinant protein molecule of fucose missing can be produced.
It can be seen that after FUT8 genes using sgRNA sequence knockouts Chinese hamster ovary celI of the present invention, (TNFR2-Fc melts the antibody of expression
Hop protein) modified without fucose, and then improve the ADCC efficiency of antibody.
2nd, the sgRNA designs of CHO-S cell DHFR genes are targetted and knock out compliance test result
1st, the sgRNA of design targeting CHO-S cell DHFR genes
It is different for First Exon (Exon1), design according to the DHFR gene orders of the Genbank Chinese hamster ovary celIs announced
SgRNA sequences, the sgRNA sequences of design are shown in Table 5：
The sgRNAs sequences of table 5
| Sequence number | SgRNA sequences (5---3) | Direction | PAM sequences | SgRNA sequence locations |
| SgRNA1‐h(SEQ ID NO：21) | GCAAGAACGGAGACCUUCCC | It is positive | TGG | Exon1 |
| SgRNA2‐h(SEQ ID NO：22) | GUCGCCGUGUCCCAGAAUAU | It is positive | GGG | Exon1 |
| SgRNA3‐h(SEQ ID NO：23) | GCCAAUGCUCAGGUACUGGC | It is positive | TGG | Exon1 |
CHO-S cell DHFR gene Exon1 partial sequences and sgRNA targetings sequence are shown in Fig. 6.
CHO-S cell genomic dnas are extracted, use the primer amplifying genom DNA of table 6, gel recovery amplified production, connection
To precut pUCA (LUC) carrier (UCA CRISPR/Cas9 rapid builds and activity detection kit of hundred Olympic Competition figure companies
Middle reagent), labeled as precut pUCA (LUC)-DHFR, Kna resistant panels are applied, 4 clones of picking are sequenced, and and
The DHFR gene orders that Genbank is announced compare, and detection sgRNA target position sequences whether there is mutation, as a result show sgRNA target spots
Sequence is without mutation.
Table 6 expands DHFR gene sgRNA target spot the primers
SEQ ID NO：26 DHFR gene SgRNA1-h, 2-h, 3-h target spot surrounding DNA sequences
2nd, the CRISPR/Cas9/sgRNA carriers of structure targeting DHFR genes
SgRNA forward and reverse complementary series is respectively synthesized, and CACC joint sequences are added at the positive end of sequence 5, anti-
AAAC joint sequences are added to the end of sequence 5, composition sequence is shown in Table 7, and double-strand sgRNA fragments are set up by being denatured method for annealing.
Table 7 sets up each sgRNA forward and reverses sequence of CRISPR/Cas9/sgRNA carriers
| Sequence number | SgRNA sequences (5---3) |
| SgRNA1-h- forward directions (SEQ ID NO：27) | CACCGCAAGAACGGAGACCTTCCC |
| Reverse (the SEQ ID NO of SgRNA1-h-：28) | AAACGGGAAGGTCTCCGTTCTTGC |
| SgRNA2-h- forward directions (SEQ ID NO：29) | CACCGTCGCCGTGTCCCAGAATAT |
| Reverse (the SEQ ID NO of SgRNA2-h-：30) | AAACATATTCTGGGACACGGCGAC |
| SgRNA3-h- forward directions (SEQ ID NO：31) | CACCGCCAATGCTCAGGTACTGGC |
| Reverse (the SEQ ID NO of SgRNA3-h-：32) | AAACGCCAGTACCTGAGCATTGGC |
With BsmB I digestion CRISPR/Cas9 carrier (the UCA CRISPR/Cas9 rapid builds of hundred Olympic Competition figure companies and work
Property detection kit in reagent), 1% Ago-Gel recovery purpose fragment, T4DNA ligases connect each sgRNA fragments and enzyme
Carrier is cut, and converts DH5a competent cells, applies Amp resistant panels, each flat board selects 5 positive colonies and carries out sequencing core
It is real.Sequencing correct plasmid is purified and preserved with high-purity kit.
3rd, for the sgRNA activity identifications of DHFR genes
Take luciferase methods to identify the activity of each sgRNA guiding Cas9 cutting target genes, concretely comprise the following steps：Passage
(293 cell culture mediums are 293 cells：High sugared DEME+10%FBS) to 24 orifice plates, secondary daily lipofection transfection
CRISPR-Cas9-sgRNA-h and SSA luciferase-cut-gene carriers (every kind of sgRNA does 3 transfections) are (wherein,
Lipofectamine 2000 transfection reagent 2ul, CRISPR-Cas9-sgRNA-h carrier 0.8ug, SSA-luciferase-
Cut-gene 0.2ug), transfected with CRISPR-Cas9 the and SSA luciferase-cut-gene carriers containing active sgRNA
As positive control (being labeled as positive).
Transfection takes supernatant 40ul after 72 hours, and adds 10ul luciferase substrates, detects luciferase intensity.
Testing result is shown in Table 8 and Fig. 7.
The fluorescein enzyme process of table 8 identifies the efficiency of each sgRNA guiding Cas9 cutting target dnas
| SgRNA1-h | SgRNA2-h | SgRNA3-h | Positive | |
| A | 435880 | 538070 | 1554960 | 858650 |
| B | 441770 | 632550 | 1455950 | 1075760 |
| C | 427220 | 515720 | 1672710 | 1011520 |
| Ave. | 434956.7 | 562113.3 | 1561207 | 981976.7 |
| sgRNA/PC | 0.44294 | 0.57243 | 1.589861 | 1 |
It can be seen that after carrying SgRNA3-h plasmid-transfected cells, caused fluorescence intensity is significantly better than positive control, explanation
Its activity is good, cuts the efficiency high of target dna, and other sgRNA's is ineffective.
4th, the intracellular DHFR genes of CHO-S are knocked out
Recovery CHO-S cells, reach 6 orifice plates, using lipofection, select CRISPR-Cas9-SgRNA3-h plasmids
Transfected CHO-S cells are (during transfection：Transfection reagent 4ul, the CRISPR-Cas9-SgRNA3-h carriers of Lipofectamine 2000
2ug), next day reaches 25cm2In square vase, from containing 750ug/ml G418,0.5mM hypoxanthine sodium, 0.08mM thymidines
Serum free growth medium carry out pressurization screening, per 2-3 days change fresh culture, pressurization 2 weeks after, to survivaling cell carry out
Limiting dilution assay monoclonal, cultivate into 4 piece of 96 orifice plate, continue culture 2 weeks.
5th, CHO-S/DHFR-/- monoclonal cell is selected
Select 30 monoclonals that cellular morphology is good, the speed of growth is fast to be digested, cell reaches one piece of 48 orifice plate, next day
Cell genomic dna on 48 orifice plates (30 DNA samples) is extracted, is expanded respectively around SgRNA3-h target spots using the primer of table 6
Sequence, Ago-Gel recovery purpose fragment, is then connected to carrier T, converts DH5a competent cells, apply Amp resistant panels
(totally 60 pieces of flat boards), next day is from 5 cloning and sequencings of picking on each flat board.It is compared, selects all with former sequence
The monoclonal that missense mutation, cell growth state are good, the speed of growth is fast carries out secondary Colony Culture.Chosen from secondary monoclonal
Select 5 monoclonals to be identified, select that all missense mutations, cell growth state are good, the speed of growth is fast and detection of mycoplasma
Negative monoclonal is amplified culture and frozen.
The speed of growth (Fig. 8), cell are carried out to the monoclonal cell (numbering CHO-S/DHFR-/-/G3, A2) finally chosen
(Fig. 9) and detection of mycoplasma (Figure 10) are analyzed in morphologic observation, and the obtained monoclonal cell speed of growth and open country are selected in as a result display
Raw type CHO-S cells are close, cellular morphology no significant difference, and do not have mycoplasma contamination.
Wherein, the catastrophe of CHO-S/DHFR-/-/G3 monoclonal cells is as follows：Double-stranded DNA breaks are different, and
Insertion/deletion bp numbers are not 3 integral multiples, wherein the more 2bp of chain (mutant A-), another chain (mutant B-) are few
4bp。
SEQ ID NO：33 CHO-S/DHFR-/-/G3 monoclonal cells sequencing (the more 2bp of mutant A-)
SEQ ID NO：34 CHO-S/DHFR-/-/G3 monoclonal cells sequencing (the few 4bp of mutant B-)
Understand, sgRNA sequences of the present invention can be used for the dihydrofolate reductase gene for knocking out Chinese hamster ovary celI, and obtain
Dihydrofolate reductase gene deletion cells strain.The cell line of acquisition has identical cell shape with wild type CHO-S cells
State and multiplication speed, the expression available for follow-up functional recombinant protein.
6th, after DHFR gene knockouts, the compliance test result of foreign protein is expressed
According to molecular cloning method, building the expression vector containing TNFR2-Fc and DHFR genes, (construction method is：Will
Neo resistance fragments on pcDNA3.1 carriers replace with DHFR genes, are named as pcDNA3.1/DHFR, synthesize TNFR2-Fc bases
Cause, insert it into the multiple cloning sites area of pcDNA3.1/DHFR carriers).And the CHO-S/ of the present invention is transfected respectively
DHFR-/-/G3 cell lines and wtCHO-S cell lines (wild type), being pressurizeed 5 days using 2ug/ml puromycin, (kill does not turn
Contaminate the Chinese hamster ovary celI of plasmid).Equal cell is taken into 6 orifice plates, the MTX for adding various dose pressurizes 2 weeks, the growth training more renewed
Base is supported, culture medium is collected after 24 hours, ELISA detects Fc expressing fusion protein situations in culture supernatant.
As a result show that the TNFR2-Fc of CHO-S/DHFR-/- cell expression is far above CHO-S cells, and as MTX is dense
The rise of degree, its TNFR2-Fc expression quantity also improve (Figure 11).
3rd, the DHFR genes of CHO-S/FUT8-/-/SD8 cells are knocked out
Recovery CHO-S/FUT8-/-/SD8 cells, reaches 6 orifice plates, using lipofection, selects CRISPR-Cas9-
SgRNA3-h plasmid transfections CHO-S/FUT8-/-/SD8 cells are (during transfection：The transfection reagent 4ul of Lipofectamine 2000,
CRISPR-Cas9-SgRNA3-h carrier 2ug), next day reaches 25cm2In square vase, from containing 750ug/ml G418,0.5mM
Hypoxanthine sodium, the serum free growth medium of 0.08mM thymidines carry out pressurization screening, and fresh cultured was changed per 2-3 days
Base, after pressurizeing 2 weeks, limiting dilution assay monoclonal is carried out to survivaling cell, cultivated into 4 piece of 96 orifice plate, continues culture 2 weeks.Afterwards
The continuous method selected DHFR and FUT8 genes while lack monoclonal cell (selects CHO-S/DHFR-/- Dan Ke with the 5 of step 2
Grand cell).
Choose DHFR and FUT8 genes simultaneously lack monoclonal cell (numbering CHO-S/FUT8-/-/DHFR-/-/B8 with
D11), detected, as a result show that its growth characteristics compares no significant difference with archaeocyte before mutation, also without mycoplasma contamination.
(see Figure 12,13)
Wherein, the catastrophe of CHO-S/FUT8-/-/DHFR-/-/B8 monoclonal cells is as follows：Double-stranded DNA breaks differ
Sample, and insertion/deletion bp numbers are not 3 integral multiples, wherein the few 7bp of a chain (mutant A-), another chain (mutant
B-) few 1bp.
SEQ ID NO：35 CHO-S/FUT8-/-/DHFR-/-/B8 monoclonal cells sequencing (the few 7bp of mutant A-)
SEQ ID NO：36 CHO-S/FUT8-/-/DHFR-/-/B8 monoclonal cells sequencing (the few 1bp of mutant B-)
Therefore, sgRNA sequences of the present invention can be used for FUT8 the and DHFR genes for knocking out Chinese hamster ovary celI, and obtain two
The cell line that kind gene lacks simultaneously.After two kinds of genes are knocked, the antibody of Chinese hamster ovary celI strain expression is modified without fucosylation,
And yield is high, application prospect is good.
SEQUENCE LISTING
<110>Sichuan Feng Xun developments in science and technology Co., Ltd
<120>A kind of kit for being used to knock out two kinds of genes of FUT8 and DHFR in Chinese hamster ovary celI
<130> GY456-17P1185
<160> 36
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> RNA
<213>SgRNA2-f sequences
<400> 1
ugggauaccc accacacugc 20
<210> 2
<211> 20
<212> RNA
<213>SgRNA4-f sequences
<400> 2
acauggacuc uggggagaag 20
<210> 3
<211> 20
<212> RNA
<213>SgRNA5-f sequences
<400> 3
guccagcuuu gcaagaaucu 20
<210> 4
<211> 20
<212> DNA
<213>Fut8-2sg12-U1 primers
<400> 4
agtggtcgag ctccccattg 20
<210> 5
<211> 21
<212> DNA
<213>Fut8-2sg12-D1 sequences
<400> 5
gcagaaggac acactgcacc a 21
<210> 6
<211> 22
<212> DNA
<213>Fut8-2sg34-U1 primers
<400> 6
tctgttgatt ccaggttccc at 22
<210> 7
<211> 23
<212> DNA
<213>Fut8-2sg34-D1 sequences
<400> 7
tgcatcagat acagctagta agg 23
<210> 8
<211> 25
<212> DNA
<213>Fut8-2sg5-U1 sequences
<400> 8
gtaagagtca tcacagtata ccaga 25
<210> 9
<211> 24
<212> DNA
<213>Fut8-2sg5-D1 sequences
<400> 9
gactatatag atgtatcact gact 24
<210> 10
<211> 413
<212> DNA
<213>FUT8 gene sgRNA2-f target spot surrounding DNA sequences
<400> 10
agtggtcgag ctccccattg tagacagcct ccatcctcgt cctccttact tacccttggc 60
tgtaccagaa gaccttgcag atcgactcct gagagtccat ggtgatcctg cagtgtggtg 120
ggtatcccag tttgtcaaat acttgatccg tccacaacct tggctggaaa gggaaataga 180
agaaaccacc aagaagcttg gcttcaaaca tccagttatt gggtaagaat ctatccccct 240
ccccttaaac agtaatatat agcagtagtt gtatgtgttt actttttact gtagatttta 300
taatatttaa tagtcatagt cccaccaaag aacaaagatt ctaaaactta aaagactatt 360
ctccctttgt tcagttaggg cagcagagca aatggtgcag tgtgtccttc tgc 413
<210> 11
<211> 621
<212> DNA
<213>FUT8 gene sgRNA4-f target spot surrounding DNA sequences
<400> 11
tctgttgatt ccaggttccc atatattctt ggatatgcca attacttttt ctgtaagcaa 60
gtgtttcata aaacttttac ttaactttca tattgacctg tactattcaa cattcagcta 120
tgttaaagta tttgtgaagt gttttgaaat gattttatat tttctaaggt gagaataaat 180
gagaaaatgt tttaatatgt ctccagtgcc cccatgacta gggatactaa ttgagtacca 240
gtacattatc agtgtgctct ccacttctcc ccagagtcca tgtcagacgc actgacaaag 300
tgggaacaga agcagccttc catcccattg aggaatacat ggtacacgtt gaagaacatt 360
ttcagcttct cgaacgcaga atgaaagtgg ataaaaaaag agtgtatctg gccactgatg 420
acccttcttt gttaaaggag gcaaagacaa agtaagttag accaacaagt ggttctgtat 480
gggattatct cttagttgaa gaaaatcctt aattctggga acttgtggtt cttgttgcta 540
actaataggt tccaaaatca aagactacat gtgcaaatat taatctaatc aagtcatacc 600
ttactagctg tatctgatgc a 621
<210> 12
<211> 524
<212> DNA
<213>FUT8 gene sgRNA5-f target spot surrounding DNA sequences
<400> 12
gtaagagtca tcacagtata ccagagagac taattttgtc tgaagcatca tgtgttgaaa 60
caacagaaac ttattttcct gtgtggctaa ctagaaccag agtacaatgt ttccaattct 120
ttgagctccg agaagacaga agggagttga aactctgaaa atgcgggcat ggactggttc 180
ctggcgttgg attatgctca ttctttttgc ctgggggacc ttattgtttt atataggtgg 240
tcatttggtt cgagataatg accaccctga ccattctagc agagaactct ccaagattct 300
tgcaaagctg gagcgcttaa aacaacaaaa tgaagacttg aggagaatgg ctgagtctct 360
ccggtaggtt tgaaatactc aaggatttga tgaaatactg tgcttgacct ttaggtatag 420
ggtctcagtc tgctgttgaa aaatataatt tctacaaacc gtctttgtaa aattttaagt 480
attgtagcag actttttaaa agtcagtgat acatctatat agtc 524
<210> 13
<211> 25
<212> DNA
<213>SgRNA2-f forward primers
<400> 13
caccgtggga tacccaccac actgc 25
<210> 14
<211> 25
<212> DNA
<213>SgRNA2-f reverse primers
<400> 14
aaacgcagtg tggtgggtat cccac 25
<210> 15
<211> 25
<212> DNA
<213>SgRNA4-f forward primers
<400> 15
caccgacatg gactctgggg agaag 25
<210> 16
<211> 25
<212> DNA
<213>SgRNA4-f reverse primers
<400> 16
aaaccttctc cccagagtcc atgtc 25
<210> 17
<211> 24
<212> DNA
<213>SgRNA5-f forward primers
<400> 17
caccgtccag ctttgcaaga atct 24
<210> 18
<211> 24
<212> DNA
<213>SgRNA5-f reverse primers
<400> 18
aaacagattc ttgcaaagct ggac 24
<210> 19
<211> 523
<212> DNA
<213>CHO-S/FUT8-/-/SD8 monoclonal cells sequencing, chain A
<400> 19
ttggtaccga gctcggatcc actagtaacg gccgccagtg tgctggaatt gcccttcagc 60
agaaggacac ctgcaccatt tgctctgctg ccctaactga acaaagggag aatagtcttt 120
taagttttag aatctttgtt ctttggtggg actatgacta ttaaatatta taaaatctac 180
agtaaaaagt aaacacatac aactactgct atatattact gtttaagggg agggggatag 240
attcttaccc aataactgga tgtttgaagc caagcttctt ggtggtttct tctatttccc 300
tttccagcct tgtggacgga tcaagtattt gacaaactgg gatacccacc acactgcagg 360
atcaccatgg actctcagga gtcgatctgc aaggtcttct ggtacagcca agggtaagta 420
aggaggacga ggatggaggc tgtctacaat ggggagcaag ggcaattctg cagatatcca 480
tcacactggc ggccgctcga gcatgcatct agagggccca att 523
<210> 20
<211> 528
<212> DNA
<213>CHO-S/FUT8-/-/SD8 monoclonal cells sequencing, chain B
<400> 20
ttggtaccga gctcggatcc actagtaacg gccgccagtg tgctggaatt gcccttcagc 60
agaaggacca ctgcaccatt tgctctgctg ctctaactga acaaagggag aatagtcttt 120
taagttttgg aatctttgtt ctttggtggg actatgacta ttaaatatta taaaatctac 180
agtaaaaagt aaacacatac aactactgct atatattact gtttaagggg agggggatag 240
attcttaccc aataactgga tgtttgaagc caagcttctt ggtggtttct tctatttccc 300
tttccagcca aaggttgtgg acggatcaag tatttgacaa actgggatac ccaccacact 360
gcaggatcac catggactct caggagtcga tctgcaaggt cttctggtac agccaagggt 420
aagtaaggag gacgaggatg gaggctgtct acaatgggga gcaagggcaa ttctgcagat 480
atccatcaca ctggcggccg ctcgagcatg catctagagg gcccaatt 528
<210> 21
<211> 20
<212> RNA
<213>SgRNA1-h sequences
<400> 21
gcaagaacgg agaccuuccc 20
<210> 22
<211> 20
<212> RNA
<213>SgRNA2-h sequences
<400> 22
gucgccgugu cccagaauau 20
<210> 23
<211> 20
<212> RNA
<213>SgRNA3-h sequences
<400> 23
gccaaugcuc agguacuggc 20
<210> 24
<211> 25
<212> DNA
<213>DHFR-EXO1-F2 primers
<400> 24
cggggcctct gatgttcaaa tagga 25
<210> 25
<211> 25
<212> DNA
<213>DHFR-EXO1-R2 primers
<400> 25
tgaggaggtg gtggtcattc tttgg 25
<210> 26
<211> 584
<212> DNA
<213>DHFR gene SgRNA1-h, 2-h, 3-h target spot surrounding DNA sequences
<400> 26
cggggcctct gatgttcaaa taggatgcta ggcttgttga gggcgtggcc tccgattcac 60
aagtgggaag cagcgccggg cgactgcaat ttcgcgccaa acttggggga agcacagcgt 120
acaggctgcc taggtgatcg ctgctgctgt catggttcga ccgctgaact gcatcgtcgc 180
cgtgtcccag aatatgggca tcggcaagaa cggagacctt ccctggccaa tgctcaggta 240
ctggctggat tgggttaggg aaaccgaggc ggttcgctga atcgggtcga gcacttggcg 300
gagacgcgcg ggccaactac ttagggacag tcatgagggg taggcccgcc ggctgcagcc 360
cttgcccatg cccgcggtga tccccatgct gtgccagcct ttgcccagag gcgctctagc 420
tgggagcaaa gtccggtcac tgggcagcac caccccccgg acttgcatgg gtagccgctg 480
agatggagcc tgagcacacg tgacagggtc cctgttaacg cagtgtttct ctaactttca 540
ggaacgaatt caagtacttc caaagaatga ccaccacctc ctca 584
<210> 27
<211> 24
<212> DNA
<213>SgRNA1-h- forward primers
<400> 27
caccgcaaga acggagacct tccc 24
<210> 28
<211> 24
<212> DNA
<213>SgRNA1-h- reverse primers
<400> 28
aaacgggaag gtctccgttc ttgc 24
<210> 29
<211> 24
<212> DNA
<213>SgRNA2-h- forward primers
<400> 29
caccgtcgcc gtgtcccaga atat 24
<210> 30
<211> 24
<212> DNA
<213>SgRNA2-h- reverse primers
<400> 30
aaacatattc tgggacacgg cgac 24
<210> 31
<211> 24
<212> DNA
<213>SgRNA3-h- forward primers
<400> 31
caccgccaat gctcaggtac tggc 24
<210> 32
<211> 24
<212> DNA
<213>SgRNA3-h- reverse primers
<400> 32
aaacgccagt acctgagcat tggc 24
<210> 33
<211> 472
<212> DNA
<213>CHO-S/DHFR-/-/G3 monoclonal cells sequencing, chain A
<400> 33
ggggcctctg atgttcaaat aggatgctag gcttgttgag ggcgtggcct ccgattcaca 60
agtgggaagc agcgccgggc gactgcaatt tcgcgccaaa cttgggggaa gcacagcgta 120
caggctgcct aggtgatcgc tgctgctgtc atggttcgac cgctgaactg catcgtcgcc 180
gtgtcccaga atatgggcat cggcaagaac ggagaccttg tccctggcca atgctcaggt 240
actggctgga ttgggttagg gaaaccgagg cggttcgctg aatcgggtcg agcacttggc 300
ggagacgcgc gggccaacta cttagggaca gtcatgaggg gtaggcccgc cggctgcagc 360
ccttgcccat gcccgcggtg atccccatgc tgtgccagcc tttgcccaga ggcgctctag 420
ctgggagcaa agtccggtca ctgggcagca ccaccccccg gacttgcatg gg 472
<210> 34
<211> 466
<212> DNA
<213>CHO-S/DHFR-/-/G3 monoclonal cells sequencing, chain B
<400> 34
ggggcctctg atgttcaaat aggatgctag gcttgttgag ggcgtggcct ccgattcaca 60
agtgggaagc agcgccgggc gactgcaatt tcgcgccaaa cttgggggaa gcacagcgta 120
caggctgcct aggtgatcgc tgctgctgtc atggttcgac cgctgaactg catcgtcgcc 180
gtgtcccaga atatgggcat cggcaagaac ggagaccctg gccaatgctc aggtactggc 240
tggattgggt tagggaaacc gaggcggttc gctgaatcgg gtcgagcact tggcggagac 300
gcgcgggcca actacttagg gacagtcatg aggggtaggc ccgccggctg cagcccttgc 360
ccatgcccgc ggtgatcccc atgctgtgcc agcctttgcc cagaggcgct ctagctggga 420
gcaaagtccg gtcactgggc agcaccaccc cccggacttg catggg 466
<210> 35
<211> 540
<212> DNA
<213>CHO-S/FUT8-/-/DHFR-/-/B8 monoclonal cells sequencing, chain A
<400> 35
ggaatctacg actgagcttg atatcgaatt cgcgtgtcgc ccttcggggc ctctgatgtt 60
caaataggat gctaggcttg ttgagggcgt ggcctccgat tcacaagtgg gaagcagcgc 120
cgggcgactg caatttcgcg ccaaacttgg gggaagcaca gcgtacaggc tgcctaggtg 180
atcgctgctg ctgtcatggt tcgaccgctg aactgcatcg tcgccgtgtc ccagaatatg 240
ggcatcggca agaacggaga ccttcaatgc tcaggtactg gctggattgg gttagggaaa 300
ccggggcggt tcgctgaatc gggtcgagca cttggcggag acgcgcgggc caactactta 360
gggacagtca tgaggggtag gcccgccggc tgcagccctt gcccatgccc gcggtgatcc 420
ccatgctgtg ccagcctttg cccagaggcg ctctagctgg gagcaaagtc cggtcactgg 480
gcagcaccac cccccggact tgcatgggta gccgctgaga tggagcctga gcacacgtga 540
<210> 36
<211> 480
<212> DNA
<213>CHO-S/FUT8-/-/DHFR-/-/B8 monoclonal cells sequencing, chain B
<400> 36
ggggctctcg actgagcttg atatcgaatt cgctgtcgcc cttgaggagg tggtggtcat 60
tctttggaag tacttgaatt cgttcctgaa agttagagaa acactgcgtt aacagggacc 120
ctgtcacgtg tgctcaggct ccatctcagc ggctacccat gcaagtccgg ggggtggtgc 180
tgcccagtga ccggactttg ctcccagcta gagcgcctct gggcaaaggc tggcacagca 240
tggggatcac cgcgggcatg ggcaagggct gcagccggcg ggcctacccc tcatgactgt 300
ccctaagtag ttggcccgcg cgtctccgcc aagtgctcga cccgattcag cgaaccgcct 360
cggtttccct aacccaatcc agccagtacc tgagcattgg ccagggaggt ctccgttctt 420
gccgatgccc atattctggg acacggcgac gatgcagttc agcggtcgaa ccatgacagc 480
Claims (9)
1. a kind of be used to knock out the kit of FUT8 genes and DHFR genes in Chinese hamster ovary celI, it is characterised in that it includes SEQ ID
NO：1 and/or SEQ ID NO：SgRNA sequences described in 3, and SEQ ID NO：SgRNA sequences described in 23.
2. a kind of be used to knock out the kit of FUT8 genes and DHFR genes in Chinese hamster ovary celI, it is characterised in that it includes SEQ ID
NO：13、SEQ ID NO：17 and SEQ ID NO：SgRNA sequences described in 31.
3. the kit of claim 1 or 2 is in the purposes for knocking out two kinds of genes of FUT8 and DHFR in Chinese hamster ovary celI.
4. purposes of the kit of claim 1 or 2 in the cell line for preparing FUT8 and DHFR genes while missing.
5. method that is a kind of while knocking out FUT8 and DHFR genes in Chinese hamster ovary celI, it is characterised in that it is to utilize CRISPR/
Cas9 systems are transformed two kinds of genes in Chinese hamster ovary celI, and step is as follows：
A, SEQ ID NO are synthesized：Sequence and its complementary strand shown in 1 and/or 3, denaturation, annealing, form double-strand, are inserted into Cas9
The multiple cloning sites of expression vector, obtain recombinant expression plasmid；
B, by step a recombinant expression carrier transfection CHO cell, picking individual cells, inoculated and cultured；
C, after individual cells propagation is monoclonal, identify and confirm that FUT8 genes are knocked, and obtain FUT8 gene delections
Cell line FUT8-/-, it is standby；
D, SEQ ID NO are synthesized：Sequence and its complementary strand described in 23, denaturation, annealing, form double-strand, are inserted into Cas9 expression
The multiple cloning sites of carrier, obtain recombinant expression plasmid；
E, step c FUT8-/- cell line is taken, is transfected with step d recombinant expression carrier, picking individual cells, inoculated and cultured；
F, after individual cells propagation is monoclonal, identify and confirm that DHFR genes are knocked, and obtain FUT8 genes and DHFR
The cell line that gene lacks simultaneously.
6. according to the method for claim 5, it is characterised in that the Chinese hamster ovary celI is CHO-S sub-types of cells.
7. according to the method for claim 5, it is characterised in that the Cas9 expression vectors are CRISPR/Cas9 carriers.
8. the FUT8 deletion cells strains that claim 5-7 any one methods describeds are prepared.
9. FUT8 and DHFR genes while the cell line of missing that claim 5-7 any one methods describeds are prepared.
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| CN113061605B (en) * | 2020-07-13 | 2022-11-29 | 中山大学 | Construction method and application of fucose transferase 8 (FUT 8) function-deficient cell strain |
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