CN113355360A

CN113355360A - Construction method of GS gene knockout CHO-K1 cell strain and suspension cell monoclonality

Info

Publication number: CN113355360A
Application number: CN202110588787.8A
Authority: CN
Inventors: 焦鹏; 徐琦; 陈少勇; 林文龙; 李睿; 黄启龙
Original assignee: Shanghai Bibo Biomedical Technology Co ltd
Current assignee: Shanghai Bibo Biomedical Technology Co ltd
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2021-09-07

Abstract

The invention discloses a construction method of a GS gene knockout CHO-K1 cell strain, which comprises the steps of culturing CHO-K1 cells in an adherent manner, designing and selecting sgRNA, forming a transfection mixture by the selected sgRNA, EGFP mRNA and Cas9mRNA to carry out cotransfection on CHO-K1, and obtaining the GS gene knockout CHO-K1 cell strain. The method uses the RNA transfection mixture to carry out cotransfection on the CHO-K1 cell, is an RNA-based GS knockout method, and simultaneously combines a CRISPR/Cas9 gene editing means and GFP screening, thereby not only avoiding genome reformation on a DNA layer, reducing the risk of genome pollution, but also improving GS gene knockout efficiency and reducing the workload of cell screening; the CHO-K1 cell strain with the GS gene knocked out obtained by the invention is verified, and the GS gene knocking out efficiency is expected.

Description

Construction method of GS gene knockout CHO-K1 cell strain and suspension cell monoclonality

Technical Field

The invention relates to the technical field of biology, in particular to a construction method of a GS gene knockout CHO-K1 cell strain and suspension cell monoclonality.

Background

Chinese hamster ovary cells (CHO cells) have become one of the most widely used host cells for the production of therapeutic recombinant proteins. Can quickly identify rare high-yield cell lines in a large number of low-yield and non-productive cells, achieves an effective and efficient clinical cell line development process, and has great significance for accelerating the development of biological medicines.

Two of the most common CHO expression systems used for recombinant protein production utilize either a selection method of Methotrexate (MTX) based on dihydrofolate reductase (DHFR) or a selection method of methylthio-Methylenemethoxypyrimidine (MSX) based on Glutamine Synthetase (GS). Among them, the CHO-K1 cell line-based GS gene knockout cell line is a widely used expression system at present. The screening principle is as follows: the CHO-K1 cell with the GS gene knockout does not have the function of L-glutamine synthetase and can survive only by strictly depending on L-glutamine provided by an external source, the CHO-K1 cell with the GS gene knockout is transfected and then placed in a culture medium without L-glutamine, only the cell which is integrated with the exogenous GS gene and the integration site of which is transcribed into an active region in a genome can survive, and the target protein gene is positioned at the upstream or the downstream of the GS gene, so that the cell strain with higher GS and protein expression can be screened.

Therefore, how to obtain a GS gene knockout CHO-K1 cell line becomes an important research topic in the field. Although conventional gene knockout by homologous recombination has been used in basic research and production of industrial cell lines, conventional gene knockout by homologous recombination is time-consuming, labor-consuming and inefficient, and is not conducive to development of CHO cell engineering; at present, the more common method is to knock out the GS gene by a CRISPR/Cas gene editing technology, but the method is generally carried out at the DNA level, the genome is inevitably reformed, the genome has certain pollution risk, and meanwhile, the GS gene knock-out efficiency needs to be improved; the CHO-K1 cell screening of GS gene knockout requires the use of compounds and has large workload.

Disclosure of Invention

The invention aims to provide a construction method of a GS gene knockout CHO-K1 cell strain, which avoids genome reformation at a DNA level in a GS gene knockout process, improves GS gene knockout efficiency and reduces workload of GS gene knockout CHO-K1 cell screening.

In order to solve the technical problems, the invention provides a construction method of a GS gene knockout CHO-K1 cell strain, which comprises the following steps:

step A: designing and selecting sgRNA-GS57 for knocking out a GS gene based on a nucleotide sequence of a CHO-K1 cell GS exon-5;

the nucleotide sequence of exon-5 is shown as SQE ID NO. 1;

the nucleotide sequence of sgRNA-GS57 is shown in SQE ID NO. 2.

And B: the sgRNA-GS57, EGFP mRNA and Cas9mRNA form a transfection mixture, CHO-K1 cells are subjected to co-transfection, FACS is used for sorting out co-transfected CHO-K1 cells, then the co-transfected CHO-K1 cells are subjected to single cell sorting, single co-transfected CHO-K1 cells are inoculated, and standing culture is carried out, so that a monoclonal GS gene knockout CHO-K1 cell strain is obtained.

Preferably, the method further comprises the steps of:

and C: c, detecting the GS expression of the GS gene knockout CHO-K1 monoclonal cell strain obtained in the step F;

step D: and F, performing second-generation sequencing on the GS gene knockout CHO-K1 monoclonal cell strain obtained in the step F, and checking the GS gene knockout effect.

Preferably, in step B, the molar weight ratio of EGFP mRNA to Cas9mRMA of the transfection mixture satisfies the following relationship: EGFP mRNA: Cas9mRMA ═ 1: 10.

Preferably, the medium used for the transfection mixture is a reduced serum medium, and the transfer mixture further comprises a transfection reagent.

Preferably, in step B, the CHO-K1 cells to be transfected are cultured adherently, and the culture medium used is F12K medium containing 10% fetal bovine serum.

Preferably, in step B, the transfection time is three days, wherein the medium is cultured for one day using F12K medium containing 10% fetal bovine serum, and then the medium is replaced for two days, and the replaced medium is F12K medium containing 10% fetal bovine serum and 4mM L-glutamine.

Preferably, in step B, FACS sorted co-transfected CHO-K1 cells were GFP positive co-transfected CHO-K1 cells.

Preferably, after single cell sorting of GFP positive co-transfected CHO-K1 cells, the specific steps for culturing the monoclonal cell strain are as follows:

step B1: after single cell sorting is carried out on GFP positive co-transfected CHO-K1 cells, the cells are inoculated to a 96-well plate, cell imaging is carried out on the 96-well plate, clone cells grown from the single cells are selected and cultured;

step B2: when the clone cells grown from the single cells grow to 40% -50% confluence, the cells are expanded and cultured to a 24-well plate, and then the cells are frozen and stored when the clone cells grow to 70% -80% confluence.

Preferably, in step B1, the culture medium used in the 96-well plate is F12K medium containing 10% fetal bovine serum, and the culture medium further comprises a 1% content of streptomycin qing solution;

in step B2, the medium used in the 24-well plate was F12K medium containing 10% fetal bovine serum.

Preferably, the steps for testing the GS expression of the GS gene knockout CHO-K1 monoclonal cell strain are as follows:

step C1: cell lysis is carried out on the CHO-K1 monoclonal cell strain with the knockout GS gene by using cell lysate, then freeze thawing treatment is carried out on the cell lysate, and then the cell lysate after freeze thawing treatment is mixed with loading buffer solution and boiled for later use;

step C2: the product obtained in step F1 was analyzed for GS expression by western blotting.

Preferably, the cell lysate used in step C1 is RIPA lysate.

Preferably, the loading buffer used in step C1 is 2xLaemmli buffer and contains 5% β -mercaptoethanol.

In order to solve the technical problems, the invention also provides a CHO-K1 suspension cell monoclonal cell bank with GS gene knockout, and the monoclonal cell bank is obtained by the following steps:

step E: suspending and domesticating GS knockout cells and forming a cryopreserved cell bank;

step F: GS knockout CHO-K1 suspension cells were monoclonalized and cell banks were established.

The transfection mixture containing sgRNA, EGFP mRNA and Cas9mRNA is used for carrying out cotransfection on CHO-K1 cells to obtain a GS gene knockout CHO-K1 cell strain, the GS gene knockout method is based on RNA, and the CRISPR/Cas9 gene editing means and GFP screening are combined, so that not only is genome reformation at a DNA level avoided, the risk of genome pollution reduced, but also the GS gene knockout efficiency is improved, and meanwhile, the CHO-K1 cells subjected to GS gene knockout by using compounds are not required to be screened, and the workload of cell screening is also reduced; the CHO-K1 cell strain with the GS gene knocked out obtained by the invention is verified, and the GS gene knocking out efficiency is expected. The CHO-K1 cell strain with the knockout GS gene is subjected to suspension domestication and monoclonality, and the requirement of the pharmaceutical industry on high-density cell culture is met.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the present invention are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flowchart of a method for constructing a GS gene knockout CHO-K1 cell line according to an embodiment of the present invention;

FIG. 2 is a schematic diagram showing the relative relationship between GS exon-5 of CHO-K1 cells, four sgRNAs and two pairs of PCR primers according to an embodiment of the present invention;

FIG. 3 is an electrophoretogram of amplified products of GS exon-5 by two pairs of PCR primers according to an embodiment of the present invention;

FIG. 4A is an electrophoretogram of a first round of GS exon-5 amplification product of an example of the present invention;

FIG. 4B is an electrophoretogram of a second round of GS exon-5 amplification product of an example of the present invention;

FIG. 4C is a graph of a sequencing analysis corresponding to the electrophoretic band of DNA from the co-transfected CHO-K1 cell of FIG. 4B;

FIG. 5 is a GS expression electrophoretogram of a GS gene knockout CHO-K1 monoclonal cell strain by the Western blotting method of the present invention;

FIG. 6 is a graph of cell sealing and cell viability for subculture of GS knockout CHO-K1 cells of the present invention.

Detailed Description

The technical solutions in the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example one

FIG. 1 shows a construction method of a GS gene knockout CHO-K1 cell strain and a suspension cell monoclonal cell bank construction method.

A construction method of a CHO-K1 cell strain with GS gene knockout comprises the following steps:

step A: based on the nucleotide sequence of GS exon-5 of CHO-K1 cells, sgRNA-GS57 is designed and selected for knocking out the GS gene, and the specific operations are as follows:

step A1: determining the gene sequence of CHO-K1 cells and selecting a GS knockout gene, comprising the steps of:

step A11 adherent culture of a CHO-K1 cell line purchased from American Type Culture Collection (ATCC) under a lot number of 62960170;

in the step a11, the culture conditions are as follows: the culture bottle is a T75 culture bottle, the culture medium is F12K culture medium containing 10% fetal calf serum, and the amount of the culture medium is 15 ml; the culture time is 2 days;

step A12: extracting RNA of CHO-K1 cells cultured by adherence by using an RNeasy kit, and carrying out reverse transcription on the extracted RNA;

in the step a12, the reverse transcription conditions: the reverse transcription primer adopts oligo dT, the reverse transcription kit adopts Omniscript reverse transcription kit, the reverse transcription temperature is 37 ℃, and the reverse transcription time is 1 hr;

step A13: purifying the reverse transcription product by using a PCR purification kit;

step A14: carrying out PCR amplification on the purified reverse transcription product;

in step A14, the PCR amplification primers are as follows:

forward primer sequence: GS-F5'-ATGGCCACCTCAGCAAGTTC-3' (SQE ID NO. 3);

reverse primer sequence: GS-R:5'-TTAGTTTTTGTATTGGAAGGGC-3' (SQE ID NO. 4);

step A15: purifying the PCR amplification product by using a PCR purification kit, and carrying out DNA gene sequencing on the amplification product to obtain a gene sequence which comprises 6 exon genes, wherein the exon-5 nucleotide sequence is as follows:

exon-5:

TGGGAATTCCAAATAGGACCCTGTGAAGGAATCCGCATGGGAGATCATCTCTGGGTGGCCCGTTTCATCTTGCATCGAGTATGTGAAGACTTTGGGGTAATAGCAACCTTTGACCCCAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTTAGCACCAAGGCCATGCGGGAGGAGAATGGTCTGAA(SQE ID NO.1)

step A2: designing sgNRA aiming at the coding sequence of the GS gene detected in the step A1, wherein four sgRNAs aiming at the coding sequence of the exon-5 gene are selected, and the four selected sgRNAs are sgRNA-GS34, sgRNA-GS41, sgRNA-GS57 and sgRNA-GS116 respectively;

in step a2, the chopchopchopchopchop gRNA design program was used to design total 171 sgrnas, of which 26 were targeted to exon-5 gene coding sequences, and the 4 selected sgrnas were the higher scoring ones of the 26 designed sgrnas, namely sgRNA-GS34, sgRNA-GS41, sgRNA-GS57, and sgRNA-GS116, respectively, of which sgRNA-GS34 and sgRNA-GS57 were targeted sense strands and sgRNA-GS41 and sgRNA-GS116 were targeted antisense strands, and fig. 2 shows the relationship between four sgrnas and GS exon-5.

Step A3: designing and synthesizing two pairs of PCR primers for PCR amplification of the exon-5, wherein the two pairs of PCR primers are GS-Surveyor-F1, GS-Surveyor-R1, GS-Surveyor-F2 and GS-Surveyor-R2 respectively, and the gene sequences of the two pairs of PCR primers are as follows:

first pair of PCR primers:

a forward primer: GS-Surveyor-F1: 5'-GAGCTTGAGTTGGCCTGAACAGTTA-3' (SQE ID NO. 5);

reverse primer: GS-Surveyor-R1: 5'-AAGTCCAACTTTCCCAACCCTAACA-3' (SQE ID NO. 6);

a second pair of PCR primers:

a forward primer: GS-Surveyor-F2: 5'-GCCCAGGTAAATGGCACTATTCTGT-3' (SQE ID NO. 7);

reverse primer: GS-Surveyor-R2: 5'-ACTTCTCCAACGGGTACAAAACGAT-3' (SQE ID NO. 8);

the two pairs of PCR primers in step a3 are used to perform two rounds of amplification on GS exon-5 gene, the first round is performed using the second pair of PCR primers, and the second round is performed using the first pair of PCR primers. FIG. 2 shows the relative relationship of two pairs of primers to GS exon-5.

The step A3 further comprises verifying the amplification effect of the two pairs of designed and synthesized PCR primers, wherein the PCR reaction mixture comprises 36. mu.l of water, 10. mu.l of buffer solution, 1. mu.l of dNTP mixed solution, 1. mu.l of DNA template, 1. mu.l of DNA polymerase and 0.5. mu.M PCR primers. Wherein the DNA template is a DNA extract of a wild-type CHO-K1 cell cultured in an adherent manner, and the wild-type CHO-K1 cell is obtained by culturing in an adherent manner after being purchased from ATCC (the purchased batch number is 62960170).

In the step a3, the result of the DNA electrophoresis bands of the PCR amplification products is shown in fig. 3, wherein the result includes the DNA electrophoresis band of the first pair of PCR primer amplification products, the DNA electrophoresis band of the second pair of PCR primer amplification products, and the DNA ladder band of 100bp, and as can be seen in fig. 3, the amplification effect using the two pairs of PCR primers is expected.

Step A4: the four sgrnas designed and selected in step a3 were evaluated for their GS gene knockout efficiency: and B, forming a transfection mixture by the four sgRNAs designed and selected in the step A3, EGFP mRNA and Cas9mRNA, co-transfecting CHO-K1 cells, sorting the co-transfected CHO-K1 cells by using a flow cytometric fluorescence sorting technology (FACS for short), extracting the genome of the co-transfected CHO-K1 cells as a template, performing PCR amplification on exon-5 of the GS gene by using two pairs of PCR primers designed and synthesized in the step A3, recovering PCR amplification products, and sequencing and analyzing the knockout efficiency of the four sgRNAs.

In step a4, CHO-K1 cells were purchased from ATCC (purchased lot No. 62960170), and after adherent culture, CHO-K1 cells to be transfected were obtained, and when adherent culture was performed, using F12K containing 10% fetal bovine serum as a culture medium, and when culture was performed to 40% to 60% confluency, the cells were divided into two culture dishes for continuous culture, wherein CHO-K1 cells in one dish were used for transfection to evaluate transfection efficiency of four sgrnas, and CHO-K1 cells in the other dish were used as a control group for background of FACS sorting;

in step A4, the medium used in the transfection mixture is a reduced serum medium, and the transfection mixture further comprises a transfection reagent.

In step A4, the amounts of the components of the transfection mixture were: 1ml of serum-reduced culture medium, 15 mu l of transfection reagent, 0.11 mu g of EGFP mRNA, 5 mu g of Cas9mRNA and 25nM of each sgRNA, and the molar weight of each component satisfies the following relationship: EGFP mRNA: Cas9 mRMA: sgRNA-GS 34: sgRNA-GS 41: sgRNA-GS 57: sgRNA-GS116 ═ 1: 10;

in the step A4, the transfection time is 3 days, wherein, the medium is cultured for one day by using F12K medium containing 10% fetal calf serum, and then the medium is replaced for two days, and the replaced medium is F12K medium containing 10% fetal calf serum and 4mM L-glutamine; after completion of the culture, the cells were digested with trypsin, and then the cells were redispersed in F12K medium containing 10% fetal bovine serum to adjust the final concentration of the cells to 5X 10⁶-10×10⁶Cells/ml were sorted by FACS.

In step A4, the FACS sorted co-transfected CHO-K1 cells were 8% of the GFP fluorescence intensity of the co-transfected CHO-K1 cells.

In the step a4, the specific steps of amplifying exon-5 of the GS gene with the two pairs of PCR primers designed and synthesized in the step A3 include:

step A41: extracting genome of cotransfected CHO-K1 cells 8% before GFP fluorescence intensity as a template, taking GS-Surveyor-F2 and GS-Surveyor-R2 as PCR primers, carrying out first round of PCR amplification on exon-5 of GS gene, and recovering PCR products; meanwhile, synchronously taking the genome of a wild type CHO-K1 cell as a template, taking GS-Surveyor-F2 and GS-Surveyor-R2 as PCR primers, carrying out first round of PCR amplification on exon-5 of a GS gene, and recovering a PCR product; FIG. 4A shows the DNA electrophoresis bands of the first round PCR amplification products, wherein the "control" is the DNA electrophoresis band of the control group CHO-K1 cells, and the "GFP positive" is the DNA electrophoresis band of the co-transfected CHO-K1 cells.

Step A42: taking the recovered first round PCR amplification product as a template, and taking GS-Surveyor-F1 and GS-Surveyor-R1 as PCR primers to perform second round PCR amplification on exon-5 of the GS gene, and recovering a second round PCR amplification product; FIG. 4B shows the DNA electrophoresis bands of the second round PCR amplification products, wherein the "control" is the DNA electrophoresis band of the control CHO-K1 cell, the "GFP positive" is the DNA electrophoresis band of the co-transfected CHO-K1 cell, and the "molecular weight standard" is the DNA ladder band of 100 bp.

Step A5: the recovered second round PCR amplification products of the co-transfected CHO-K1 cells were sequenced to evaluate the knockout efficiency of the four sgrnas.

In the above step A5, DNA electrophoresis bands of the co-transfected CHO-K1 cells are recovered by gel cutting and sequenced, and the sequencing result is shown in FIG. 4C, wherein 7 electrophoresis bands are shown in FIG. 4C, wherein Band-1 is determined as an electrophoresis Band caused by sgRNA-41, Band-2 is not determined as an electrophoresis Band caused by sgRNA, Band-3 is determined as an electrophoresis Band caused by sgRNA-57, Band-wild type is also determined as an electrophoresis Band generated by wild-type CHO-K1 cells, Band-4 and Band-5 are determined as electrophoresis bands caused by sgRNA-57, and Band-6 is likely to be an electrophoresis Band generated by sgRNA-34 and sgRNA-57, so that the knockout efficiency of sgRNA-57 is the best, and sgRNA-57 and Cas9 are knocked out at position 182 of exon-5.

In step a5, the nucleotide sequence of sgRNA-57:

SgRNA-57：UAGCACCAAGGCCAUGCGGG(SQE ID NO.2)；

In the step B, the CHO-K1 cells are obtained by adherent culture after being purchased from ATCC (purchased from a lot number of 62960170), and CHO-K1 cells to be transfected are obtained after being subjected to adherent culture, when the CHO-K1 cells are subjected to adherent culture, a culture medium containing 10% fetal bovine serum F12K is used, and then the cultured CHO-K1 cells are divided into two culture dishes for continuous culture, wherein CHO-K1 cells in one culture dish are used for co-transfection, and CHO-K1 cells in the other culture dish are used as a background control group for FACS sorting;

in step B, the medium used in the transfection mixture is a serum-reduced medium, and the transfection mixture further comprises a transfection reagent.

In the step B, the amounts of the components of the transfer mixture are respectively: serum-reduced medium 600 μ l, transfection reagent 20 μ l, EGFP mRNA 0.22 μ g, Cas9mRNA 10 μ g, sgRNA-GS 5725 nM, wherein the molar amounts of EGFP mRNA and Cas9mRMA satisfy the following relationship: EGFP mRNA: Cas9mRMA ═ 1: 10.

In the step B, the transfection time is three days, wherein, F12K culture medium containing 10% fetal calf serum is used for culturing for one day, and then the culture medium is replaced for culturing for two days, and the replaced culture medium is F12K culture medium containing 10% fetal calf serum and 4mM L-glutamine; after the culture was completed, the cells were dispersed in the medium by digestion with pancreatin at 37 ℃ for 5min, and then centrifuged, and the separated cells were redispersed in a complete medium for subsequent cell sorting.

In the step B, the co-transfected CHO-K1 cells sorted by FACS are GFP positive co-transfected CHO-K1 cells;

in the step B, after single cell sorting is carried out on GFP positive co-transfected CHO-K1 cells, the specific steps for culturing the monoclonal cell strain are as follows:

step B1: after single cell sorting is carried out on GFP positive co-transfected CHO-K1 cells, the cells are inoculated to a 96-well plate, cell imaging is carried out on the 96-well plate, clone cells growing from the single cells are selected and determined, and subsequent culture is carried out;

in the step B1, the culture medium used in the 96-well plate is F12K medium containing 10% fetal bovine serum, and the culture medium further includes 1% streptomycin solution;

step B2: when the clone cells grown from the single cells grow to 40% -50% confluence, the cells are expanded and cultured to a 24-well plate, and then the cells are frozen and stored when the clone cells grow to 70% -80% confluence, so that a CHO-K1 cell strain with GS knockout is obtained through culture.

In the step B2, the medium used in the 24-well plate was F12K medium containing 10% fetal bovine serum.

In the embodiment, the transfection mixture containing sgRNA, EGFP mRNA and Cas9mRNA is used for carrying out co-transfection on CHO-K1 cells, the RNA-based GS knockout method is provided, and the CRISPR/Cas9 gene editing means and GFP screening are combined, so that the genome reformation on a DNA layer is avoided, the risk of genome pollution is reduced, the GS gene knockout efficiency is improved, meanwhile, the compound is not required to be used for CHO-K1 cell screening of GS gene knockout, and the workload of cell screening is reduced.

In this example, the method further comprises a step of verifying the GS gene knockout CHO-K1 cell: and (3) verifying GS expression of the GS gene knockout CHO-K1 cell and sequencing the gene of the GS gene knockout CHO-K1 cell.

And C: the GS expression of the CHO-K1 monoclonal cell strain with the GS gene knockout is tested, and the specific operation steps are as follows:

in the step C1, the cell lysate used is RIPA lysate; the loading buffer used in step F1 was a 2xLaemmli buffer and contained 5% β -mercaptoethanol.

Step C2: performing GS expression analysis on the product obtained in the step C1 by a western blotting method;

in the step C2, an electrophoretogram is shown in fig. 5, wherein viculin is an electrophoretic band of an internal reference protein, and GS is an electrophoretic band expressed by the sample GS; m is a standard molecular weight electrophoresis band, C is a GS expression electrophoresis band of a control group (obtained by adherent culture after purchase from ATCC, and purchased with a lot number of 62960170), the number 3 is a GS expression electrophoresis band of a GS knockout CHO-K1 cell of the invention, the molecular weight of GS protein is about 45Kda, and the molecular weight of internal reference protein is about 120 Kda; thus, as can be seen in fig. 5, the control group showed GS expression, whereas CHO-K1, which is the GS gene knockout of the present invention, showed no GS expression.

The test structure shows that: deletion and insertion mutations were detected simultaneously in the GS exon-5 region of the sample, i.e., the GS knockout CHO-K1 monoclonal cell line cultured in this example was as expected.

The invention also provides a GS gene knockout CHO-K1 suspension cell line, which comprises GS knockout CHO-K1 cell suspension domestication and frozen cell bank formation, GS knockout CHO-K1 suspension cell monoclonality and cell bank establishment:

step E: the method comprises the following specific steps of (1) suspension domestication of GS knockout cells and formation of a cryopreserved cell bank:

step E1: recovering the GS knocked-out CHO-K1 cells obtained in the step B, and performing adherent culture by using an F12K culture medium, wherein L-glutamine is added into the culture medium;

step E2: subculturing the anchorage-cultured GS knockout CHO-K1 cells to the 13 th generation;

in step E2, the medium used in the 1 st generation culture was serum-free EXCELL302 medium containing 4mM L-glutamine and 2% GS supplement.

In step E2, the medium used in the 2 nd to 6 th generations was EXCELL302 medium containing 4mM L-glutamine and 2% GS supplement.

In step E2, the generation 7 medium was 50% EXCELL302+ 50% Fusion serum-free medium containing 6mM L-glutamine.

In step E2, the medium used in the 8 th generation was 25% EXCELL302+ 75% Fusion serum-free medium containing 6mM L-glutamine.

In step E2, the medium used in the 9 th to 13 th generations was Fusion serum-free medium containing 6mM L-glutamine.

In the step E2, cell sealing and cell Viability curves are shown in fig. 6, and in fig. 6, VCD is cell density (viable cell count) and viatility is cell Viability, which shows that the GS gene knockout CHO-K1 cell prepared by the present invention can be better domesticated by suspension.

Step E3: centrifuging the GS gene knockout CHO-K1 suspension cells obtained in the step E2 for 5 minutes under the condition of 130g, taking the precipitated cells to re-disperse in 140ml of freezing medium, filling the cells into a freezing tube according to 1 ml/tube, pre-freezing at the temperature of minus 80 ℃ for 24 hours, and then transferring the cells into liquid nitrogen for storage.

In step E3, the culture medium was frozen in 126ml of EXCELL Fusion medium, 14ml of DMSO, and 6mM L-glutamine.

Step F: the method for single cloning of the GS knockout CHO-K1 suspension cells and establishing a cell bank comprises the following specific steps:

step F1: recovering and passaging the GS knockout CHO-K1 suspension cells obtained in the step E, dividing the cell passaging into two parts, wherein one part is cultured for 24 hours, centrifuged to obtain supernatant, sterilized and filtered by 0.22 mu m, and stored at 4 ℃ to serve as a conditioned medium; another aliquot was continued and the cultured cells were used for subsequent screening.

Step F2: a screening medium is composed of the conditioned medium obtained in the step F1 and a cloning medium, and 6mM L-glutamine is added, wherein the conditioned medium accounts for 20%, and the cloning medium accounts for 80%; the selection medium was used to select the cells used for selection in step F1.

In step F2, the cloning medium is EXCELL cloning medium.

Step F3: and (3) taking 10ml of the cell culture solution for screening in the step F1, transferring the cell culture solution into a 50ml centrifuge tube, sorting the cells into 5 96-well plates by using a flow cytometer, wherein each well contains 200 mu l of screening culture medium, then, photographing and observing the well plates by using cell imaging equipment, recording, selecting the cloned cells determined to be the source of the single cells, and expanding and culturing the cloned cells to 24-well plates.

Step F4: cloning cells after propagation at 1.5X 10⁵Cells/ml were inoculated into 125 shake flasks and the cell density and cell viability were observed periodically.

In the above step F4, the medium was EXCELL Fusion medium and contained 6mM L-glutamine.

Step F5: and (4) selecting clones with better cell density and cell activity, and freezing and storing.

Step F6: the clone obtained in step F5 was recovered and cloned at a cell density of 3X 10⁵-5×10⁵Inoculating the cells/ml into a 125ml shake flask for culture, wherein the used culture medium is EXCELL Advanced culture medium containing 6mM L-glutamine, and the culture volume is 30 ml; then expanding culture to 2 × 500ml shake flask, the culture volume is 2 × 100 ml; then expanding and culturing to 2 × 1000ml shake flask with the culture volume of 2 × 300ml as fermentation seeds;

step F7: inoculating the fermented seeds into a 7L bioreactor, and culturing for 14 days, wherein the used substrate is EXCELL Advanced CHO Fed-Batch culture medium added with 6mM L-glutamine for 3.5L; the Feed used was EX-CELL Advanced CHO Feed 1 and 30mM L-glutamine without glucose.

In step F7, the feed was added every other day starting on the third day, and the feed amounts on days 3 and 11 were 8mL and the feed amounts on days 5, 7, and 9 were 16 mL.

In step F7, glucose was added to a final concentration of 5g/L on day 3.

Step F8: after 14 days of culture, 500ml of culture solution is taken, 300g of culture solution is centrifuged for 10 minutes, precipitated cells are taken and suspended by using a freezing culture medium, and then the cells are subpackaged into freezing tubes according to 1 ml/tube, and are pre-frozen at the temperature of-70 ℃ and then transferred into liquid nitrogen for freezing, so that the single clone cell bank of the suspension cells of the CHO-K1 knocked out by GS is obtained.

In the above step F8, the cryopreservation medium was EX-CELL Advanced medium containing 6mM L-glutamine and 10% DMSO.

The obtained GS gene knockout CHO-K1 suspension cell monoclonal cell bank meets the requirement of the pharmaceutical industry on high-density cell culture.

In conclusion, the transfection mixture containing sgRNA, EGFP mRNA and Cas9mRNA is used for carrying out cotransfection on CHO-K1 cells to obtain a GS gene knockout CHO-K1 cell strain, the GS gene knockout method is based on RNA, and the CRISPR/Cas9 gene editing means and GFP screening are combined, so that genome reformation at a DNA level is avoided, the risk of genome pollution is reduced, the GS gene knockout efficiency is improved, meanwhile, the compound is not required to be used for screening the GS gene knockout CHO-K1 cells, and the workload of cell screening is reduced; the CHO-K1 cell strain with the GS gene knocked out obtained by the invention is verified, and the GS gene knocking out efficiency is expected. The CHO-K1 cell strain with the knockout GS gene is subjected to suspension domestication and monoclonality, and the requirement of the pharmaceutical industry on high-density cell culture is met.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> Shanghai Bibo biomedical science and technology Co., Ltd

<120> construction method of GS gene knockout CHO-K1 cell strain and suspension cell monoclonality

<130> CPC-NP-21-102512

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<170> SIPOSequenceListing 1.0

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<211> 200

<212> DNA

<213> exon-5 (exon-5)

<400> 1

tgggaattcc aaataggacc ctgtgaagga atccgcatgg gagatcatct ctgggtggcc 60

cgtttcatct tgcatcgagt atgtgaagac tttggggtaa tagcaacctt tgaccccaag 120

cccattcctg ggaactggaa tggtgcaggc tgccatacca actttagcac caaggccatg 180

cgggaggaga atggtctgaa 200

<210> 2

<211> 20

<212> RNA

<213> SgRNA-57(SgRNA-57)

<400> 2

uagcaccaag gccaugcggg 20

<210> 3

<211> 20

<212> DNA

<213> GS-F(GS-F)

<400> 3

atggccacct cagcaagttc 20

<210> 4

<211> 22

<212> DNA

<213> GS-R(GS-R)

<400> 4

ttagtttttg tattggaagg gc 22

<210> 5

<211> 25

<212> DNA

<213> GS-Surveyor-F1(GS-Surveyor-F1)

<400> 5

gagcttgagt tggcctgaac agtta 25

<210> 6

<211> 25

<212> DNA

<213> GS-Surveyor-R1(GS-Surveyor-R1)

<400> 6

aagtccaact ttcccaaccc taaca 25

<210> 7

<211> 25

<212> DNA

<213> GS-Surveyor-F2(GS-Surveyor-F2)

<400> 7

gcccaggtaa atggcactat tctgt 25

<210> 8

<211> 25

<212> DNA

<213> GS-Surveyor-R2(GS-Surveyor-R2)

<400> 8

acttctccaa cgggtacaaa acgat 25

Claims

1. A construction method of a CHO-K1 cell strain with GS gene knockout, which is characterized by comprising the following steps:

the nucleotide sequence of exon-5 is shown as SQE ID NO. 1;

the nucleotide sequence of the sgRNA-GS57 is shown as SQE ID NO. 2;

2. The method for constructing a GS gene knockout CHO-K1 cell line according to claim 1, further comprising the steps of:

3. The method for constructing the GS knockout CHO-K1 cell line, according to claim 1, wherein in the step B, the molar weight ratio of EGFP mRNA to Cas9mRMA of the transfection mixture satisfies the following relationship: EGFP mRNA: Cas9mRMA ═ 1: 10.

4. The method for constructing the GS knockout CHO-K1 cell line according to claim 3, wherein the medium used in the transfection mixture is a serum-reduced medium, and the transfer mixture further comprises a transfection reagent.

5. The method for constructing a GS gene knockout CHO-K1 cell line according to claim 1, wherein in the step B, the CHO-K1 cell to be transfected is cultured adherently in a culture medium of F12K containing 10% fetal bovine serum.

6. The method for constructing a GS knockout CHO-K1 cell line according to claim 1, wherein in the step B, the transfection time is three days, wherein the cell line is cultured in F12K medium containing 10% fetal bovine serum for one day, and then the medium is replaced for two days, and the replaced medium is F12K medium containing 10% fetal bovine serum and 4mM L-glutamine.

7. The method for constructing a GS knock-out CHO-K1 cell line according to claim 1, wherein in the step B, the co-transfected CHO-K1 cells selected by FACS are GFP-positive co-transfected CHO-K1 cells.

8. The method for constructing a GS gene knockout CHO-K1 cell line according to claim 7, wherein the specific steps of culturing a monoclonal cell line after single cell sorting of GFP positive co-transfected CHO-K1 cells are as follows:

9. The method for constructing a GS gene knockout CHO-K1 cell line according to claim 8,

in the step B1, the culture medium used by the 96-well plate is F12K culture medium containing 10% fetal bovine serum, and the culture medium also comprises 1% content of streptomycin qing;

10. The method for constructing a GS gene-knockout CHO-K1 cell line according to claim 2, wherein the step of examining GS expression of a GS gene-knockout CHO-K1 monoclonal cell line is as follows:

11. The method for constructing a GS knock-out CHO-K1 cell line according to claim 10, wherein the cell lysate used in the step C1 is RIPA lysate.

12. The method for constructing a GS knock-out CHO-K1 cell line according to claim 10, wherein the loading buffer used in the step C1 is 2xLaemmli buffer and contains 5% β -mercaptoethanol.

13. A GS gene-knockout CHO-K1 suspension cell monoclonal cell bank obtained by suspension acclimation and monoclonality of a GS gene-knockout CHO-K1 cell strain according to claim 1, comprising the steps of: