CN116083484A - Construction method of HEL-S-283 gene knockout human colon cancer HCT116 cell strain - Google Patents
Construction method of HEL-S-283 gene knockout human colon cancer HCT116 cell strain Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, and particularly discloses a construction method of a human colon cancer HCT116 cell strain with HEL-S-283 gene knocked out. The invention constructs the sgRNA sequence containing the specific targeting HEL-S-283 gene: the recombinant lentiviral plasmids of CRISPR-V2-HEL-S-283-gRNA1 and CRISPR-V2-HEL-S-283-gRNA2 of TCATGGTAGGATTGTGACAAAGG and AACCATGAAGTCATCTAGCAGGG can introduce HEL-S-283gRNA1 and gRNA2 specificity into host cells and perform functions through a CRISPR-Cas9 system, and finally the human colon cancer HCT116 cell strain for knocking out HEL-S-283 genes is obtained, the knocking-out result is stable, and the knocking-out efficiency is high.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a construction method of a human colon cancer HCT116 cell strain with HEL-S-283 gene knocked out.
Background
Colon cancer is a common malignancy of the digestive tract. The global incidence of colon cancer in 2020 was as high as 10.0% and was secondary to lung and breast cancer, and its mortality rate in cancer was secondary to all malignant tumors (9.4%), and secondary to lung cancer. Along with the progress of the aging of society and the change of dietary structures and life styles of people, the incidence rate of colon cancer is continuously increased. About 60% of patients are already in the middle and late stages of the course of the disease at the time of initial diagnosis, losing the opportunity for radical surgical treatment. And once recurrence and metastasis occur after radical operation of early patients, the survival of the patients can be seriously threatened. The deep exploration of the occurrence and metastasis mechanism of colon cancer and the exploration of novel recurrence and metastasis related markers become research hot spots in recent years, and the deep exploration of the occurrence and metastasis mechanism of colon cancer is helpful for optimizing individual and accurate treatment of colon cancer patients. Therefore, the early diagnosis marker of the intestinal cancer with high specificity, good sensitivity and strong stability is searched, the important role of the early diagnosis marker in the tumorigenesis and metastasis process is clarified, the clinical-patient treatment mode is guided, the postoperative survival rate of malignant tumor patients is improved, and the life quality of the patients is improved.
HEL-S-283, also known as REGγ, PA28 γ or 11Sγ, is a member of the 11S proteasome activator family. HEL-S-283 can exert the ability to regulate cell cycle, transcription, signaling, death, and immune response by activating the 20S proteasome to degrade multiple target proteins in a non-ubiquitin and ATP-independent manner. Researchers have detected the expression level of HEL-S-283 in tissue chips containing lung cancer, thyroid cancer, colon cancer, liver cancer and corresponding tissues beside the cancer by using an immunohistochemical method, found that HEL-S-283 is obviously highly expressed in the tumor tissues (the number of cases exceeds 50%), and further found that HEL-S-283 acts on each signal channel to participate in the occurrence and development of various tumors by further analysis. Therefore, HEL-S-283 has become one of the hot spots in the field of basic research of tumors.
The CRISPR-Cas9 system was developed from the adaptive immune system of bacteria and archaea against foreign viruses or plasmids, and includes three different types, of which the Type ii CRISPR/Cas system has only one subunit of DNA endonuclease Cas9, the simplest structure, and therefore the most widely used. In addition to the Cas9 protein, the system also includes two short bars CRISPRRNAs (crRNAs) and trans-activating crRNAs (tracrRNA). The CRISPR-Cas9 system has the characteristics of simple operation, high cutting efficiency and the like, and is considered to have better application prospect.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provide a HEL-S-283 gene knockout human colon cancer HCT116 cell strain and a construction method thereof. The HEL-S-283 gene knockout human colon cancer HCT116 cell strain provides important significance for clinically preventing and treating colon cancer, researching pathogenesis and screening anti-colon cancer drugs.
The invention provides a construction method of HEL-S-283 gene knockout human colon cancer HCT116 cell strain, which comprises the following steps:
(1) Designing a sequence of sgRNAs aiming at a coding sequence of HEL-S-283 gene of human colon cancer HCT116 cells, wherein the sgRNAs comprise sgRNA1 and sgRNA2;
(2) Cloning the sgRNAs obtained in the step (1) onto a CRISPR-V2 vector to obtain CRISPR-V2-HEL-S-283-gRNA1 and CRISPR-V2-HEL-S-283-gRNA2 plasmids;
(3) Transfecting the CRISPR-V2-HEL-S-283-gRNA1 and the CRISPR-V2-HEL-S-283-gRNA2 plasmids obtained in the step (2) into human colon cancer HCT116 cells by using a liposome transfection method;
(4) Culturing the transfected HCT116 cells for 36-48 hours in a conventional way;
(5) First screening: carrying out limiting dilution on the cells cultured in the step (4) to obtain monoclonal cells, and culturing to obtain monoclonal cell strains;
(6) Second screening: obtaining monoclonal cells from the monoclonal cell strain in the step (5) by limiting dilution method, and continuously culturing to obtain HCT116HEL-S-283 -/- A monoclonal cell line;
(7) Extraction of HCT116HEL-S-283 -/- Monoclonal cell strain genome DNA, PCR amplifying sgRNA sequence, analyzing amplified product; the cell strain incapable of amplifying fragments with theoretical length is HCT116HEL-S-283 -/- A cell line.
In step (1), the sgRNAs sequences are:
sgRNA1：TCATGGTAGGATTGTGACAAAGG(SEQ ID NO.1)
sgRNA2：AACCATGAAGTCATCTAGCAGGG(SEQ ID NO.2)
in the step (3), the transfection reagent used in the transfection is Lipofectamine TM 3000(Thermo Scientific TM ，Cat No.：L3000150)。
The plating cell density in the limiting dilution method described in the steps (5) and (6) is preferably 0.3 to 0.5 cells/well.
In the step (5), the culture time is 7-14 days; preferably from 12 to 14 days.
In the step (6), the culture time is 7 days.
In the step (7), the PCR amplification primer sequence is as follows:
primers for Region1 (annealing temperature 62.0 ℃ C.).
Upstream primer GGGTATCTTGTGATGGAGATGTGG (SEQ ID NO. 3)
Downstream primer GCACTTCAGGTGGGGTACTT (SEQ ID NO. 4)
Primers for Region2 (annealing temperature 62.0 ℃ C.).
Upstream primer TCCTCCTCTTCTCTCTCTTTCCA (SEQ ID NO. 5)
Downstream primer TGAGATGGAGTTTCGCTCTTGT (SEQ ID NO. 6)
Primers for Region3 (annealing temperature 62.0 ℃ C.).
Upstream primer GGGTATCTTGTGATGGAGATGTGG (SEQ ID NO. 7)
Downstream primer TGAGATGGAGTTTCGCTCTTGT (SEQ ID NO. 8)
In one specific embodiment, the construction method of the HEL-S-283 gene knockout human colon cancer HCT116 cell strain comprises the following steps:
(1) Aiming at the coding sequence of the HEL-S-283 gene of the human colon cancer HCT116 cell, the sgRNAs sequence is designed;
(2) Cloning the sgRNA obtained in the step (1) onto a CRISPR-V2 vector to obtain CRISPR-V2-HEL-S-283-gRNA2 and CRISPR-V2-HEL-S-283-gRNA2 plasmids;
(3) By Lipofectamine TM 3000(Thermo Scientific TM CatNo.: l3000150) transfection reagent CRISPR-V2-HEL-S-283-gRNA2 and CRISPR-V2-HEL-S-28 in (2)3-gRNA2 plasmid is co-transferred into human colon cancer HCT116 cell strain;
(4) Culturing the transfected HCT116 cell strain for 36-48 hours in a conventional way;
(5) Discarding the culture medium supernatant, washing 2 times with phosphate buffer solution, performing pancreatin digestion, stopping digestion, centrifuging, and re-suspending with the whole culture medium;
(6) Diluting the resuspended cells into a monoclonal antibody, and inoculating the monoclonal antibody into a 96-well plate for culture;
(7) After single cells form cell clusters with proper size, carrying out passage in 96-well plates, and continuing the culture of the monoclonal cells;
(8) Performing amplification culture and genotyping;
(9) Sending to gene sequencing company for whole genome detection.
The transfection method in the step (3) is a liposome transfection method;
the method for obtaining the monoclonal cell strain by separation in the step (6) and the step (7) is a limiting dilution method, and the density of the plated cells is 0.3-0.5 cells/hole;
the time of the culture in the step (8) is 1 to 2 weeks.
The genotyping PCR identifying primer sequence in the step (8) is as follows:
primers for Region1 (annealing temperature 62.0 ℃ C.).
Upstream primer GGGTATCTTGTGATGGAGATGTGG (SEQ ID NO. 3)
Downstream primer GCACTTCAGGTGGGGTACTT (SEQ ID NO. 4)
Primers for Region2 (annealing temperature 62.0 ℃ C.).
Upstream primer TCCTCCTCTTCTCTCTCTTTCCA (SEQ ID NO. 5)
Downstream primer TGAGATGGAGTTTCGCTCTTGT (SEQ ID NO. 6)
Primers for Region3 (annealing temperature 62.0 ℃ C.).
Upstream primer GGGTATCTTGTGATGGAGATGTGG (SEQ ID NO. 7)
Downstream primer TGAGATGGAGTTTCGCTCTTGT (SEQ ID NO. 8)
The invention also provides the HEL-S-283 gene knockout human colon cancer HCT116 cell strain constructed by the method.
The invention also provides application of the HEL-S-283 gene knockout human colon cancer HCT116 cell strain in clinical prevention and treatment of colon cancer, research of pathogenesis and screening of anti-colon cancer drugs.
The invention also provides a sgRNA for knocking out the HEL-S-283 gene of human colon cancer, and the target sequence of the sgRNA is as follows: TCATGGTAGGATTGTGACAAAGG and AACCATGAAGTCATCTAGCAGGG.
The invention also provides application of the sgRNA for knocking out the HEL-S-283 gene of the human colon cancer in construction of HEL-S-283 gene knocked out human colon cancer HCT116 cell strain.
The invention also provides a primer pair, which comprises:
primers for Region1 (annealing temperature 62.0 ℃ C.).
Upstream primer GGGTATCTTGTGATGGAGATGTGG (SEQ ID NO. 3)
Downstream primer GCACTTCAGGTGGGGTACTT (SEQ ID NO. 4)
Primers for Region2 (annealing temperature 62.0 ℃ C.).
Upstream primer TCCTCCTCTTCTCTCTCTTTCCA (SEQ ID NO. 5)
Downstream primer TGAGATGGAGTTTCGCTCTTGT (SEQ ID NO. 6)
Primers for Region3 (annealing temperature 62.0 ℃ C.).
Upstream primer GGGTATCTTGTGATGGAGATGTGG (SEQ ID NO. 7)
Downstream primer TGAGATGGAGTTTCGCTCTTGT (SEQ ID NO. 8)
The invention also provides application of the primer pair in constructing HEL-S-283 gene knockout human colon cancer HCT116 cell strain.
Compared with the prior art, the invention has the following advantages and effects:
the invention constructs the sgRNA sequence containing the specific targeting HEL-S-283 gene: the recombinant lentiviral plasmids of CRISPR-V2-HEL-S-283-gRNA1 and CRISPR-V2-HEL-S-283-gRNA2 of TCATGGTAGGATTGTGACAAAGG and AACCATGAAGTCATCTAGCAGGG can introduce HEL-S-283gRNA1 and gRNA2 specificity into host cells and perform functions through a CRISPR-Cas9 system, and finally, the human colon cancer HCT116 cell strain for knocking out HEL-S-283 genes is obtained, the knocking-out result is stable, the knocking-out efficiency is high, and the research of the obtained knocking-out cell strain lays a foundation for the occurrence and development of human colon cancer.
Drawings
FIG. 1 is a schematic diagram of PCR identification sites and primer sequences of HEL-S-283 gene of the invention.
FIG. 2 is a schematic diagram of HCT116HEL-S-283 in accordance with the present invention -/- Positive clone PCR identification results.
FIG. 3 is a Western Blot result chart of a human colon cancer cell HCT116 gene cell strain with HEL-S-283 gene knocked out.
FIG. 4 is a schematic diagram of HCT116HEL-S-283 in accordance with the invention -/- Positive clone sequencing results.
FIG. 5 is a schematic diagram of HCT116HEL-S-283 in accordance with the invention -/- Positive clone cell morphology picture.
FIG. 6 is a HCT116HEL-S-283 of the invention -/- Cloning experiments of Positive clone cells, knocking out HEL-S-283 -/- After that, the proliferation ability of the cells becomes weak.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
Example 1
Construction of sgRNA expression vector by CRISPR-Cas9 technology
1) Searching human HCT116HEL-S-283 gene sequence on an ensable website (http:// asia. Ensembl. Org/index. Html), finding 6-exon sequences of most transcripts, inputting the 6-exon sequences into a CRISPR design website (http:// crispor. Tefor. Net /) for analysis, and obtaining related sgRNA sequences according to two evaluation principles of sgRNA: if PAM (NGG) exists downstream of the A sequence, the B off-target efficiency is low, and off-target is not easy to occur. 120 bp sgRNA sequence with higher score and fewer off-target sites was selected as the target site.
2) According to the designed sgRNA, the forward primer and the reverse primer are synthesized according to the method of Addgene website (https:// media. Addgene. Org/data/plasmids/52/52961/52961-attribute_B3xTpla0bkYD. Pdf), and CRISPR-V2-HEL-S-283-gRNA1 and CRISPR-V2-HEL-S-283-gRNA2 expression plasmids are constructed according to the description.
Example 2
The monoclonal cells are obtained by adopting a limiting dilution method, and the specific operation is as follows:
1) Removing the cultured cell plates/dishes from the incubator, and discarding the culture medium supernatant;
2) Aiming at the coding sequence of the HEL-S-283 gene of the human colon cancer HCT116 cell, the sgRNAs sequence is designed; the target sequences of the sgRNAs are respectively: TCATGGTAGGATTGTGACAAAGG and AACCATGAAGTCATCTAGCAGGG;
3) Cloning the sgRNA obtained in the step 2) onto a CRISPR-V2 vector to obtain CRISPR-V2-HEL-S-283-gRNA1 and CRISPR-V2-HEL-S-283-gRNA2 plasmids;
4) By Lipofectamine TM 3000(Thermo Scientific TM CatNo.: l3000150) transfection reagent the CRISPR-V2-HEL-S-283-gRNA1 and CRISPR-V2-HEL-S-283-gRNA2 plasmids in 3) were co-transferred into human colon cancer HCT116 cell lines;
5) Conventionally culturing the transfected HCT116 cell strain to about 90% of cell density; discarding the culture medium supernatant, washing 2 times with phosphate buffer solution, digesting with pancreatin, stopping digestion, centrifuging, re-suspending with the whole culture medium, and blowing the cells into single cell suspension;
6) Counting the number of cells by using a cell counter, then adjusting the cell concentration to 100 cells/ml by using a gradient dilution method, adding 10 microliter of single cell suspension into each well of a 96-well plate, culturing for one week in a culture box conventionally by using 15% fetal bovine serum culture medium, observing the monoclonal cell holes by using a microscope, and marking;
7) After the cells grow out of the monoclonal cells, digesting the monoclonal cells by using a pancreatin solution, and transferring the monoclonal cells into a 48-well plate for continuous culture;
8) After the cells grow fully, digesting the monoclonal cells by using a pancreatin solution, and transferring the monoclonal cells into a 24-well plate for continuous culture;
9) After a period of time, taking part of cells to extract DNA for genotyping, sending to a sequencing company for knockout identification, and sequencing results are shown in figure 4; there was a base mutation at the location of the sgRNA, indicating a knockout.
Example 3
1. Extraction of HCT116 cell strain holoprotein and knock-out identification by Western Blot experiment
The whole protein extraction comprises the following specific steps:
1) Cell plating: HCT116 cells were plated at a density of about 20% in 6 cm dishes.
2) When the cells grow to 80% density, the cells are harvested according to the following procedure:
a, removing the culture medium in a 6 cm culture dish, adding 1 ml of precooled phosphate buffer solution, gently washing cells, and removing the phosphate buffer solution;
b re-adding 1 ml of phosphate buffer, gently scraping off the cells with a cell scraper, and transferring the cells into a 1.5 ml EP tube;
c2000 rotation, centrifuging at 4 ℃ for 8 minutes;
d removing the supernatant, placing the cells on ice for later use or freezing briefly in liquid nitrogen, and storing in a refrigerator at-80 ℃.
3) Preparing a protein lysate, adding a protease inhibitor and a phosphatase inhibitor into each 1 ml of cold cell lysate according to a certain proportion, uniformly mixing and placing on ice.
4) The mixture of 3) was added to 1) a 1.5 ml EP tube with cell pellet, 400. Mu.l of protein lysate was added to each tube, and the mixture was lysed on ice for 30 minutes.
5) Centrifuge at 4℃for 10 min at 12000 rpm.
6) The supernatant was transferred to a new pre-chilled EP tube and protein concentration was determined using BCA method.
7) The protein is adjusted to proper concentration by adopting 6 times of loading buffer solution, heated for 10 minutes at 100 ℃, split charging and preservation at-80 ℃.
2. The Western Blot specifically comprises the following steps:
1) Placing the prepared glued plate in a groove, adding into the groove1 Xrunning buffer was added to prepare a sample, and 1. Mu.l and 3. Mu.l protein markers (Thermo Scientific) were added to the wells on each side TM CatNo.:26616 About 10. Mu.l of protein samples were taken and filled with 1 Xloading buffer.
2) At the beginning, the voltage is constant at 80 volts, and the voltage is adjusted to 120 volts after the sample runs out of the concentrated glue.
3) When the indicator bromophenol blue runs to the bottom, electrophoresis is terminated.
4) Preparing a1×transfer buffer: 70% double distilled water, 20% methanol, 10%10 Xtransfer buffer.
5) Transferring: the transfer film is clamped in a 1X transfer film buffer solution, and sponge, 2-3 layers of filter paper, NC film, albumin glue, 2-3 layers of filter paper and sponge are sequentially placed from the black surface to the white surface. After being clamped, the materials are placed in a film rotating groove, the white surface is close to the positive electrode, and the black surface is close to the negative electrode. The transfer time was 1 hour.
6) After completion of transfer, NC membrane was removed (at this time, the protein on the protein gel had been transferred to NC membrane)
7) The 1 XPBS buffer was washed three times, 5 minutes/time, 3 times, followed by the addition of 7% milk solution at room temperature for 1 hour.
8) Milk was decanted, 1 XPBS buffer, 5 min/time, 3 times.
9) The membrane (according to the molecular weight of the target protein) was cut, and the cut membrane was placed in a cassette, 1 XPBS buffer for 5 minutes. The corresponding antibody was then added overnight at 4 ℃.
10 Primary antibody was recovered and washed with 1 XPBST buffer for 3X 5 min.
11 Fluorescent secondary antibody, 4 ℃ for 1 hour.
12 Film scanning by using an Odessy machine, and data processing. The Western Blot result of the HCT116 cell strain of the human colon cancer HEL-S-283 gene is shown in figure 3; FIG. 4 shows that there is a sequence deletion at the location of the sgRNA, indicating that there is a knockout, and that Western Blot has been performed to verify the success of the knockout, and FIG. 3 shows that the knocked-out cell line does not express HEL-S-283 relative to the wild type, indicating that the knocked-out cell line was successfully obtained.
Example 4
Cloning formation experiments
1) Cell inoculation: cell passaging was performed according to cell passaging technique, and control cells and HCT116HEL-S-283 were used -/- Cells were inoculated in 6-well plates, 1500 cells/well, three wells were set, and mixed well in a cell incubator.
2) And observing the clone formation condition every day, continuously culturing for 14 days, stopping culturing when the clone is formed and is visible to the naked eye, taking out the incubator, and placing the incubator on an external test bed for subsequent operation.
3) Fixing: the original medium was aspirated, and washed with phosphate buffer for 5 minutes, three times. 4% paraformaldehyde is fixed at normal temperature for 15 minutes. After fixation, 4% paraformaldehyde was discarded, and 700. Mu.l of phosphate buffer was added to each well for washing, 5 minutes/time, and washing was repeated three times.
4) Dyeing: 700 microliters of 0.2% crystal violet dye was added to each well and stained at room temperature for 15 minutes.
5) Cleaning: the staining solution was discarded, and the solution was washed with 700. Mu.l of phosphate buffer for 5 minutes/time and repeated three times.
6) And (3) airing: and reversely buckling the pore plate on the water absorbing paper, draining the water in the pore plate, and naturally airing the 6 pore plate.
7) Photographing: and (5) photographing and recording, calculating the clone number, and analyzing the experimental result.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (10)
1. The preparation method for constructing the HEL-S-283 gene knockout human colon cancer HCT116 cell strain is characterized by comprising the following steps:
(1) Aiming at the coding sequence of the HEL-S-283 gene of the human colon cancer HCT116 cell, the sgRNAs sequences comprising sgRNA1 and sgRNA2 are designed;
(2) Cloning the sgRNAs obtained in the step (1) onto a CRISPR-V2 vector to obtain CRISPR-V2-HEL-S-283-gRNA1 and CRISPR-V2-HEL-S-283-gRNA2 plasmids;
(3) Co-transferring the CRISPR-V2-HEL-S-283-gRNA1 and the CRISPR-V2-HEL-S-283-gRNA2 plasmids in the step (2) into a human colon cancer HCT116 cell strain by using a liposome transfection method;
(4) Culturing the transfected HCT116 cell strain for 36-48 hours in a conventional way;
(5) First screening: obtaining monoclonal cells by a cell limiting dilution method after culturing in the step (4), and culturing to obtain monoclonal cell strains;
(6) Second screening: obtaining monoclonal cells by limiting dilution method of the monoclonal cell strain obtained in the step (5), and continuously culturing to obtain HCT116HEL-S-283 -/- A monoclonal cell line;
(7) Extraction of HCT116HEL-S-283 -/- Monoclonal cell strain genome DNA, PCR amplifying sgRNA sequence, analyzing amplified product; the cell strain incapable of amplifying fragments with theoretical length is HCT116HEL-S-283 -/- A cell line.
2. The method according to claim 1, wherein in step (1), the target sequences of the sgRNAs are respectively: TCATGGTAGGATTGTGACAAAGG and AACCATGAAGTCATCTAGCAGGG.
3. The method of claim 1, wherein the step of determining the position of the substrate comprises,
in the step (3), the transfection reagent used in the transfection is Lipofectamine TM 3000；
And/or, the density of the plated cells in the limiting dilution method in the step (5) and the step (6) is 0.3 to 0.5 cells/well;
and/or, in the step (5), the culture time is 7-14 days;
and/or, in the step (6), the culture time is 7 days.
4. The method of claim 1, wherein in step (7), the PCR amplification primer sequences are:
primers for Region1 upstream primer GGGTATCTTGTGATGGAGATGTGG
Primers for Region1 downstream primer GCACTTCAGGTGGGGTACTT;
primers for Region2 upstream primer TCCTCCTCTTCTCTCTCTTTCCA
Primers for Region2 downstream primer TGAGATGGAGTTTCGCTCTTGT;
primers for Region3 upstream primer GGGTATCTTGTGATGGAGATGTGG
Primers for Region3 downstream primer TGAGATGGAGTTTCGCTCTTGT.
5. The HEL-S-283 gene knockout human colon cancer HCT116 cell line constructed according to the method of any one of claims 1-4.
A sgRNA characterized in that the target sequence of said sgRNA is as follows: TCATGGTAGGATTGTGACAAAGG and AACCATGAAGTCATCTAGCAGGG.
7. The use of sgRNA according to claim 6 in the construction of HEL-S-283 knockout human colon carcinoma HCT116 cell line.
8. A primer pair, characterized in that the primer pair comprises:
primers for Region1 upstream primer GGGTATCTTGTGATGGAGATGTGG
Primers for Region1 downstream primer GCACTTCAGGTGGGGTACTT;
primers for Region2 upstream primer TCCTCCTCTTCTCTCTCTTTCCA
Primers for Region2 downstream primer TGAGATGGAGTTTCGCTCTTGT;
primers for Region3 upstream primer GGGTATCTTGTGATGGAGATGTGG
Primers for Region3 downstream primer TGAGATGGAGTTTCGCTCTTGT.
9. The use of the primer pair according to claim 8 in constructing HEL-S-283 gene knockout human colon cancer HCT116 cell line.
10. The use of the HEL-S-283 gene knockout human colon cancer HCT116 cell line according to claim 5 for the prevention and treatment of colon cancer, the study of pathogenesis and the screening of anti-colon cancer drugs.
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