Preparation method of efficient and stable gene knockout cell line

Abstract

The invention relates to the technical field of gene engineering technology, in particular to a preparation method of an efficient and stable gene knockout cell line. According to the AKAP11 gene knockout cell line provided by the invention, the 6th exon of the AKAP11 gene in the cell line is subjected to 3395bp frameshift deletion mutation, so that a coding frame is shifted, and protein translation is terminated in advance; after continuous subculture, the homozygous knockout phenotype is still maintained through PCR sequencing verification. A CRISPR / Cas9 system is combined with puromycin screening, the positive cloning rate reaches 30% or above, the construction period is shortened to 45 days, the constructed cell line can be directly used for AKAP11 related cell signal path research, disease model construction and targeted drug screening, and a reliable tool is provided for basic research and clinical transformation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering technology, and in particular to a preparation method of a high-efficiency and stable gene knockout cell line. BACKGROUND

[0002] AKAP11 (A-kinase anchoring protein 11) is an important member of the A-kinase anchoring protein family. By combining with signal molecules such as protein kinase A (PKA) and phosphatase, AKAP11 participates in the spatiotemporal regulation of intracellular signal transduction and plays a key role in physiological processes such as cell proliferation, apoptosis, chromosome separation and neuronal differentiation. In recent years, research has found that AKAP11 gene abnormalities are closely related to a variety of diseases: in the field of tumors, high expression of AKAP11 can promote the invasion and metastasis of lung cancer cells; in neurological diseases, loss of AKAP11 function may lead to synaptic formation disorders in neurons, which is related to the pathogenesis of Alzheimer's disease.

[0003] At present, the function research of AKAP11 mainly depends on RNA interference technology, but this technology has the limitations of unstable silencing efficiency (usually less than 70%), significant off-target effect and reversible effect, which makes it difficult to accurately reveal the physiological function of the gene. The existing AKAP11 knockout cell lines are mostly constructed by zinc finger nuclease (ZFN) or TALEN technology, which has the following defects: (1) low knockout efficiency, mostly heterozygous knockout, which cannot completely eliminate the function of the gene; (2) poor genetic stability, some cells have gene sequence recovery after 10 generations of subculture; (3) long construction period (usually 3-6 months), and the positive clone rate is less than 5%. Therefore, it is of great significance to construct an AKAP11 KO cell line with gene homozygous knockout, high genetic stability and high efficiency for in-depth study of the biological function of AKAP11 and the mechanism of related diseases. SUMMARY

[0004] In order to solve the problems in the prior art, the present application aims to overcome the defects of incomplete knockout, poor genetic stability and low construction efficiency of the existing AKAP11 knockout cell line, and provides an AKAP11 gene homozygous knockout cell line which still maintains the knockout state after continuous subculture, and a high-efficiency and stable construction method.

[0005] The first aspect of the present application provides an AKAP11 gene knockout cell line, wherein the 6th exon of the AKAP11 gene in the cell line has a 3395bp frameshift deletion mutation, which causes a shift in the coding frame and premature termination of protein translation; after continuous subculture, the homozygous knockout phenotype is still maintained by PCR sequencing verification.

[0006] Further, the host cell of the cell line is a 293T cell.

[0007] Further, the number of continuous passage culture is not less than 10 generations.

[0008] The second aspect of the application provides a method for constructing the AKAP11 gene knockout cell line, comprising the following steps:

[0009] (1) sgRNA design and screening

[0010] Two sgRNAs are designed for the 6th exon of AKAP11 gene, and the sequences are as follows:

[0011] sgRNA1: 5'-ttaaagattctcattagctc-3',

[0012] sgRNA2: 5'-agatcggtaggcaagcctat-3';

[0013] (2) Vector construction

[0014] The sgRNA screened in step (1) is cloned into the BbsI enzyme cutting site of pSpCas9(BB)-2A-Puro vector (Addgene#62988) to construct sgRNA-Cas9-puro recombinant expression vector, and the vector construction is verified by sequencing to be correct;

[0015] (3) Cell transfection

[0016] The logarithmically growing host cells are inoculated in a 6-well plate, and after 24 hours of culture, the cells reach 70%-80% of the fusion degree, and the recombinant expression vector is transfected into the host cells by using a transfection reagent;

[0017] (4) Positive clone screening

[0018] After 48 hours of transfection, the screening medium containing puromycin is replaced, and the resistant cell clones are obtained by continuous screening;

[0019] (5) Single clone culture and genotype identification

[0020] The resistant cell clones are inoculated in a 96-well plate by using the limited dilution method, and the single clone cell strain is obtained by culture; the genomic DNA of each single clone cell is extracted, and the identification primers F / R are used for PCR amplification,

[0021] The PCR product is verified by Sanger sequencing, and the cell strain with homozygous knockout of AKAP11 gene is screened, which is the target AKAP11 KO cell line;

[0022] The sequence of the identification primer F is 5'-ctaacatgcgtgacagcatgca-3', and the sequence of the identification primer R is: 5'-agtatctgcaagcttgacctgtaagag-3';

[0023] (6) Stability and function verification

[0024] After the AKAP11 KO cell line obtained in step (5) is continuously passaged, sampling is performed, genotyping is verified by PCR sequencing, and it is confirmed that the homozygous knockout phenotype is maintained.

[0025] Further, in step (3), the host cell is 293T cell, the inoculation density is 1×10 5 Cells / hole, the transfection reagent used is Biyun Tian lipo8000, and the amount of recombinant expression vector used is 2 ug / hole.

[0026] Further, in step (3), the DMEM culture medium containing 10% fetal bovine serum by mass fraction is used for the culture of the host cell, and the culture is carried out at 37 DEG C in a CO2 incubator containing 5% by volume for 24 hours.

[0027] Further, in step (4), the concentration of puromycin in the screening medium is 1-10 ug / ml, and the continuous screening time is 5-7 days.

[0028] Further, in step (5), the monoclonal cell strain is obtained after 14 days of culture; the PCR amplification system is 50 uL, including: DNA template 1 uL (50-200 ng), F primer (10 uM) 1 uL, R primer (10 uM) 1 uL, GXL DNA Polymerase 25 uL, GXL pcr buffer 10 uL, 2.5 mM dNTP 4 uL, and the rest is dd H2O.

[0029] Further, in step (5), the PCR amplification program is: ① 98 DEG C pre-denaturation for 3 min; ② 98 DEG C denaturation for 10 s, 60 DEG C annealing for 15 s, 68 DEG C extension for 30 s, cycle 40 times; ③ 68 DEG C final extension for 5 min.

[0030] Further, in step (6), the number of continuous passages is not less than 10 generations.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] (1) Knockout thoroughness: the AKAP11 KO cell line constructed by the present application is homozygous knockout, and no wild type allele is left after sequencing verification;

[0033] (2) Construction efficiency: The CRISPR / Cas9 system combined with puromycin screening is used, the positive clone rate is more than 30%, and the construction cycle is shortened to 45 days, which is significantly better than the prior art (ZFN / TALEN technology positive rate <5%, cycle 3-6 months);

[0034] (3) Wide application: The cell line can be directly used for AKAP11 related cell signal pathway research, disease model construction and targeted drug screening, and provides a reliable tool for basic research and clinical transformation. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Identification results of the AKAP11 KO cell line constructed for the implementation column. DETAILED DESCRIPTION

[0036] The application will be described in detail below in conjunction with the specific embodiments.

[0037] The application will be described in detail below in conjunction with the specific embodiments. The following examples will help those skilled in the art to further understand the application, but do not limit the application in any form. It should be noted that for those skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made. These all belong to the protection scope of the application.

[0038] Example 1:

[0039] Construction of AKAP11 gene knockout cell line

[0040] (1) sgRNA design and screening

[0041] (1) Based on the sequence of the 6th exon of human AKAP11 gene, 2 sgRNAs were designed using online tool CRISPR OR, and the oligo sequence of sgRNA was synthesized by GenScript:

[0042] SgRNA1: 5'-ttaaagattctcattagctc-3'

[0043] SgRNA2: 5'-agatcggtaggcaagcctat-3'

[0044] (2) Vector construction

[0045] The sgRNA sequence screened in step (1) was cloned into the BbsI enzyme cutting site of pSpCas9(BB)-2A-Puro vector (Addgene #62988) to construct recombinant vectors pSpCas9-AKAP11-sgRNA1 and pSpCas9-AKAP11-sgRNA1; Sanger sequencing verification, sgRNA sequence insertion is correct, no base mutation.

[0046] (3) Cell transfection

[0047] 1) Host cell preparation: Take the logarithmic growth period of HEK293T cells, inoculate 1×10 5 cells / well in a 6-well plate, use DMEM medium containing 10% fetal bovine serum, cultivate in a 37℃, 5% CO2 incubator for 24h, until the cell confluence reaches 70%-80%;

[0048] 2) Transfection: according to the operation instruction of lipo8000 (Biyun Tian, C0533), cultivate at 37℃ for 48h.

[0049] (4) Positive clone screening

[0050] After transfection for 48h, replace the screening medium containing 2μg / mL puromycin (Bi Yun Tian, ST551), replace the medium every 3 days, continue screening for 10 days, and the surviving cells are resistant clones.

[0051] (5) Single clone identification

[0052] 1) Single clone culture: use limited dilution method to inoculate resistant cells to 96-well plate (1 cell / well), cultivate for 14 days, pick single clone cell mass to 24-well plate for expansion culture;

[0053] 2) DNA extraction: use Tengen genomic DNA extraction kit (DP304) to extract cell DNA, the operation steps are as follows:

[0054] ① Collect 1×10 6 cells, add 200μL Buffer GA and 20μL Proteinase K, incubate at 56℃ overnight until the cells are completely lysed;

[0055] ② Add 200μL Buffer GB, incubate at 70℃ for 10min, the solution becomes clear;

[0056] ③ Add 200μL anhydrous ethanol, shake well and transfer to the adsorption column CB3, centrifuge at 12000rpm for 30s, discard the waste liquid;

[0057] ④ Add 500μL Buffer GD, centrifuge at 12000rpm for 30s, discard the waste liquid;

[0058] V. Add 600 μL rinse solution PW, centrifuge at 12000 rpm for 30 s, repeat 1 time;

[0059] VI. Dry the adsorption column at room temperature for 5 min, add 50 μL ddH2O, stand at room temperature for 5 min, centrifuge at 12000 rpm for 2 min, collect the DNA solution;

[0060] 3) PCR amplification and sequencing:

[0061] I. Primer design: design identification primers (amplification fragment length 1005 bp) F: 5'-ctaacatgcgtgacagcatgca-3'

[0062] R: 5'-agtatctgcaagcttgacctgtaagag-3'

[0063] II. PCR system (50 μL):

[0064] The PCR amplification system is as follows:

[0065] Component Volume DNA template 1 μl (50-200 ng) F1 primer (10 μM) 1 μl R1 primer (10 μM) 1 μl GXL DNA Polymerase 25 μl GXL pcr buffer 10 μl 2.5 mM dNTP 4 μl ddH2O Up to 50 μl

[0066] The PCR reaction program is as follows:

[0067]

[0068] Example 2:

[0069] Stability verification of AKAP11-KO-#6 cell line

[0070] (1) Subculture: AKAP11-KO-#5 cells were continuously subcultured and sampled;

[0071] (2) Genotype identification: detection according to the PCR sequencing method of Example 1, the results show that each generation is a homozygous deletion genotype, and there is no wild type sequence;

[0072] Figure 1 The identification results of the AKAP11 KO cell line constructed in the example are shown in the following table: Figure 1 As can be seen from the sequencing, the cell line with a deletion of 3395 bp of the sixth exon of the AKAP11 gene is finally obtained, and is stably subcultured. Compared with the sequencing of the wild type cell, the constructed KO cell line has a deletion of 3395 bp.

[0073] The specific embodiments of the present application are described above. It needs to be understood that the present application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which do not affect the essence of the present application.

Claims

1. An AKAP11 gene knockout cell line, characterized in that, In this cell line, a 3395bp frameshift deletion mutation occurred in exon 6 of the AKAP11 gene, resulting in a shift of the coding frame and premature termination of protein translation. After continuous passage culture, the homozygous knockout phenotype was still maintained by PCR sequencing.

2. The AKAP11 gene knockout cell line according to claim 1, characterized in that, The host cells of the cell line are 293T cells.

3. The AKAP11 gene knockout cell line according to claim 1, characterized in that, The number of generations of continuous subculturing shall not be less than 10.

4. A method for constructing an AKAP11 gene knockout cell line as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) sgRNA design and screening Two sgRNAs were designed targeting exon 6 of the AKAP11 gene, with the following sequences: sgRNA1: 5'-ttaaagattctcattagctc-3', sgRNA2: 5'-agatcggtaggcaagcctat-3'; (2) Carrier construction The sgRNA obtained in step (1) was cloned into the BbsI restriction site of the pSpCas9(BB)-2A-Puro vector (Addgene#62988) to construct the sgRNA-Cas9-puro recombinant expression vector. Sequencing confirmed that the vector was constructed correctly. (3) Cell transfection Host cells in the logarithmic growth phase were seeded into 6-well plates and cultured for 24 hours until the cell confluence reached 70%-80%. The recombinant expression vector was then transfected into the host cells using a transfection reagent. (4) Screening of positive clones 48 hours after transfection, the selection medium was replaced with puromycin-containing medium, and resistant cell clones were obtained through continuous screening. (5) Single-clone culture and genotype identification Resistant cell clones were seeded into 96-well plates using a limiting dilution method to obtain monoclonal cell lines. Genomic DNA was extracted from each monoclonal cell line and PCR amplification was performed using identification primers F / R. The PCR products were verified by Sanger sequencing, and cell lines with homozygous knockout of the AKAP11 gene were selected as the target AKAP11KO cell lines. The sequence of the identification primer F is 5'-ctaacatgcgtgacagcatgca-3', and the sequence of the identification primer R is 5'-agtatctgcaagcttgacctgtaagag-3'. (6) Stability and Functionality Verification After continuous passage of the AKAP11 KO cell line obtained in step (5), samples were taken and the genotype was verified by PCR sequencing to confirm that it maintained the homozygous knockout phenotype.

5. The method for constructing the AKAP11 gene knockout cell line according to claim 4, characterized in that, In step (3), the host cells are 293T cells, and the seeding density is 1×10⁻⁶ cells. 5 Cells / well, the transfection reagent used was Beyotime lipo8000, and the amount of recombinant expression vector used was 2 μg / well.

6. The method for constructing the AKAP11 gene knockout cell line according to claim 4, characterized in that, In step (3), the host cells were cultured in DMEM medium containing 10% fetal bovine serum and cultured at 37°C in a CO2 incubator containing 5% CO2 for 24 hours.

7. The method for constructing the AKAP11 gene knockout cell line according to claim 4, characterized in that, In step (4), the concentration of puromycin in the screening medium is 1-10 μg / mL, and the screening time is 5-7 days.

8. The method for constructing the AKAP11 gene knockout cell line according to claim 4, characterized in that, In step (5), a single-clone cell line is obtained after culturing for 14 days; the PCR amplification system is 50 μL and includes: 1 μL DNA template (50-200 ng), 1 μL F primer (10 μM), 1 μL R primer (10 μM), 25 μL GXL DNA Polymerase, 10 μL GXL pcr buffer, 4 μL 2.5 mM dNTP, and the remainder is ddH2O.

9. The method for constructing the AKAP11 gene knockout cell line according to claim 4, characterized in that, In step (5), the PCR amplification program is as follows: ① 98℃ pre-denaturation for 3 min; ② 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 68℃ extension for 30 s, cycled 40 times; ③ 68℃ final extension for 5 min.

10. The method for constructing the AKAP11 gene knockout cell line according to claim 4, characterized in that, In step (6), the number of consecutive generations is no less than 10.

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