CN110184302B - Construction method of SIRT1 gene knockout IPEC-J2 cell line - Google Patents
Construction method of SIRT1 gene knockout IPEC-J2 cell line Download PDFInfo
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Abstract
The invention discloses a construction method of an IPEC-J2 cell line with SIRT1 gene knockout, which comprises the following steps: (1) Designing and synthesizing a gRNA probe aiming at a first exon of the SIRT1 gene, wherein the base sequence of the gRNA is shown as SEQ ID NO.1, and the reverse complementary sequence of the gRNA is shown as SEQ ID NO. 2; (2) Cloning the double-stranded gRNA probe which forms reverse complementation after annealing into a pSpCas9-2A-PuroV2.0 vector shown in figure 2, and constructing a plasmid which simultaneously expresses sgRNA shown in SEQ ID NO.1 and Cas9 protein; (3) Transfecting the plasmid constructed in the step (2) into IPEC-J2 cells; (4) And (4) screening a monoclonal mutant cell line from the IPEC-J2 cell transfected in the step (3) to obtain a SIRT1 gene knockout IPEC-J2 cell line which is lack of 5 bases in the first exon of the SIRT1 gene compared with a wild type cell line. The invention constructs the SIRT1 gene knockout IPEC-J2 cell line for the first time, and obtains the stable monoclonal SIRT1 gene knockout IPEC-J2 homozygous cell line by screening, which can be used as a cell model for researching piglet intestinal oxidative stress.
Description
Technical Field
The invention relates to the technical field of gene knockout, in particular to a construction method of an IPEC-J2 cell line with SIRT1 gene knockout.
Background
IPEC-J2 cells are porcine intestinal columnar epithelial cells isolated from the jejunum of newborn piglets. When cultured on 0.4 μm pore size filters, this cell line forms a polarized monolayer with a high trans-epithelial resistance. The IPEC-J2 cell is unique in that it is derived from porcine small intestine tissue and is not transformed (compared to IPI-2I) compared to the common human colon derived lines HT-29, T84 and Caco-2. Porcine intestinal epithelial cells mimic the physiology of human intestinal epithelium much more closely than rodent intestinal epithelial cell lines (e.g., IEC-6 or IEC-18), which is particularly important in the study of zoonotic infections. In addition, IPEC-J2 cells are increasingly used in gut oxidative stress studies, which provide a specific model for studying weaned piglet gut health, to detect the interaction between various exogenous antioxidants and gut epithelial cells, and are used as an initial screening tool for potential feed antioxidants.
SIRT1 (Silent formation type formation regulation 2 homologue 1) is a highly conserved Nicotinamide Adenine Dinucleotide (NAD) in mammals + ) The dependent protein deacetylase is important in the physiological and pathological processes of metabolic regulation, development, tumorigenesis, aging and the like. SIRT1, the major nuclear Sirtuin in mammalian tissues, is a key metabolic sensor that responds to external environmental signals by regulating cellular stress responses, energy metabolism, apoptosis, inflammation, and other biological processes. In the past years, SIRT1 has been identified as a key repressor of oxidative stress and inflammation in a variety of tissues/cells. Several recent studies have shown that SIRT1 plays a key role in regulating intestinal inflammation and tissue oxidation-reduction homeostasis. However, whether SIRT1 stimulates or inhibits intestinal stress remainsAt issue, it is unclear how the intestinal epithelial SIRT1 regulates complex environment-host interactions to regulate intestinal epithelial integrity.
Disclosure of Invention
Based on the problems, the invention aims to overcome the defects of the prior art and provide a construction method for constructing a SIRT1 gene knockout IPEC-J2 cell line, and the stable monoclonal SIRT1 gene knockout IPEC-J2 homozygous cell line can be obtained by screening and can be used as a cell model for researching piglet intestinal oxidative stress.
In order to achieve the purpose, the invention adopts the technical scheme that:
a construction method of an IPEC-J2 cell line with SIRT1 gene knockout comprises the following steps:
(1) Designing and synthesizing a gRNA probe aiming at a first exon of the SIRT1 gene, wherein the base sequence of the gRNA is shown as SEQ ID NO.1, and the reverse complementary sequence of the gRNA is shown as SEQ ID NO. 2;
(2) The double-stranded gRNA probe which is formed by annealing the DNA chains shown in SEQ ID NO.1 and 2 and is reversely complementary is cloned into a pSpCas9-2A-Puro (PX 459) V2.0 vector shown in figure 2, and a plasmid which can simultaneously express the sgRNA shown in SEQ ID NO.1 and the Cas9 protein is constructed;
(3) Transfecting the plasmid constructed in the step (2) into IPEC-J2 cells;
(4) Screening a monoclonal mutation cell line from the IPEC-J2 cell transfected in the step (3) to obtain a SIRT1 gene knocked-out IPEC-J2 cell line, wherein the IPEC-J2 cell line lacks 5 basic groups (5 '-GCGG-3') in a first exon of a SIRT1 gene compared with a wild cell line, so that the SIRT1 protein generates frame shift mutation in the translation process and terminates translation in advance. The construction method of the invention has the difficulty that functional gRNAs are screened from a plurality of gRNA sites, then a SIRT1 gene knockout homozygous cell line is screened from a mutant IPEC-J2 chimeric cell population, and the process is repeatedly tested, and finally an IPEC-J2 cell line which is successfully constructed and lacks 5 bases (5 '-GCGGC-3') in the first exon of the SIRT1 gene compared with a wild-type cell line is constructed.
As a further optimization of the above technical solution, in the step (3), lipo3000 (Lipofectamine 3000) is used for transfection.
As a further optimization of the above technical scheme, in the step (4), the method for screening the monoclonal mutant cell line comprises a purine toxin combined limiting dilution method.
As a further optimization of the technical scheme, the concentration of the purotoxin is 2 mug/mL.
As a further optimization of the above technical solution, the use method of the purine toxin comprises: the cells were cultured with the cell culture medium containing the purotoxin and replaced with a new cell culture medium containing the purotoxin every day for a total of 7 days.
As a further optimization of the above technical solution, in the step (4), the method for screening monoclonal mutant cell lines further comprises high resolution melting curve analysis (HRMA).
As a further optimization of the above technical solution, the analyzing of the high resolution melting curve includes:
1) HRMA amplification conditions were: 2min at 98 ℃; 40 cycles of 98 ℃ 15sec,60 ℃ 20sec (signal collected); 10sec at 95 ℃;
2) After the amplification, melting curve analysis was performed, and signals were collected from 55 ℃ to 95 ℃ at 0.2 ℃ per 10 sec.
As another aspect of the invention, the invention provides a SIRT1 gene knockout IPEC-J2 cell line constructed by the method.
As a further aspect of the invention, the invention provides a SIRT1 gene knock-out IPEC-J2 cell line that lacks the 5 bases in the first exon (GCGGC) of the SIRT1 gene compared to a wild-type IPEC-J2 cell line.
In conclusion, the beneficial effects of the invention are as follows:
(1) Constructing a SIRT1 gene knockout IPEC-J2 cell line by using a CRISPR/Cas9 gene editing technology for the first time, and screening to obtain a stable monoclonal SIRT1 gene knockout IPEC-J2 homozygous cell line which can be used as a cell model for researching piglet intestinal oxidative stress;
(2) The function of SIRT1 in intestinal epithelial cells is researched by using an IPEC-J2 cell line with the SIRT1 gene knocked out, so that a signal conduction path of intestinal stress reaction is expected to be analyzed, and a new research model and thought are provided for relieving piglet weaning stress.
Drawings
FIG. 1 is a schematic diagram of a porcine SIRT1 genomic structure and a CRISPR/Cas9 target site;
FIG. 2 is a map of the pSpCas9-2A-Puro (PX 459) V2.0 vector;
FIG. 3 is a PX459 (SIRT 1-sgRNA) plasmid sequencing map;
FIG. 4 is a CRISPR target site sequencing peak diagram of a SIRT1 wild type IPEC-J2 cell line;
FIG. 5 is a CRISPR target site sequencing peak diagram of SIRT1 mutant IPEC-J2 cell line;
FIG. 6 is a sequence alignment chart of IPEC-J2 cell SIRT1 wild type and mutant monoclonal cell lines.
Detailed Description
The invention discloses a construction method for screening a SIRT1 gene knockout single-cell IPEC-J2 homozygous cell line by combining a CRISPR/Cas9 gene editing technology with a puroxin, which comprises the steps of firstly designing a plurality of SIRT1 gene sgRNA (single guide RNA) recognition sites aiming at a first exon in an SIRT1 gene Open Reading Frame (ORF); designing and synthesizing a gRNA probe sequence according to the recognition site, cloning the gRNA probe into a pSpCas9-2A-Puro (PX 459) V2.0 vector, and constructing a plasmid PX459 (SIRT 1-sgRNA) for simultaneously expressing sgRNA and Cas9 protein; the constructed plasmids are respectively transfected into IPEC-J2 cells by using Lipo3000 (Lipofectamine 3000), a monoclonal mutant cell line is screened by combining a purine toxin (Puromycin) with a limiting dilution method, and a mutation result is detected by HRMA (High-Resolution Melt Analysis).
The construction method can cause the frame shift mutation of the genome SIRT1 target site of the chimeric cell line, and achieves the purpose of stably knocking out the SIRT1 gene in IPEC-J2 cells through purine toxin screening and single cell cloning culture; the established SIRT1 gene knockout small intestinal epithelial cell line can provide a model for deeply disclosing the intestinal epithelial barrier function damage and the protection mechanism thereof caused by piglet weaning stress, and can also provide a research model for the action mechanism of novel antioxidant and antibiotic substitute products.
In some embodiments, the method for constructing the SIRT1 knock-out IPEC-J2 cell line of the present invention comprises the steps of:
(1) Designing a SIRT1 gene sgRNA target site on the first exon according to the gene sequence of SIRT1 (as shown in fig. 1);
(2) Cloning a double-stranded gRNA probe which forms a reverse complementary sequence after annealing into a pSpCas9-2A-Puro (PX 459) V2.0 vector (see figure 2) to construct a plasmid PX459 (SIRT 1-sgRNA) for simultaneously expressing sgRNA and Cas9 protein;
(3) Cloning and screening PX459 (SIRT 1-sgRNA) positive plasmids, and sequencing and identifying a gRNA probe insertion result (see figure 3);
(4) Transfecting the constructed plasmid into IPEC-J2 cells by using Lipo3000 (Lipofectamine 3000);
(5) Screening a monoclonal mutant cell line by using a purine toxin (Puromycin) in combination with a limiting dilution method;
(6) Extracting cell genome DNA, and identifying mutation results by HRMA and sequencing.
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. The materials, reagents and the like in the present invention are commercially available or publicly available, unless otherwise specified. Unless otherwise specified, the experimental methods in the present invention are all conventional methods.
Example 1
One embodiment of the construction method of the SIRT1 gene knockout IPEC-J2 cell line comprises the following steps:
1. SIRT1 target site design and sgRNA sequence synthesis
The ORF sequences of all transcripts of the pig SIRT1 Gene (Gene ID: NM-001145750.2) were obtained from the NCBI database, and the first exon where the ORF region was located was found for target site design.
The sgRNA guide sequence was designed and screened using an online tool (http:// tools. Genome engineering. Org): 5' CTCCGCGGTTTCTTGCGGAG-3', wherein the sgRNA action site is positioned in the first exon of the pig SIRT1 gene, and CACCG is added at the 5' end of the sgRNA action site to form forward Oligo DNA.
Simultaneously synthesizing reverse complementary sequences of sgRNA sequences:
5 'CTCCGCAAGAAACCGCGGAG-3', and adding AAAC at the 5 'end and C at the 3' end to form reverse Oligo DNA. The base sequence of SIRT1-sgRNA is shown as follows:
5’-CACCG CTCCGCGGTTTCTTGCGGAG-3’(SEQ ID NO.1)
3’-C GAGGCGCCAAAGAACGCCTCCAAA-5’(SEQ ID NO.2)
2. construction of PX459 (SIRT 1-sgRNA) plasmid
Annealing the above plus strand Oligo DNA (i.e., SEQ ID NO. 1) and minus strand Oligo DNA (i.e., SEQ ID NO. 2) to form dsDNA:
TABLE 1 reaction System
The reaction system of the table 1 is mixed uniformly by finger bomb, and is placed in a PCR reactor after instantaneous centrifugation to react according to the following reaction program: 30min at 37 ℃; 5min at 95 ℃, gradient cooling to 25 ℃,5 ℃/min.
mu.L of annealed dsDNA was ligated with Bbs I digested linearized pSpCas9-2A-Puro (PX 459) V2.0 vector, the linker system is shown in Table 2 below:
TABLE 2 connection System
The reaction system was left at room temperature for 10min to react.
3. Transformation and screening identification of PX459 (SIRT 1-sgRNA) positive clone
Adding 10 μ L of the ligation product into Stbl3 competent cells, and standing on ice for 30min; water bath at 42 ℃ for 90sec; placing on ice for 3-5 min, adding 900 mu L LB liquid culture medium without Amp, and performing shaking culture at 200rpm for 1h; 200 mu L of bacterial liquid is uniformly coated on Amp + On an LB solid culture plate of (1); culturing at 37 deg.C for 1hAfter that, the cells were cultured in an inverted state overnight.
Picking several single colonies with sterilized toothpick, inoculating to 1ml amp + Culturing the strain in the LB liquid culture medium at 37 ℃ and 200rpm for 2 to 3 hours; positive clones were identified by PCR, and the reaction system is shown in Table 3 below:
TABLE 3 reaction System
The PCR reaction conditions were: pre-denaturation at 94 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and amplification for 29 cycles; finally, extension is carried out for 5min at 72 ℃. After the PCR is finished, 3 microliter of the amplified PCR product is taken for agarose gel analysis, and positive clone bacteria are selected for sequencing.
The sequencing result is shown in fig. 3, the sequence of the insert fragment is matched with the sequence of the sgRNA locus, and the obtained plasmid is proved to be a positive plasmid and can be used for a subsequent knockout experiment.
After the positive plasmids are subjected to amplification culture, plasmids are extracted by using an endotoxin-free plasmid extraction kit, and cell transfection is carried out.
4. Cell transfection
The IPEC-J2 cells in good state are treated according to the method 10 6 The amount of individual cells/well was added to a 6-well plate and transfection was started with a cell density of around 80%. The medium was changed to antibiotic-free complete medium (2 mL/well) before transfection.
Preparation of transfection complexes:
a: 2. Mu.g// well plasmid PX459 (SIRT 1-sgRNA) for transfection was diluted with 150. Mu.L of Opti-MEM;
b: 8 μ L Lipofectamine 3000/well diluted with 150 μ L of Opti-MEM;
a and B are mixed according to a volume ratio of 1:1, and incubating at room temperature for 5min; adding 300. Mu.L of transfection complex per well, 37 ℃,5% 2 Culturing in an incubator.
5. IPEC-J2 single cell clone for screening and identifying SIRT1 positive mutation
Turn over then6h, the cell culture medium containing the transfection complex was removed and the cell culture medium was replaced with fresh one. After 24 hours of incubation, puromycin was added at a concentration of 2. Mu.g/mL for screening. Changing the complete medium containing 2. Mu.g/mL of purotoxin daily, digesting the selected cells 7 days later, inoculating into a number of 96-well cell culture plates by limiting dilution, 37 ℃,5% 2 And (5) performing static culture in an incubator for 7-14 days, observing the growth condition of the single cell clone during the static culture, and marking.
After 14 days, transferring the grown single cell clone to a 24-pore plate for continuous amplification culture, inoculating the cells into two 12-pore cell culture plates when the cells reach about 80%, wherein one plate is used for continuous amplification culture and cryopreservation, the other plate is used for extracting cell DNA, screening potential single cell clone mutant strains by using HRMA, and sending PCR products to a worker for sequencing analysis.
Designing specific HRMA primers near the knockout site according to the SIRT1 gene sequence:
f primer: 5 'AGGCGGCGCTCGCCCTTCAG-3' (SEQ ID NO. 3)
R primer: 5 'TCCAAGGCCGCCCCACCAGGCTC-3' (SEQ ID NO. 4)
The HRMA reaction system is shown in table 4 below:
TABLE 4 HRMA reaction System
HRMA amplification conditions were: 2min at 98 ℃; 40 cycles of 98 ℃ for 15sec,60 ℃ for 20sec (signal collected); 10sec at 95 ℃; after the amplification was completed, melting curve analysis was performed, and signals were collected from 55 ℃ to 95 ℃ at 0.2 ℃ per 10 sec. After the reaction is finished, analyzing the result, and purifying the PCR product of the potential mutation clone cell strain and then sending the PCR product to a manufacturer for sequencing analysis.
The potential positive clone sequencing peak diagram is shown in FIG. 5, and compared with the wild type (see FIG. 4), the mutant cell strain SIRT1 gene lacks 5bp base (see FIG. 6), causes frame shift mutation in the first exon region, and introduces a stop codon in advance.
And (3) further expanding and culturing the screened and identified mutant cell line, freezing and storing a part of the mutant cell line, and performing subsequent related experimental research on the rest mutant cell line.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> animal science institute of academy of agricultural sciences, guangdong province
<120> construction method of IPEC-J2 cell line with SIRT1 gene knockout
<130> 2019
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213> Artificial sequence
<400> 1
caccgctccg cggtttcttg cggag 25
<210> 2
<211> 25
<212> DNA
<213> Artificial sequence
<400> 2
cgaggcgcca aagaacgcct ccaaa 25
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<400> 3
aggcggcgct cgcccttcag 20
<210> 4
<211> 23
<212> DNA
<213> Artificial sequence
<400> 4
tccaaggccg ccccaccagg ctc 23
Claims (5)
1. A construction method of an IPEC-J2 cell line with SIRT1 gene knockout is characterized by comprising the following steps:
(1) Designing and synthesizing a gRNA probe aiming at a first exon of a piglet SIRT1 gene, wherein the base sequence of the gRNA is shown as SEQ ID NO.1, and the reverse complementary sequence of the gRNA is shown as SEQ ID NO. 2;
(2) The DNA chains shown in SEQ ID NO.1 and 2 are annealed to form a double-stranded gRNA probe with reverse complementarity, and the double-stranded gRNA probe is cloned into a pSpCas9-2A-Puro (PX 459) V2.0 vector to construct a plasmid for simultaneously expressing the sgRNA shown in SEQ ID NO.1 and the Cas9 protein;
(3) Transfecting the plasmid constructed in the step (2) to a piglet IPEC-J2 cell;
(4) Screening a monoclonal mutation cell line from the IPEC-J2 cell transfected in the step (3) to obtain an IPEC-J2 cell line knocked out by the SIRT1 gene, wherein compared with a wild cell line, 5 basic groups in a first exon of the SIRT1 gene are lacked, so that the SIRT1 protein generates frame shift mutation in the translation process, and the translation is terminated in advance;
in the step (3), lipo3000 is used for transfection;
in the step (4), the method for screening the monoclonal mutant cell line comprises a purine toxin combined with a limiting dilution method;
the method of screening monoclonal mutant cell lines also includes high resolution melting curve analysis.
2. The method of constructing according to claim 1, wherein the concentration of the purine toxin is 2 μ g/mL.
3. The method of constructing according to claim 1, wherein the purine toxin is used by: the cells were cultured with the cell culture medium containing the purotoxin and replaced with a new cell culture medium containing the purotoxin every day for a total of 7 days.
4. The construction method of claim 1, wherein the high resolution melting curve analysis comprises:
1) The amplification conditions for the high resolution melting curve analysis were: 2min at 98 ℃; 40 cycles at 98 ℃ for 15sec,60 ℃ for 20 sec; 10sec at 95 ℃;
2) After the amplification was completed, melting curve analysis was performed, and signals were collected from 55 ℃ to 95 ℃ at 0.2 ℃ per 10 sec.
5. A SIRT1 gene knock-out IPEC-J2 cell line constructed by the method of any one of claims 1 to 4.
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| Fish oil alleviates liver injury induced by intestinal ischemia/reperfusion via AMPK/SIRT-1/autophagy pathway;Hui-rong Jing等;《World Journal of Gastroenterology》;20180221(第07期);全文 * |
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