CN117904206A - Construction method and application of HSPB6 gene knockout cell line - Google Patents
Construction method and application of HSPB6 gene knockout cell line Download PDFInfo
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Abstract
The invention provides a construction method and application of an HSPB6 gene knockout cell line, wherein the method utilizes CRISPR-Cas9 technology to construct the HSPB6 gene knockout cell line, and the method uses SEQ ID NO in the sequence of the HSPB6 gene: 1 to 3 are target sequences. The method adopts three different sgRNAs with target sequences of 3 rd exons of human HSPB6 genes, and the sgRNAs can be used for specifically recognizing the HSPB6 genes, knocking out the HSPB6 genes and constructing cell line products with the knocked out HSPB6 genes, thereby being beneficial to researching the influence of the expression regulation of the HSPB6 on the cell functions; the constructed HSPB6 gene knockout cell line can be used for researching the molecular mechanism of the HSPB6 in GSD occurrence by influencing chondrocyte autophagy.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a construction method and application of a HSPB6 gene knockout cell line.
Background
Hereditary bone diseases (GENETIC SKELETAL identifiers, GSD) are a type of cartilage dysplasia diseases with genetic heterogeneity, mainly including achondroplasia (achondroplasia, ACH), hypochondrogenesis (hypochondroplasia, HCH) and lethal bone hypoplasia (thanatophoric dysplasia, TD), resulting in serious physical disability, even premature death, and serious harm in the infant. Therefore, it is important to understand the pathogenesis of GSDs and to explore effective molecular targets.
Heat shock protein 6 (Heat shock protein B, HSPB 6), also known as HSP20, is a 17kda protein belonging to the small heat shock protein family. HSPB6 is constitutively expressed in various tissues, while it is expressed at high levels in skeletal, cardiac and smooth muscles. Currently, the molecular mechanisms of HSPB6 in GSD development, in particular its role in mediating chondrocyte autophagy, remain to be elucidated.
The CRISPR/Cas9 system is a natural immune system of prokaryotes. The genetic means of genome targeted knockout and knockout is a third generation gene editing technology following ZFN, TALENs and other technologies. The target genome sequence is identified through the sgRNA (guide RNA) which is designed artificially, and Cas9 protease is guided to effectively cut DNA double chains to form double chain breaks, and gene knockout or knock-in and the like can be caused by repair after DNA damage, so that the aim of modifying genome DNA is finally achieved. The CRISPR/Cas9 system has the characteristics of high efficiency, simple operation, low cost and the like, and becomes the gene editing system with the most application prospect at present. However, no related studies have been reported whether targeted knockout of HSPB6 gene using CRISPR/Cas9 technology would affect chondrocyte autophagy.
Disclosure of Invention
The present invention aims to solve at least to some extent one of the technical problems in the above-described technology. Therefore, the invention provides a construction method and application of an HSPB6 gene knockout cell line.
For this reason, in a first aspect of the present invention, the present invention provides a method for constructing a HSPB6 gene knockout cell line using CRISPR-Cas9 technology, using the sequence of the HSPB6 gene as set forth in SEQ ID NO:1 to 3 are target sequences.
According to the embodiment of the invention, three different sgRNAs with target sequences of 3 rd exons of human HSPB6 genes are adopted, and can be used for specifically recognizing the HSPB6 genes, knocking out the HSPB6 genes and constructing cell line products with the knocked-out HSPB6 genes, so that the influence of the expression regulation of the HSPB6 on the cell functions can be studied; the constructed HSPB6 gene knockout cell line can be used for researching the molecular mechanism of the HSPB6 in GSD occurrence by influencing chondrocyte autophagy.
According to an embodiment of the invention, the method comprises the steps of:
(1) Constructing HSPB6 gene knockout plasmids HSPB6-sgRNA1, HSPB6-sgRNA2 and HSPB6-sgRNA3;
(2) Packaging the plasmids obtained in the previous step by using slow viruses to obtain HSPB6 gene knockout slow viruses;
(3) And infecting ATDC5 target cells by using the HSPB6 gene knockout lentivirus to obtain an HSPB6 gene knockout cell line ATDC5-HSPB6-sgRNA1, ATDC5-HSPB6-sgRNA2 and ATDC5-HSPB6-sgRNA3.
Further, the plasmid is selected from LENTILCRISPR V2; the lentivirus is selected from HEK293T, and the plasmid used for lentivirus packaging is selected from psPAX2 and pMD2.G.
In a second aspect of the present invention, the present invention provides a plasmid, which is the above-mentioned HSPB6-sgRNA1, HSPB6-sgRNA2, HSPB6-sgRNA3.
In a third aspect of the invention, the invention proposes a set of plasmids consisting of psPAX plasmid, pMD2.G plasmid and the above-mentioned HSPB6-sgRNA1 plasmid; or comprises psPAX plasmid, pMD2.G plasmid and HSPB6-sgRNA2 plasmid; or consists of psPAX plasmid, pMD2.G plasmid and the above HSPB6-sgRNA3 plasmid.
In a fourth aspect of the invention, the invention proposes the use of the above-described set of plasmids for knocking out the HSPB6 gene in ATDC5 target cells based on CRISPR-Cas9 technology.
In a fifth aspect of the invention, the invention provides a cell line in which the HSPB6 gene is knocked out, the cell line being obtained by the method of constructing a cell line as described above. Thus, the constructed HSPB6 gene knockout cell line can be used for researching the molecular mechanism of the HSPB6 in GSD generation by influencing chondrocyte autophagy.
In a sixth aspect of the invention, the invention proposes the use of the above described cell lines for studying the molecular mechanism of HSPB6 in GSD development.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a schematic diagram of the design of an sgRNA according to an embodiment of the present invention;
Figure 2 is a diagram of successful verification of construction of a HSPB6 knockout plasmid according to an embodiment of the present invention;
figure 3 is a graph showing the change in HSPB6 protein expression of an embodiment of the invention;
FIG. 4 is a graph showing the staining results of the cell line constructed in the example of the present invention by microscopic observation.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
The following disclosure provides many different embodiments, or examples, for implementing different embodiments of the invention. In order to simplify the present disclosure, specific embodiments or examples are described below. Of course, they are merely examples and are not intended to limit the invention. In addition, one of ordinary skill in the art will recognize the applicability of other processes and/or the use of other materials, as examples of the various specific processes and materials provided by the present invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques in the fields of chemistry, molecular biology, etc., which are within the ability of a person skilled in the art. In addition, unless otherwise indicated, herein, nucleic acids are written in a5 'to 3' direction from left to right, and amino acid sequences are written in an amino-to carboxy-terminal direction from left to right.
In the following examples of the present invention,
LENTICRISPR V2 vector, available from Addgene Corp.
Specifically described are: the reagents and materials used in the present invention are commercially available unless otherwise specified.
The invention is described below by way of illustrative specific examples, which are not intended to limit the scope of the invention in any way.
Example 1 construction of HSPB6 Gene knockout plasmid Using CRISPR/Cas9 technology
Selection and design of sgrnas targeting HSPB6 genes
The gene sequence of human HSPB6 was found in Genebank, and 3 different sgRNA"GGAGGTGCACGCGCGCCA CG(SEQ ID NO：1)"、"GCGGCAGGCGGTAGCGACGG(SEQ ID NO：2)"、"G CAGCCGGATCCACGCCAGG(SEQ ID NO：3)", evaluation principles were designed by selecting the higher scoring sequence on the HSPB6 sequence by the Crispr design tool and the design principle of sgRNA, including: whether there is a P AM (NGG) downstream of the sequence; the off-target efficiency is low, and the off-target is not easy to occur. Adding CACC to the 5 'end of sgRNAF to obtain forward oligonucleotide, adding AAAC to the 5' end of sgRNAR to obtain reverse oligonucleotide, synthesizing forward oligonucleotide and reverse oligonucleotide respectively, denaturing and annealing the forward oligonucleotide and the reverse oligonucleotide to form double-stranded DNA fragment. The design schematic is shown in figure 1.
TABLE 1 primer names and sequences
The annealed oligo double-stranded fragment and the lentiviral vector LENTILCRISPR V2 after cleavage with Esp I were ligated in the following manner: 4. Mu.L of annealed oligo double-stranded fragment, 20ng lentilCRISPR v2,1. Mu. L T4DNA ligase, 2. Mu. L T4DNA ligase buffer, and filled to 20. Mu.L with enzyme-free water. Ligation was performed at 16℃for 12h, the ligation products were transformed into E.coli DH5a competent cells, plated on ampicillin-resistant LB plates, positive colonies were screened, positive colony plasmids were extracted for analysis and sequencing, and sequencing primer names were as shown in Table 2 below to confirm successful construction of CRISPR-Cas9 targeted HSPB6 gene knockout plasmids (designated as HSPB6-sgRNA1, HSPB6-sgRNA2, HSPB6-sgRNA3, respectively, as shown in FIG. 2).
TABLE 2 sequencing primer names and sequences
EXAMPLE 2 construction of HSPB6 Gene knockout cells
Culture of engineered cells HEK293T and chondrocyte ATDC 5: and (3) rapidly taking out the engineering cells HEK293T and the chondrocyte ATDC5 stored in the liquid nitrogen tank, immediately putting the engineering cells HEK293T and the chondrocyte ATDC5 into a water bath kettle at 37 ℃ to completely melt, transferring the engineering cells HEK293T and the chondrocyte ATDC5 into a 4mL centrifuge tube, adding 1mL of RMPI-1640 complete culture medium, uniformly mixing, centrifuging at 600 Xg for 5min, discarding the supernatant, re-suspending the cells by using 2mL of RMPI-1640 complete culture medium, adding the re-suspended cells into a 10cm cell culture dish, supplementing the required culture medium, and continuously culturing in a CO 2 incubator at 37 ℃ and with 5% CO 2 and saturated humidity.
Viral infection of engineered cell HEK293T viral packaging and chondrocyte ATDC 5:
① HEK293T cells were seeded in 10cm dishes at about 1.5X10 6 cells/dish and transfected at 40-50% cell fusion.
② After 24h of cell adherence, virus packaging plasmid psPAX, pMD2.G and target gene plasmid HSPB6-sgRNA are mixed with X-TREMEGENEHP DNA transfection reagent according to the ratio of=4:3:1 respectively, and then lentiviral packaging and HEK293T transfection are carried out.
③ After 24h transfection, fresh RPMI-1640 (10% FBS+/P & S-) medium is replaced for continuous culture, after 24h, the supernatant of HEK293T cells is filtered to a 15mL centrifuge tube by a 0.45 μm filter to obtain virus particles, and the virus particles are subpackaged and frozen at-80 ℃ for standby.
④ A6 cm dish was inoculated with 3X 10 5 ATDC5 target cells per dish for infection with the plasmid of interest. The virus particles in ③ (containing polybrene at a final concentration of 1. Mu.g/mL) were infected with ATDC5 cells for 12h and fresh RPMI-1640 (10% FBS+/P & S-) medium was changed.
⑤ After 24 hours, the cells were digested with 0.25% trypsin and inoculated into a new 6cm dish, while uninfected normal cells were used as a control, and 1.0ug/mL puromycin added to kill uninfected cells, and the culture was changed every 24 hours. Finally screening to obtain a monoclonal cell line named ATDC5-HSPB6-sgRNA1, ATDC5-HSPB6-sgRNA2 and ATDC5-HSPB6-sgRNA3.
Example 3 Western blot experiments to identify HSPB6 knockouts
Total protein extraction: the culture broth was aspirated and 1mL of pre-chilled 1 XPBS was added to each well and washed 3 times, cells were collected, RIPA, PMSF and phosphatase inhibitors were added to fill the disruption ice bath for 30min, and vortexed at high speed for 10s every 10min. The supernatant was then collected into a new 1.5mL centrifuge tube pre-chilled by centrifugation at 15,000Xg for 20min at 4 ℃. The BCA method measures the protein concentration, and the protein is denatured for 10min at 100 ℃ in a metal bath.
Gel preparation and Western blot: a Bio-Rad gel making system was used to prepare 12% of the separation gel and 5% of the concentrate gel. The denatured protein samples should be mixed and centrifuged prior to loading. The first well was a protein Marker well and the 10-well comb protein loading was 20 μg per well. Electrophoresis at 200V for 45min;200mA on-ice film transfer for 120min; the blocking solution is 5% skimmed milk prepared from 1 XTBST, and the mixture is placed in a horizontal shaking table to shake the blocking film for 1h at room temperature. After the sealing, the membrane is washed 3 times by 1 XTBE on a horizontal shaking table for 5min each time; incubating the primary antibody at 4 ℃ overnight; after incubation of primary antibody was completed, membranes were washed 3 times with 1 XTBE for 5min each. The secondary antibody is incubated by gentle shaking on a room-temperature flat shaking table for 1h, and the membrane is washed by 1 XTBST for 3 times, each time for 5min; preparing ECL working solution under the light-shielding condition, and mixing A, B solutions in equal volume; and uniformly covering the film with the chemiluminescent liquid, reacting for 2min, and placing the film in a chemiluminescent instrument for detecting an exposure strip to detect the change of the expression of the HSPB6 protein. As shown in FIG. 3, constructed ATDC5-HSPB6-sgRNA1, ATDC5-HSPB6-sgRNA2 and ATDC5-HSPB6-sgRNA3 cell lines were identified at the protein level. There was a different degree of reduction in HSPB6 protein levels in the different knockdown cells compared to control cells, with the knockdown efficiency being most pronounced in ATDC5-HSPB6-sgRNA2 cells.
Example 4 immunofluorescence experiments to observe autophagy
① Cell plating culture: cell climbing sheets with corresponding specifications are paved, 1X 10 5 cells/hole are inoculated in a 12-hole plate, lysosome-Tracker probes are added after 24 hours, and cell staining incubation treatment is carried out according to the product specification.
② Fixing: the culture broth was discarded, 1 XPBS was added to each well and washed 3 times, the retentate was removed by pipetting, and the wells were fixed with 4% paraformaldehyde for 1h at room temperature.
③ Permeabilization: the fixative was discarded, washed 3 times with 1 XPBS, and 1mL of 1 XPBS plus 0.5% Triton X-100 ice was allowed to stand for 5min.
④ Closing: the permeabilization solution was discarded, PBSB was washed 3 times, and 1mL of PBSB was taken and blocked by shaking slowly for 30min.
⑤ Incubation resistance: the blocking solution was discarded and washed 3 times with PBST. 400. Mu.L of the diluted primary antibody was added to the well to cover the slide, and the slide was slowly shaken overnight at 4 ℃.
⑥ Secondary antibody incubation: wash 3 times with PBST. 400 mu L of the diluted secondary antibody is added into a hole to cover a slide, and the slide is incubated for 1h at room temperature in a light-proof mode.
⑦ Nuclear dyeing: 400. Mu.L of diluted DAPI was added to the wells and the nuclei were slowly transfected in the dark for 5min.
⑧ Sealing piece: the nuclear dye was removed by blotting and washed 3 times with PBST in the dark. The slide was gently picked up with forceps, the edge gently touched the filter paper to drain the residual liquid, and about 5. Mu.L of a 95% glycerol sealed sheet was taken and stored in a dark place.
⑨ And (5) microscopic examination: the staining results are observed by a Zessi 880 laser confocal microscope, images are shot, representative cell mapping is intercepted by using Photoshop, as shown in fig. 4, a FGFR3 (G382D) cell model with low cartilage development (HCH) is adopted, the influence of gene targeting intervention HSPB6 on autophagy function is explored, and immunofluorescence experiment results show that compared with WT cells, the LC3II protein expression amount and the lysosome quantity in the FGFR3 (G382D) cells are reduced, and the yellow fluorescence focal distribution of the co-localization of LC3II and lysosomes is reduced. Furthermore, targeting HSPB6 intervention increased expression of LC3II protein and number of lysosomes, and yellow fluorescence focal profile co-localized to LC3II and lysosomes increased compared to corresponding control cells, suggesting that targeting intervention HSPB6 is effective in rescuing autophagy inhibition in FGFR3 (G382D) cells.
In summary, according to the embodiment of the invention, three different sgrnas with target sequences of the 3 rd exon of the human HSPB6 gene are adopted, and can be used for specifically recognizing the HSPB6 gene, knocking out the HSPB6 gene and constructing cell line products for knocking out the HSPB6 gene, compared with the shRNA interference method reported in the traditional literature, the CRISPRCas9 method obtains the sgRNA molecule which targets the HSPB6 gene efficiently, specifically and accurately, regulates and controls the expression of the target gene HSPB6 from the genome level, remarkably improves the inhibition efficiency of the HSPB6 gene, and is beneficial to researching the influence of the regulation of the expression of the HSPB6 on the cell function; the constructed HSPB6 gene knockout cell line can be used for researching the molecular mechanism of the HSPB6 in GSD occurrence by influencing chondrocyte autophagy.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be understood as necessarily being directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (8)
1. A method for constructing an HSPB6 gene knockout cell line by using a CRISPR-Cas9 technology, which is characterized in that the sequence of the HSPB6 gene is represented by SEQ ID NO:1 to 3 are target sequences.
2. The method of claim 1, comprising the steps of:
(1) Constructing HSPB6 gene knockout plasmids HSPB6-sgRNA1, HSPB6-sgRNA2 and HSPB6-sgRNA3;
(2) Packaging the plasmids obtained in the previous step by using slow viruses to obtain HSPB6 gene knockout slow viruses;
(3) And infecting ATDC5 target cells by using the HSPB6 gene knockout lentivirus to obtain an HSPB6 gene knockout cell line ATDC5-HSPB6-sgRNA1, ATDC5-HSPB6-sgRNA2 and ATDC5-HSPB6-sgRNA3.
3. The method of claim 2, wherein the plasmid is selected from LENTILCRISPR V; the lentivirus is selected from HEK293T, and the plasmid used for lentivirus packaging is selected from psPAX2 and pMD2.G.
4. A plasmid, characterized in that the plasmid is HSPB6-sgRNA1, HSPB6-sgRNA2, HSPB6-sgRNA3 as described in claim 2.
5. A set of plasmids consisting of psPAX plasmid, pmd2.G plasmid and HSPB6-sgRNA1 plasmid according to claim 2; or consist of psPAX plasmid, pMD2.G plasmid and HSPB6-sgRNA2 plasmid as described in claim 2; or consist of psPAX plasmid, pMD2.G plasmid and HSPB6-sgRNA3 plasmid as described in claim 2.
6. Use of the set of plasmids according to claim 5, wherein the use is based on CRISPR-Cas9 technology to knock out the HSPB6 gene in ATDC5 target cells.
7. A cell line, characterized in that the HSPB6 gene is knocked out in said cell line, said cell line being obtained by the method of any one of claims 1 to 3.
8. Use of a cell line according to claim 7 for studying the molecular mechanism of HSPB6 in GSD development.
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