CN117126855A - Method for constructing Igf2bp1 gene knockout mouse embryonic stem cell line - Google Patents
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for constructing an Igf2bp1 gene knockout mouse embryonic stem cell line, which comprises the steps of designing an sgRNA sequence through a first exon of Igf2bp1DNA, synthesizing and annealing an oligonucleotide chain 1 and an oligonucleotide chain 2 to obtain double-stranded DNA. Cloning the DNA fragment into a LeniCRISPR v2 vector to construct a LeniCRISPR-bp 1 recombinant plasmid. Transfecting the recombinant plasmid into 293T cells, and collecting lentiviruses; and (3) taking virus supernatant to infect a mouse embryo stem cell, adding puromycin to screen positive cells, and separating monoclonal cells for culturing to obtain the mouse ESC cell strain with Igf2bp1 gene stably knocked out. The constructed Igf2bp1 gene-deleted mouse embryo stem cell strain can be used as a cell model for researching the aspects of stem cell proliferation and differentiation, transcription and translation regulation and the like of Igf2bp1, and explores the regulation and control effect of the Igf2bp1 gene in development related diseases.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for constructing an Igf2bp1 gene knockout mouse embryonic stem cell line.
Background
Embryonic stem cells (Embryonic Stem Cell, ESCs) are derived from the inner cell mass of early blasts, are a class of pluripotent stem cells with dual capacities of self-renewal and differentiation. ESCs can be directionally differentiated into various cell types, such as myocardial cells, nerve cells, muscle cells and the like, and have wide application prospects in tissue repair and regeneration medicine. Through the study of mouse ESCs, we can get a better understanding of the molecular mechanisms of cell fate decisions and differentiation regulation during embryonic development. In addition, embryonic stem cell lines can also be used to create disease models and drug screening. By using the established disease model, the occurrence mechanism of the disease can be better understood, and the development of new drugs and the progress of drug effect evaluation can be accelerated. In general, related research on mouse ESCs has important scientific significance and application value for deep study on developmental biology, disease occurrence mechanism, drug screening and the like.
The Igf2bps (Insulin-like growth factor 2mRNA-binding proteins, igf2 bps) family is a class of RNA binding proteins, including Igf2bp1, igf2bp2, and Igf2bp3.Igf2bps are involved in posttranscriptional regulation by binding to the 3' untranslated region of mRNA (3'untranslated region,3'UTR). Research also found that Igf2bps recognizes RNAm 6 A(N 6 -methyladenosine, m 6 A) To regulate mRNA stability and translation efficiency, and plays a key role in physiological or pathological processes such as stem cell self-renewal, cell differentiation, tumorigenesis and development, etc. Wherein Igf2bp1 is mainly expressed in embryo development stage and adult tissues and participates in embryo growth, organ formation and differentiation; the Igf2bp1 knockout mice are obviously damaged in development, the overall size of each organ and organism is obviously smaller, and the survival rate is obviously reduced, which indicates that the Igf2bp1 knockout mice play an important role in the embryo development process. However, at present, for Igf2bp1, self-renewal and targeting of stem cells is dividedThere are relatively few studies in chemistry. Therefore, the construction of the stem cell line with the Igf2bp1 knockdown is of great importance, can be used for researching the regulation and control effects of the Igf2bp1 in the aspects of cell proliferation, differentiation, tumor progression and the like, and has great significance for deeply understanding the molecular mechanism of cell fate decision.
Currently, researchers mainly use methods such as small interfering RNAs (sirnas) and antisense RNAs (shrnas) to mine new functions and regulatory mechanisms of specific genes, however, these methods often have problems of inefficiency and instability. In particular, in stem cell research, exogenous gene transfection efficiency is very low due to its special cell membrane characteristics, DNA modification enzyme activity and immune response, making research of gene function more challenging.
Disclosure of Invention
In order to solve the technical problems, the CRISPR/Cas9 gene editing system is utilized, and the CRISPR/Cas9 gene editing system utilizes Cas9 protein and specific guide RNA (sgRNA) to be co-located at a specific position of a target gene so as to induce double-strand break. The cell is repaired by using a self repair mechanism, the repair process often causes base insertion or deletion, and frame shift mutation is caused, so that the target gene knockout effect is realized, a mouse embryonic stem cell strain with high specificity and stability and Igf2bp1 gene deletion is obtained, and an effective cell model is provided for researching the action mechanism of Igf2bp1 in stem cell proliferation and differentiation and related disease progression.
The invention not only provides a CRISPR/Cas9 system-based sgRNA sequence for targeted knockout of Igf2bp1 genes, but also provides a detailed scheme for constructing Igf2bp1 gene-deleted mouse embryonic stem cell strains.
The aim of the invention is achieved by the following technical scheme:
in a first aspect, the invention provides a sgRNA sequence for knocking out a mouse Igf2bp1 gene, wherein the sgRNA sequence is shown as SEQ ID NO. 1.
In a second aspect, the invention provides a method for knocking out Igf2bp1 genes of mouse embryonic stem cells, which is to modify the Igf2bp1 genes in the mouse embryonic stem cells by using a CRISPR/Cas9 lentiviral system, and specifically comprises the following steps:
step S1, artificially synthesizing oligonucleotide sequences sg-Oligo1 and sg-Oligo2 containing an sgRNA sequence to form double-stranded DNA, wherein the sg-Oligo1 sequence is shown as SEQ ID NO. 2, and the sg-Oligo2 sequence is shown as SEQ ID NO. 3; inserting the synthesized double-stranded DNA into a polyclonal site of an sgRNA skeleton expression plasmid vector and transforming, selecting a monoclonal strain, extracting the sgRNA recombinant plasmid, and obtaining the sgRNA recombinant plasmid with correct sequencing through sequencing identification, wherein the sgRNA skeleton expression plasmid vector also expresses Cas9 nuclease;
s2, transfecting the sgRNA recombinant plasmid into HEK 293T cells, and then harvesting virus supernatant to infect the mouse embryonic stem cells to obtain the mouse embryonic stem cells with Igf2bp1 gene knocked out.
Preferably, the sgRNA backbone expression plasmid vector is LentiCRISPR v2; the correct sgRNA recombinant plasmid is called LentiCRISPR-bp1 for short;
as a preferred embodiment of the present invention, the specific embodiment of the method for knocking out Igf2bp1 gene of mouse embryonic stem cells provided in the second aspect comprises the steps of:
designing an sgRNA sequence aiming at an exon of the Igf2bp1 gene, and synthesizing sg-Oligo1 and sg-Oligo2; the LeniCRISPR v2 plasmid is subjected to BsmBI single enzyme digestion, and after agarose gel electrophoresis separation, DNA fragments are purified by using a gel recovery kit; annealing the synthesized sg-Oligo1 and sg-Oligo2 to form double-stranded DNA, and phosphorylating by T4 kinase; then connecting the carrier with the annealed double-stranded DNA fragment under the action of T4 DNA ligase, converting the connection product into competent cells, selecting a monoclonal strain, extracting the sgRNA recombinant plasmid, and obtaining the sgRNA recombinant plasmid LentiCRISPR-bp1 with correct sequencing through sequencing identification;
the recombinant plasmid LentiCRISPR-bp1 is transfected into HEK 293T cells, the virus supernatant is collected for 48 hours, a proper amount of virus supernatant is taken to infect the mouse embryonic stem cells, and after 24 hours, puromycin is added for screening for one week. Preparing the screened positive cells into cell suspension, screening monoclonal cells by using a limiting dilution method, collecting the monoclonal cells, extracting genome DNA, amplifying DNA products by using PCR, and sequencing to identify genotype changes;
the steps of collecting the 48h virus supernatant by transfecting HEK 293T cells with the recombinant plasmid specifically comprise: cell confluence reached about 70%, lentiCRISPR-bp1, packaging plasmid VSVG and PSPAX were co-transfected into cells, the complete medium was changed for 24h, and after 48h the virus supernatant was collected and filtered with 0.45 μm filter.
The mouse embryo stem cell is a J1 cell line, is purchased from a cell bank of China academy of sciences and is cultured by adopting feeder-free cells. The steps of taking a proper amount of virus supernatant to infect the embryo stem cells of the mice, adding puromycin after 24 hours and screening for one week specifically comprise: mouse embryonic stem cells ESC were grown to 30% -40% density, added with viral supernatant for 24h, 8 μg/ml of polybrene was added during infection, and after 48h, 1.0 μg/ml of puromycin was added for one week to screen, and protein samples were collected for preliminary verification by Western blotting.
The specific steps of screening monoclonal cells by utilizing a limiting dilution method, collecting monoclonal cells and extracting genome DNA, amplifying DNA products by adopting PCR and carrying out sequencing to identify genotype changes include: the mixed knocked-out cell suspension is inoculated into a 96-well plate for 10-15 days by a limiting dilution method, and the monoclonal is picked and inoculated into a 24-well plate for continuous culture. Cells are collected in the passage process and used for genome extraction, upstream and downstream primers are designed at 200bp at the front end and the rear end according to sgRNA loci, PCR products are further cloned into pUCm-T vectors, and genotype-changed clones are obtained after sequencing and comparison.
In a third aspect, the invention provides a method for constructing an Igf2bp1 gene knockout mouse embryonic stem cell line, which is characterized in that the mouse embryonic stem cell of the Igf2bp1 gene knockout obtained in the second aspect is subjected to passage screening by adopting a limiting dilution method, so as to obtain a stable Igf2bp1 gene knockout mouse embryonic stem cell line.
In a fourth aspect, the invention provides a mouse embryonic stem cell strain with Igf2bp1 gene deletion, which is obtained by adopting the method for constructing the Igf2bp1 gene knockout mouse embryonic stem cell line in the third aspect.
In a fifth aspect, the present invention provides a kit for site-directed knockout of an Igf2bp1 gene in a mouse genome, comprising any one of the following (1) to (2):
(1) The coding strand template and the non-coding strand template comprising an sgRNA sequence of the second aspect form a double-stranded DNA;
(2) The sgRNA recombinant plasmid of the second aspect.
Compared with the prior art, the invention has the following advantages and effects: the invention provides a sgRNA sequence design for knocking out a mouse Igf2bp1 gene, which is a sgRNA design scheme aiming at a first exon of Igf2bp1, and can target the Igf2bp1 gene to realize effective knocking out of the Igf2bp1 gene and functional loss of encoding proteins thereof; the method for knocking out the Igf2bp1 gene of the mouse embryonic stem cell provided by the invention realizes the silencing effect on the gene at the genome level, effectively improves the defect that the silencing of the mRNA or protein level by adopting siRNA is incomplete or the gene expression cannot be silenced in the prior art, and also provides a construction method for obtaining the mouse embryonic stem cell strain with continuous passage Igf2bp1 gene deletion and a mouse embryonic stem cell strain model with Igf2bp1 gene deletion. The Igf2bp1 gene has potential regulation and control effect in stem cell biology, and the mouse embryo stem cell strain model with the Igf2bp1 gene deletion constructed by the invention can be used for researching the functions of Igf2bp1 in stem cell proliferation and differentiation, transcription and translation regulation and control and the like. The embryonic stem cells can be directionally differentiated in vitro and used for constructing disease models. Therefore, the regulation and control effect of the Igf2bp1 gene in development related diseases can be explored by utilizing the mouse embryonic stem cell strain model with the Igf2bp1 gene deletion.
Drawings
FIG. 1 is a schematic diagram of the construction of a recombinant plasmid of LentiCRISPR-bp1;
FIG. 2 shows the genomic DNAPCR amplification product of monoclonal Igf2bp1-B2 (bp 1-B2). Lane M: DNAMaroker; lane WT: wild-type genomic DNAPCR products; lanes bp1-B2: genomic DNAPCR product of clone No. B2;
FIG. 3 shows Sanger sequencing of the monoclonal bp1-B2 genomic DNAPCR product.
FIG. 4 is a genomic alignment analysis of wild type and monoclonal bp 1-B2. WT represents wild-type ESC genomic DNA sequence; bp1-B2 represents knockout Igf2bp1 monoclonal B2 genome DNA sequence; the dashed line represents the deleted fragment.
FIG. 5 shows the protein level change of Igf2bp1 gene knockout by Westernblot verification. WT represents Igf2bp1 protein expression from wild-type ESCs; bp1-B2 represents the expression of Igf2bp1 protein of knockout clone B2.
FIG. 6 shows the mRNA expression level of qPCR assay for monoclonal bp 1-B2.
FIG. 7 shows the genomic DNAPCR amplification product of monoclonal Igf2bp1-C1 (bp 1-C1). Lane M: DNAMaroker; lane WT: wild-type genomic DNAPCR products; lanes bp1-C1: genomic DNAPCR product of clone No. C1.
FIG. 8 shows Sanger sequencing of the monoclonal bp1-C1 genomic DNAPCR product.
FIG. 9 is a genomic alignment analysis of wild type and monoclonal bp 1-C1. WT represents wild-type ESC genomic DNA sequence; bp1-C1 represents knockout Igf2bp1 monoclonal C1 genome DNA sequence; the dashed line represents the deleted fragment.
FIG. 10 shows the protein level change of Igf2bp1 gene knockout by Westernblot verification. WT represents Igf2bp1 protein expression from wild-type ESCs; bp1-C1 represents the expression of Igf2bp1 protein of knockout clone C1.
FIG. 11 shows qPCR detection of mRNA expression levels of monoclonal bp 1-C1.
Detailed Description
The following is a detailed description of specific embodiments of the invention. The examples are provided for clarification of the invention only and do not limit the scope of the invention. The examples are intended as references for further improvement by those of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The reagent and consumable materials used in the embodiment of the invention are all commercial products unless specified otherwise.
The technical scheme of the invention can be realized by the following embodiments:
1. sgRNA sequence design
According to NCBI database, query and download Igf2bp1 genomic DNA sequence: species: murine, gene name: igf2bp1, gene ID:140486 the sgRNA sequence, indicated by SEQ ID NO. 1, was designed on the basis of the first exon of Igf2bp1 and was GGATTGCCCCGACGAGCACT.
2. Construction of recombinant plasmid LentiCRISPR-bp1
(1) Designing an oligonucleotide chain according to the sgRNA sequence, namely a sequence shown in SEQ ID NO. 1, wherein the designed oligonucleotide chain sequence is shown in Table 1, specifically, the sgRNA oligonucleotide chains sg-Oligo1 and sg-Oligo2 are synthesized by adding CACCG at the 5' -end of a coding chain template, adding AAAC at the 5' -end of a non-coding chain template and C at the 3' -end so as to be complementary with a sticky end formed after BsmBI digestion;
TABLE 1 Igf2bp1 targeting site and sgRNA oligonucleotide sequence
| Oligo name | Oligo sequences |
| sg-Oligo1 | The sequence shown in SEQ ID NO. 2: 5'-CACCGGGATTGCCCCGACGAGCACT-3' |
| sg-Oligo2 | The sequence shown in SEQ ID NO. 3: 5'-AAACAGTGCTCGTCGGGGCAATCCC-3' |
(2) Preparation of LenigirSPR v2 vector cleavage System the LenigirSPR v2 vector cleavage System specifically included 1. Mu.g of LenigirSPR v2, 2.5. Mu.l of BsmBI (available from NEB), 5. Mu.l of NEBufferTM r3.1 (available from NEB) and a complement of 50. Mu.l of ddH 2 The conditions of the enzyme digestion reaction are as follows: incubating for 3 hours at the temperature of 55 ℃ to obtain a LeniCRISPR v2 digested plasmid product;
(3) Purifying the digested plasmid product by using a norfloxacin recovery kit DC301-01, wherein the specific steps are operated according to the instructions of the norfloxacin recovery kit DC 301-01;
(4) Preparing a sg-Oligo phosphorylation and annealing sgRNA reaction system, namely a primer annealing system, wherein the primer annealing system specifically comprises: 1 μl of sg-Oligo1 at a concentration of 100 μM, 1 μl of sg-Oligo2 at a concentration of 100 μM, 1 μl of 10×T DNA ligase Reaction buffer (from NEB), 1 μl of T4 PNK enzyme (from NEB) and the complement of 10 μl of ddH 2 0; the primer annealing conditions were: incubation at 37 ℃ for 30min, maintaining at 95 ℃ for 5min, reducing the temperature to 40 ℃ every minute for 6min, and annealing to form sg-Oligo double chains;
(5) Diluting the sg-Oligo double chain formed by annealing for 100 times, and incubating the LeniCRISPR v2 digested plasmid product obtained in the step (2) for 2 hours at room temperature according to a ligation reaction system to obtain a ligation product of the LeniCRISPR v2 digested plasmid product and the sgRNA ligation product; the specific connection reaction system comprises: 50ng of LentiCRISPR v2 digested plasmid product, 1. Mu.l of the above 100-fold diluted sg-Oligo duplex, 1. Mu.l of XT 4 DNA Ligase Buffer (from NEB), 1. Mu.l of T4 DNA Ligase (from NEB) and the complement of 10. Mu.l of ddH20;
(6) The connection product is transformed into competent cells DH5 alpha (purchased from the Optimaceae) and evenly spread on LB solid medium plates with ampicillin resistance, and the plates are inversely cultured in a culture box at 37 ℃ for 12-16 hours;
(7) Single colony is picked for expansion culture, a Reuzan plasmid extraction kit DC201-01 is used for plasmid extraction, the operation is carried out according to instructions, the single colony is sent to Nanjing qinghao biological company for sanger sequencing, and after sequencing comparison, the strain containing the LentiCRISPR-bp1 recombinant plasmid is placed at the temperature of minus 80 ℃ for preservation. The diagram is shown in figure 1, namely, the construction mode diagram of the LentiCRISPR-bp1 recombinant plasmid.
3. Lentivirus packaging and infection
(1) Lentivirus package
Inoculating HEK 293T to a 60mm cell culture dish one day before transfection, and growing until the confluency of the HEK 293T reaches 60% -70%; the following day, a 1.5ml EP tube was prepared, 500. Mu.l DMEM high sugar medium (from Kaiki's organism) was added, 0.625. Mu.g VSVG, 2.5. Mu.g PSPAX and 5. Mu.g LentiCRISPR-bp1 vector were added, and the mixture was gently flicked with fingers and incubated at room temperature. Another 1.5ml EP tube was prepared, 500. Mu.l DMEM medium was added, 35. Mu.l transfection reagent ExFect Transfection Reagent (ex Novazab) was added, and mixed gently by finger flick.
After 5min incubation at room temperature, the DMEM medium containing the plasmid to be transfected was added dropwise to the EP tube containing the transfection reagent and gently flicked to mix, and the mixture was incubated at room temperature for 15min. Taking out cells from the incubator, carefully dripping the DNA mixed solution into a cell culture medium, placing the cells into the incubator for continuous culture after uniform mixing, and replacing the cells into a basic culture medium for continuous culture after 4 hours of transfection, wherein the components of the culture medium are as follows: 445ml DMEM high-sugar medium (from Kyodo organism) +50ml FBS (from Shanghai Dattschil biosciences) +5ml PS (from Gibco). The virus product was collected 48h after transfection and filtered through a 0.45 μm pdf filter, and the virus supernatant was frozen at-80 ℃.
(2) Infection of target cells
Target cells, namely mouse embryonic stem cells ESC, are J1 cell lines purchased from a cell bank of China academy of sciences (catalog number: SCSP-219) and cultured by feeder-free cells;
taking out frozen lentivirus from the refrigerator at-80 ℃ and thawing on ice; the target cell stock broth was discarded, the virus supernatant was added, at the same time 10mg/ml of a polybrene solution (from MCE) was added to a final concentration of 8. Mu.g/ml, and the petri dish was placed in an incubator at 37℃with 5% CO 2 For 24 hours, discarding the lentivirus-containing medium and replacing the desired stem cell medium, medium composition: 410ml KnockOut TM DMEM (available from Gibco) +75ml KnockOut) TM Serum replacement (from Gibco) +5ml Glutamax TM Supplement (from Gibco) +5ml MEM Non-Essential Amino Acids Solution (from Life) +5ml PS (Gibco) +3.5ul 2-mercaptoethanol (from Sigma) +50ul Recombinant Mouse LIF Protein (from millipore), and the dishes were placed in an incubator at 37℃with 5% CO 2 The culture was continued for 24 hours under the conditions of (C).
After 48h from the above infection process, cells were removed from the incubator and the culture broth was discarded, and a stem cell culture broth containing 1. Mu.g/ml puromycin solution (available from MCE) was added at 37℃with 5% CO 2 Is cultured under the condition of (2); after one week of screening, a small number of cells were collected for Western blotting detection, and Igf2bp1 eggs were initially observedWhether the white level reduction effect reaches 80 percent or not, and the rest cells are continuously used for passage seed reservation.
4. Screening stable monoclonal cell strain and genotyping
(1) Screening of stable monoclonal cell lines
Digesting the ESC cells which are knocked out by mixing, and diluting the cells to a proper concentration by using a stem cell culture medium after cell counting; taking a 96-well plate, adding 4000 cells into a first hole A1 at the left side, and sequentially doubling-diluting to a last hole H1 in a first row; then the multichannel pipettes are used to dilute the solution from left to right to the last column. Adding 100 μl of stem cell culture solution into each well, placing in a cell culture box, and culturing at 37deg.C under 5% CO2 for 10-15 days; observing the condition of cells in a 96-well plate under a microscope, selecting a cell culture hole with only one cell as a monoclonal, digesting the monoclonal by pancreatin, and inoculating the monoclonal into a 24-well plate for continuous culture. After the 24-well plate is full, cells are collected, one part of the cells are passaged into the 6-well plate for culture, the other part of the cells are used for extracting protein by using 1X SDS Loadingbuffer, and Western blotting is used for detecting whether the Igf2bp1 protein is completely knocked out.
(2) Genotyping
Selecting clones with complete deletion of Igf2bp1 protein level according to Western blotting detection results, collecting cells, continuously subculturing one part of the cells, extracting genomic DNA from the other part of the cells according to a Noruzab genomic DNA extraction kit (DC 102-01), and performing the specific steps according to instructions; PCR primers were designed 200bp before and after the sgRNA target sequence on the Igf2bp1 gene as shown in Table 2. The PCR reaction system for amplifying the target gene sequence by taking the extracted genome DNA as a template comprises the following specific steps of: mu.l of 5 XHF buffer, 1.25. Mu.l of Igf2bp1-F (10. Mu.M), 1.25. Mu.l of Igf2bp1-R (10. Mu.M), 0.5. Mu.l of dNTP (10 mm), 20ng of DNA, 0.5ul of Phusion enzyme, and 25. Mu.l of ddH20, the PCR procedure being: incubating at 98 ℃ for 30s; incubating for 5s at 98 ℃, 30s at 58 ℃ and 30s at 72 ℃, and amplifying for 30 cycles; maintaining at 72deg.C for 10min; enriching gene fragments, performing agarose gel electrophoresis on the amplified sequence, cutting out target DNA bands, recovering DNA according to the instruction book of a Norwegian gel recovery kit, delivering to Nanjing department biotechnology Co., ltd for sequencing verification, and comparing the sequencing result with a wild Igf2bp1 gene to determine the genotype of the cell strain.
Table 2 sgRNA target sequence PCR primer on Igf2bp1 gene
| Igf2bp1-F | The sequence shown in SEQ ID NO. 4: 5'-GAGCTCCGGACAACTTCAGG-3' |
| Igf2bp1-R | The sequence shown in SEQ ID NO. 5: 5'-AGGAGCTGCAGGTCCCCGTTG-3' |
Through sequencing comparison, finally, 2 stable cell strains with Igf2bp1 gene deletion mutation are identified. The genomic DNAPCR amplification products of the monoclonal Igf2bp1-B2 (bp 1-B2) are shown in FIG. 2. Lane M: DNAMaroker; lane WT: wild-type genomic DNAPCR products; lanes bp1-B2: genomic DNAPCR product of clone No. B2. FIG. 4 shows genomic alignment analysis of wild type and monoclonal bp 1-B2. WT represents wild-type ESC genomic DNA sequence; bp1-B2 represents knockout Igf2bp1 monoclonal B2 genome DNA sequence; the dashed line represents the deleted fragment. FIG. 7 shows the genomic DNAPCR amplification product of monoclonal Igf2bp1-C1 (bp 1-C1). Lane M: DNAMaroker; lane WT: wild-type genomic DNAPCR products; lanes bp1-C1: genomic DNAPCR product of clone No. C1. FIG. 9 shows genomic alignment analysis of wild type and monoclonal bp 1-C1. WT represents wild-type ESC genomic DNA sequence; bp1-C1 represents knockout Igf2bp1 monoclonal C1 genome DNA sequence; the dashed line represents the deleted fragment.
FIG. 3 shows the Sanger sequencing of the monoclonal bp1-B2 genomic DNAPCR product, which shows a deletion of 2 bases in the first exon of Igf2bp1 in the clone with the knock-out number bp 1-B2. Another Sanger sequencing result of the monoclonal bp1-C1 genomic DNAPCR product is shown in FIG. 8. Sequencing results showed that the first exon of Igf2bp1 of clone with the number bp1-C1 was deleted by 128 bases.
Western blotting results also show that compared with wild type ESC cells, the complete knockout of Igf2bp1 protein level in the bp1-B2 clone and the bp1-C1 clone is realized. Specifically, as shown in FIG. 5, the protein level change of Igf2bp1 gene knockout is verified by Western blot. WT represents Igf2bp1 protein expression from wild-type ESCs; bp1-B2 represents the expression of Igf2bp1 protein of knockout clone B2. FIG. 10 shows protein level changes of Igf2bp1 gene knockout by Western blot verification. WT represents Igf2bp1 protein expression from wild-type ESCs; bp1-C1 represents the expression of Igf2bp1 protein of knockout clone C1.
From the analysis of the mRNA expression level, as shown in FIG. 6, the mRNA expression level of the single clone bp1-B2 was detected by qPCR, and as shown in FIG. 11, the mRNA expression level of the single clone bp1-C1 was detected by qPCR, and it was found that the levels of the mRNA knockdown of the clone bp1-B2 and the clone bp1-C1 reached about 90% and about 70%, respectively.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The sgRNA sequence for knocking out the mouse Igf2bp1 gene is characterized by being shown as SEQ ID NO. 1.
2. The method for knocking out the Igf2bp1 gene of the mouse embryonic stem cells is characterized by modifying the Igf2bp1 gene in the mouse embryonic stem cells by utilizing a CRISPR/Cas9 lentiviral system, and specifically comprises the following steps:
step S1, artificially synthesizing oligonucleotide sequences sg-Oligo1 and sg-Oligo2 containing an sgRNA sequence to form double-stranded DNA, wherein the sg-Oligo1 sequence is shown as SEQ ID NO. 2, and the sg-Oligo2 sequence is shown as SEQ ID NO. 3; inserting the synthesized double-stranded DNA into a polyclonal site of a sgRNA skeleton expression plasmid vector and transforming, selecting a monoclonal strain, extracting a sgRNA recombinant plasmid, and obtaining the sgRNA recombinant plasmid with correct sequencing through sequencing identification, namely LentiCRISPR-bp1, wherein the sgRNA skeleton expression plasmid vector also expresses Cas9 nuclease;
s2, transfecting the sgRNA recombinant plasmid into HEK 293T cells, and then harvesting virus supernatant to infect the mouse embryonic stem cells to obtain the mouse embryonic stem cells with Igf2bp1 gene knocked out.
3. The method for knocking out the Igf2bp1 gene of the mouse embryonic stem cell according to claim 2, wherein the sgRNA skeleton expression plasmid vector is LentiCRISPRv2.
4. The method for knocking out the Igf2bp1 gene of the mouse embryonic stem cell according to claim 2, wherein the specific embodiment of the method for knocking out the Igf2bp1 gene of the mouse embryonic stem cell comprises the steps of:
designing an sgRNA sequence aiming at an exon of the Igf2bp1 gene, and synthesizing sg-Oligo1 and sg-Oligo2; the LeniCRISPR v2 plasmid is subjected to BsmBI single enzyme digestion, and after agarose gel electrophoresis separation, DNA fragments are purified by using a gel recovery kit; annealing the synthesized sg-Oligo1 and sg-Oligo2 to form double-stranded DNA, and phosphorylating by T4 kinase; then connecting the carrier with the annealed double-stranded DNA fragment under the action of T4 DNA ligase, converting the connection product into competent cells, selecting a monoclonal strain, extracting the sgRNA recombinant plasmid, and obtaining the sgRNA recombinant plasmid LentiCRISPR-bp1 with correct sequencing through sequencing identification;
transfecting HEK 293T cells with recombinant plasmid LentiCRISPR-bp1, collecting 48h virus supernatant, taking a proper amount of virus supernatant to infect mouse embryonic stem cells, adding puromycin after 24h, and screening for one week; preparing the screened positive cells into cell suspension, screening monoclonal cells by using a limiting dilution method, collecting monoclonal cells, extracting genome DNA, amplifying DNA products by using PCR and sequencing to identify genotype changes.
5. The method for knocking out Igf2bp1 gene in mouse embryonic stem cells according to claim 4, wherein the step of collecting 48h virus supernatant by transfecting HEK 293T cells with the recombinant plasmid comprises the following steps: cell confluence reached about 70%, lentiCRISPR-bp1, packaging plasmid VSVG and PSPAX were co-transfected into cells, the complete medium was changed for 24h, and after 48h the virus supernatant was collected and filtered with 0.45 μm filter.
6. The method for knocking out the Igf2bp1 gene of the mouse embryonic stem cell according to claim 5, wherein the mouse embryonic stem cell is a J1 cell line, is purchased from a cell bank of China academy of sciences and is cultured by adopting feeder-free cells; the steps of taking a proper amount of virus supernatant to infect the embryo stem cells of the mice, adding puromycin after 24 hours and screening for one week specifically comprise: mouse embryonic stem cells ESC were grown to 30% -40% density, added with viral supernatant for 24h, 8 μg/ml of polybrene was added during infection, and after 48h, 1.0 μg/ml of puromycin was added for one week to screen, and protein samples were collected for preliminary verification by Western blotting.
7. The method for knocking out Igf2bp1 gene in mouse embryonic stem cells according to claim 6, wherein the specific steps of screening monoclonal cells by limiting dilution method, collecting monoclonal cells and extracting genomic DNA, amplifying DNA products by PCR and sequencing to identify genotype changes comprise: inoculating the mixed knocked-out cell suspension into a 96-well plate by using a limiting dilution method, culturing for 10-15 days, and selecting a monoclonal to be inoculated into a 24-well plate for continuous culture; cells are collected in the passage process and used for genome extraction, upstream and downstream primers are designed at 200bp at the front end and the rear end according to sgRNA loci, PCR products are further cloned into pUCm-T vectors, and genotype-changed clones are obtained after sequencing and comparison.
8. The method for constructing the Igf2bp1 gene knockout mouse embryonic stem cell line is characterized in that the method for constructing the Igf2bp1 gene knockout mouse embryonic stem cell line is to adopt a limiting dilution method to carry out passage screening on the mouse embryonic stem cells of the Igf2bp1 gene knockout mouse embryonic stem cell obtained by the Igf2bp1 gene knockout method of claim 2, so as to obtain the stable Igf2bp1 gene knockout mouse embryonic stem cell line.
9. The Igf2bp1 gene-deleted mouse embryonic stem cell strain, which is characterized in that the Igf2bp1 gene-deleted mouse embryonic stem cell strain is obtained by adopting the method for constructing the Igf2bp1 gene-deleted mouse embryonic stem cell line according to claim 8.
10. A kit for site-directed knockout of an Igf2bp1 gene in a mouse genome, comprising any one of the following (1) to (2):
(1) The sgRNA sequence-containing oligonucleotide sequences sg-Oligo1 and sg-Oligo2 as claimed in claim 2, forming double stranded DNA;
(2) The sgRNA recombinant plasmid Lentidispr-bp 1 as claimed in claim 2.
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