CN111304259A - Method for constructing ABCC4 gene knockout mouse by CRISPR/Cas9 technology and application thereof - Google Patents
Method for constructing ABCC4 gene knockout mouse by CRISPR/Cas9 technology and application thereof Download PDFInfo
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Abstract
The invention discloses a method for constructing an ABCC4 gene knockout mouse by using a CRISPR/Cas9 technology, which mainly comprises three parts, namely gRNA target site selection, microinjection, F0 generation mouse identification, homozygote propagation and identification, and can successfully obtain an ABCC4 gene systemically knocked homozygous mouse; the ABCC4 gene knockout homozygous mouse obtained by the invention can be used for making a slow obstructive pulmonary disease model, and provides a reliable animal model for researches on the aspects that ABCC4 influences the functional action of cilia growth in the development of slow obstructive pulmonary disease and the like; the gRNA target site sequence provided by the invention has a low off-target effect, can be successfully cut, and has a key effect in the process of constructing an ABCC4 gene knockout mouse; compared with the traditional gene editing technology, the CRISPR/Cas9 can realize gene site-directed modification, is simple, easy and efficient to operate, has higher probability of homologous recombination, shortens the time required by constructing a model mouse, and improves the accuracy and efficiency of gene knockout.
Description
Technical Field
The invention relates to the technical field of gene editing, in particular to a method for constructing an ABCC4 gene knockout mouse by using a CRISPR/Cas9 technology and application thereof.
Background
ABCC4 is a member of ATP-binding transporter family, is expressed in various blood cells, neurons, epithelial cells and endothelial cells, and can be localized on the basolateral or apical membrane to play the role of active transport of substances. ABCC4 transports substances widely, including key molecules in cell signaling pathways such as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), prostaglandins, and the like. ABCC4 is involved in regulating intracellular cAMP concentrations. cAMP, as a second messenger, plays a key role in diverse cellular functions, including regulation of the endothelial barrier, contraction of smooth muscle cells and activation of inflammatory cells, among others. Increasing intracellular cAMP levels is currently considered a strategy for treating inflammatory lung diseases. In addition, the ABCC4 protein can directly transport prostaglandin and participate in the role of prostaglandin signaling pathway in cilia growth. Decreased ABCC4 expression in both human retinal pigment epithelial cells and mouse kidney inner medullary collecting duct cells showed a phenotype of decreased cilia number and shortened length. 2017, the global advocate for chronic obstructive pulmonary disease indicates that smoking or other harmful factors can cause the reduction of the cilia clearance function of the airway of a patient with chronic obstructive pulmonary disease, so that bacteria are planted in the respiratory tract, mucus secretion is increased, the immune response of the body is continuously stimulated, and the progress of the chronic obstructive pulmonary disease is promoted. Our previous studies found that the SNP site of ABCC4 gene is related to the risk of chronic obstructive pulmonary disease and causes the decrease of ABCC4 expression level in blood of patients with chronic obstructive pulmonary disease. In order to further prove the influence of the decrease of the expression level of ABCC4 on the generation and development of the chronic obstructive pulmonary disease, an ABCC4 gene knockout mouse needs to be constructed, and a model for the model preparation of the chronic obstructive pulmonary disease is made, so that an animal model is provided for researching the influence of ABCC4 on a cilium growth mechanism and promoting the generation and development of the chronic obstructive pulmonary disease.
Compared with the traditional zinc finger nuclease and transcription activator-like effector nuclease gene editing technology, the CRISPR/Cas9 carries out fixed-point modification on genes by designing different guide RNAs, has simple operation, easy implementation and high efficiency, has heritability, no species restriction, short experimental period and lower cost for gene modification, and has higher probability of homologous recombination than the naturally occurring homologous recombination.
Animal models are an important part of disease research, and any disease research requires an experiment on the animal model. The CRISPR/Cas9 technology can be used for carrying out gene editing on animals artificially and preparing disease models. At present, various animal disease models, such as hemophilia B mouse genetic disease model, targeted editing immunodeficient mouse model, mouse tumor animal models of liver cancer and the like, autism cynomolgus monkey model, Huntington chorea gene knock-in pig model and the like, are established by using CRISPR/Cas9 technology. The establishment of these animal disease models lays a solid foundation for drug screening and therapeutic research. In conclusion, in order to research the mechanism of ABCC4 for causing the development of chronic obstructive pulmonary disease, it is essential to prepare ABCC4 knockout mice and is a prerequisite for preparing mouse models of chronic obstructive pulmonary disease.
Disclosure of Invention
The invention adopts CRISPR/Cas9 technology to carry out gene targeting on mouse fertilized eggs, realizes ABCC4 gene knockout, and then obtains ABCC4 homozygous mouse with systemic gene knockout through microinjection and mating propagation. The mouse can be further smoked to prepare a chronic obstructive pulmonary disease model, and a foundation is laid for researching the action mechanism of ABCC4 in the development of chronic obstructive pulmonary disease.
The CRISPR/Cas9 system is matched to target DNA via guide rna (grna) to direct Cas9 protein to bind to a specific genetic locus and cleave. The designed gRNA may bind to a sequence of a non-target site, resulting in a gene mutation, i.e., off-target effect. The invention provides a group of gRNA target site sequences, and through NCBI database analysis, the gRNA off-target sites designed in the sequence are fewer, the score is higher, and the off-target effect of CRISPR/Cas9 in ABCC4 gene editing is effectively reduced.
The ABCC4 gene transports prostaglandins that play a role in cilia growth, and abnormalities in cilia structural function can affect the development of chronic obstructive pulmonary disease. In order to explore a detailed mechanism, the invention provides a method for constructing an ABCC4 gene knockout mouse by using a CRISPR/Cas9 technology, and a homozygous mouse with an ABCC4 gene knockout systemically can be successfully obtained. The ABCC4 gene knockout homozygous mouse obtained by the invention can be used for making a slow obstructive pulmonary disease model, and provides a reliable animal model for researches on the aspects that ABCC4 influences the functional action of cilia growth in the development of slow obstructive pulmonary disease and the like. Since the ABCC4 gene research is mainly focused on the fields of tumor drug resistance and treatment at present, the research in the field of chronic obstructive pulmonary diseases is still the first time.
Because a method for constructing an ABCC4 knockout mouse is lacked at present, the invention discloses and provides a method for constructing an ABCC4 knockout mouse by using a CRISPR/Cas9 technology for the first time.
The first purpose of the invention is that: a method for constructing an ABCC4 systemic gene knockout mouse by using a CRISPR/Cas9 technology is provided, and the specific implementation process mainly comprises three parts of gRNA target site selection, microinjection, identification of F0 mouse generation, homozygote reproduction and identification.
gRNA target site selection
The Abcc4 gene (NCBI Reference Sequence: NM — 001033336.3; Ensembl: ensusg 00000032849) is located on mouse chromosome 14 and contains 31 exons in total, the ATG initiation codon is located in exon 1, and the TGA termination codon is located in exon 31 (Transcript:
ensust 00000036554). Exon 2 starts at 1.89% of the coding region, and exons 2 and 3 cover 5.84% of the coding region and do not contain other known genes, so exons 2 and 3 are selected as knock-out regions, as shown in fig. 1. A gRNA target site is selected from non-tandem repeat sequences in 2000bp upstream of the exon 2 and 2000bp downstream of the exon 3, gRNA is designed and synthesized, a selected target site sequence is analyzed on an NCBI database, a gRNA target site with few off-target sites is selected, and a company synthesizes the complementary gRNA. The gRNA target site sequences are seen in table 1.
TABLE 1 gRNA sequences
Microinjection and identification of F0-generation mice
The synthetic gRNA and Cas9 protein (purchased from NEB under stock number M0386M) were injected directly into fertilized eggs of C57BL/6N mice and then transplanted into oviducts of surrogate mother mice. After the mouse is born, the mouse tail is cut to carry out PCR identification, and a positive F0 mouse is obtained.
Wherein genomic DNA of mouse rat tail is extracted
High purity Genomic DNA was extracted using the MiniBEST Universal Genomic DNA Extraction kit (Ver.5.0_ Code No.9765) kit, according to the instructions. The method comprises the following steps:
⑴ cut 2-5mm rat tail tissue into 1.5mL centrifuge tubes, add 180. mu.L Buffer GL, 20. mu.L proteinase K and 10. mu.L RNase A.
⑵ 56 ℃ overnight.
⑶ 12000 at 12000rpm for 2 minutes, and the impurities were discarded.
⑷ mu.L of Buffer GB and 200 mu.L of absolute ethanol are added and mixed well.
⑸ placing the centrifugal column matched with the kit in a collecting tube, transferring the sample to the centrifugal column, centrifuging at 12000rpm for 2 minutes, and discarding the liquid in the collecting tube.
⑹ mu.L of Buffer WA WAs added to the spin column, centrifuged at 12000rpm for 1min, and the liquid in the collection tube WAs discarded.
⑺ mu.L of Buffer WB was added to the column, centrifuged at 12000rpm for 1min, and the collection tube was discarded (Note: ensure that Buffer WB was first mixed with 100% ethanol, when Buffer WB was added, residual salts were washed along the tube wall)
⑻ repeat step ⑺.
⑼ the column was placed in a collection tube and centrifuged at 12000rpm for 2 min.
⑽ placing the centrifugal column in a new 1.5mL centrifuge tube, adding 50-200 μ L sterile water to the center of the centrifugal column membrane, standing for 5min (note: preheating sterile water to 65 ℃ in advance can improve DNA dissolution rate)
⑾ 12000 at 12000rpm for 2min to increase the DNA yield, step ⑽ can be repeated.
⑿ agarose gel electrophoresis detection.
Performing PCR identification
The PCR identification strategy is shown in FIG. 2, and the sequences of primers required for identification are shown in Table 2. The band size amplified by the primer F1R1 is 434bp, and the band size amplified by the primer F1R2 is 592 bp. Endogenous mouse Rgs7 gene is used as internal reference, and the electrophoresis band size of the product obtained by PCR reaction is 335 bp. For agarose gel electrophoresis, 100bp DNA Ladder Marker (Thermoscientific, SM0242) was used as a reference.
A25-mu LPCR reaction system was prepared according to Table 3, PCR amplification was performed using the extracted genomic DNA of mouse tail as a template and using the primer F1R1 according to Table 4, and the resultant was subjected to agarose gel electrophoresis to obtain a band of 434bp in size, which was identified as positive.
TABLE 2 PCR primer sequences
TABLE 3 PCR reaction System
TABLE 4 PCR amplification procedure
Reproduction and characterization of homozygotes
The sexually mature positive F0 generation mice are respectively bred with wild mice for one generation to obtain offspring, and the offspring is cut to extract genomic DNA from rat tails for PCR identification. Positive mice identified by PCR were scored as heterozygous for the F1 generation. The obtained sexually mature F1 generation heterozygotes were selfed and homozygote propagation was performed. When PCR identification is carried out, electrophoretic bands with sizes of 434bp and 592bp are identified as heterozygotes, and electrophoretic bands with sizes of 434bp are identified as homozygotes.
According to the method provided by the invention, 7 ABCC4 gene knockout homozygous mice are constructed, the numbers are 50, 51, 55, 28, 29, 31 and 38, and the PCR identification electrophoresis results are shown in FIGS. 3 and 4.
The invention also aims to provide an ABCC4 gene knockout mouse constructed by the method for manufacturing a chronic obstructive pulmonary disease model, and provides a reliable animal model for researching the function of the ABCC4 gene in cilium development, the influence of maintaining airway homeostasis and the effect of the ABCC4 gene in the development of chronic obstructive pulmonary disease and the like.
The invention has the beneficial effects that: 1. the gRNA target site sequence provided by the invention can reduce off-target effect, is successfully cut by gRNA, and has a key effect in the process of constructing an ABCC4 gene knockout mouse;
2. compared with the traditional gene editing technology, the method for constructing the ABCC4 knockout mouse by the CRISPR/Cas9 technology has the advantages that the CRISPR/Cas9 can realize gene site-specific modification, the operation is simple, feasible and efficient, the probability of homologous recombination is high, the time required for constructing the model mouse is shortened, and the accuracy and the efficiency of gene knockout are improved;
3. the mouse obtained by the method provided by the invention can be used for manufacturing a slow obstructive pulmonary disease model, and provides a basis for researching a mechanism of the ABCC4 gene in the process of influencing cilia growth and promoting the generation and development of the slow obstructive pulmonary disease.
Drawings
FIG. 1 is a schematic diagram of ABCC4 gene targeting provided by the present invention;
FIG. 2 is a schematic representation of the PCR identification provided by the present invention;
FIG. 3 is an electrophoretogram of a genome product amplified by the primer F1R1 provided by the invention;
FIG. 4 is the electrophoresis chart of the F1R2 and internal reference primer amplified genome product provided by the invention.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The first embodiment is as follows:
a method for constructing an ABCC4 systemic gene knockout mouse by using a CRISPR/Cas9 technology mainly comprises three parts, namely gRNA target site selection, microinjection, identification of an F0 mouse generation, homozygote propagation and identification.
gRNA target site selection
The Abcc4 gene (NCBI Reference Sequence: NM — 001033336.3; Ensembl: ensusg 00000032849) is located on mouse chromosome 14 and contains 31 exons in total, the ATG initiation codon is located in exon 1, and the TGA termination codon is located in exon 31 (Transcript:
ensust 00000036554). Exon 2 starts at 1.89% of the coding region, and exons 2 and 3 cover 5.84% of the coding region and do not contain other known genes, so exons 2 and 3 are selected as knock-out regions, as shown in fig. 1. A gRNA target site is selected from non-tandem repeat sequences in 2000bp upstream of No.2 exon and 2000bp downstream of No.3 exon, gRNA is designed and synthesized, the sequence of the selected gRNA target site is analyzed on an NCBI database, a gRNA target site with few off-target sites is selected, and a company synthesizes the complementary gRNA. The gRNA target site sequences are seen in table 1.
TABLE 1 gRNA sequences
Microinjection and identification of F0-generation mice
The synthetic gRNA and Cas9 protein (purchased from NEB under stock number M0386M) were injected directly into fertilized eggs of C57BL/6N mice and then transplanted into oviducts of surrogate mother mice. After the mouse is born, the mouse tail is cut to carry out PCR identification, and a positive F0 mouse is obtained.
Wherein genomic DNA of mouse rat tail is extracted
High purity Genomic DNA was extracted using the MiniBEST Universal Genomic DNA Extraction kit (Ver.5.0_ Code No.9765) kit, according to the instructions. The method comprises the following steps:
⑴ cut 2-5mm rat tail tissue into 1.5mL centrifuge tubes, add 180. mu.L Buffer GL, 20. mu.L proteinase K and 10. mu.L RNase A.
⑵ 56 ℃ overnight.
⑶ 12000 at 12000rpm for 2 minutes, and the impurities were discarded.
⑷ mu.L of Buffer GB and 200 mu.L of absolute ethanol are added and mixed well.
⑸ placing the centrifugal column matched with the kit in a collecting tube, transferring the sample to the centrifugal column, centrifuging at 12000rpm for 2 minutes, and discarding the liquid in the collecting tube.
⑹ mu.L of Buffer WA WAs added to the spin column, centrifuged at 12000rpm for 1min, and the liquid in the collection tube WAs discarded.
⑺ mu.L of Buffer WB was added to the column, centrifuged at 12000rpm for 1min, and the collection tube was discarded (Note: ensure that Buffer WB was first mixed with 100% ethanol, when Buffer WB was added, residual salts were washed along the tube wall)
⑻ repeat step ⑺.
⑼ the column was placed in a collection tube and centrifuged at 12000rpm for 2 min.
⑽ placing the centrifugal column in a new 1.5mL centrifuge tube, adding 50-200 μ L sterile water to the center of the centrifugal column membrane, standing for 5min (note: preheating sterile water to 65 ℃ in advance can improve DNA dissolution rate)
⑾ 12000 at 12000rpm for 2min to increase the DNA yield, step ⑽ can be repeated.
⑿ agarose gel electrophoresis detection.
Performing PCR identification
The PCR identification strategy is shown in FIG. 2, and the sequences of primers required for identification are shown in Table 2. The band size amplified by the primer F1R1 is 434bp, and the band size amplified by the primer F1R2 is 592 bp. Endogenous mouse Rgs7 gene is used as internal reference, and the electrophoresis band size of the product obtained by PCR reaction is 335 bp. For agarose gel electrophoresis, 100bp DNA Ladder Marker (Thermoscientific, SM0242) was used as a reference.
A25-mu LPCR reaction system was prepared according to Table 3, PCR amplification was performed using the extracted genomic DNA of mouse tail as a template and using the primer F1R1 according to Table 4, and the resultant was subjected to agarose gel electrophoresis to obtain a band of 434bp in size, which was identified as positive.
TABLE 2 PCR primer sequences
TABLE 3 PCR reaction System
TABLE 4 PCR amplification procedure
Reproduction and characterization of homozygotes
The sexually mature positive F0 generation mice are respectively bred with wild mice for one generation to obtain offspring, and the offspring is cut to extract genomic DNA from rat tails for PCR identification. Positive mice identified by PCR were scored as heterozygous for the F1 generation. The obtained sexually mature F1 generation heterozygotes were selfed and homozygote propagation was performed. When PCR identification is carried out, electrophoretic bands with sizes of 434bp and 592bp are identified as heterozygotes, and electrophoretic bands with sizes of 434bp are identified as homozygotes.
According to the method provided by the invention, 7 ABCC4 gene knockout homozygous mice are constructed, the numbers are 50, 51, 55, 28, 29, 31 and 38, and the PCR identification electrophoresis results are shown in FIGS. 3 and 4.
The sequence of the 2000 upstream to 2000 downstream exon 3 fragment of exon 2 is shown as follows, wherein the exon marks the bottom line, the genome sequence without knockout is black lower case letters, the gRNA target site sequence is capital letters, the knockout region is oblique font, the sequence of the 2000 upstream to 2000 downstream exon 3 fragment of exon 2 is shown as SEQ ID NO.8 in the sequence table,
catagatgatcaaagatagcttcttatcctagtgtgctttgcttgacagcaaattaaaccatgtgtctcagcctggggctttttgagatttgggtagtgagagagcctgggagaagcaatctgtggtatcttatgaaaaaaagtttgagttgagcgtgtgttgagttcatatattgaatttaacactactccctctttataatgttgctacatacgatgcaaatgcagttaactgtgcggtctcattggacagggttaccagtgctttcctacgtcatggggcttgggtcttgagtttgaactcaggacctgtatatgctaagcacttgtttggctacttttctatatacacccaaggccctttcttaactattatatttcttccccaagctgattttacctgctgattgaaaacaaagctcaccacaaaccatattaagcctttgtaagcctaaaaaatgctaataaattccacttagaagtaccggatggaaaatacccgatgttctactggaagagagggctcaggcgtgtaggtagtgtctcagacatgtctgggagcccacctacattgatccaaggcttgcaaggtgacaaatgtcctgaaaactcttggggagggtgctaagagagaggtgagctggttggttcctcagtgatcaactcactgatccctcagaaTCTACTGGGTGGACGAGGCAGGGgcattggcacctcttgggacccccaaatttactttggaagtccttgtaatgtctgctatggtcttggggagctgtcatagctctgagccaaaaagtctgtgcctttggagtgtcctgagcaagttgcgtggaggttgactgaacaccttgctccctggagctgctgggtgctctcctctacagaaacccctgagcagcagcttctccaaggggagagcttcccagagtatgcagtcccctgtaaagtgtgcactctcaagagagacaagaggaaagacatctgcttaattcagtaatctcatctatcaaggctttctagaaggacccacatttaattgcccaagagagacacatgtatattccctggccttctgaagctttcgcacaaaagatgatagattgttcctgtgagtgggggtgggggtgctgggtgggtcggtgggtgggggacagacaggagagggctgagcttccatggacagtggctgacatttgtcatcccagaggatctgcagcatctttagatgtccttccatttgcccaaggctgagagagagagagagagacagacagacagacagagaaggagacagagagagacagagaccactcactggttagcagtcatatatccagaaagcagccacatctatgtagacattaaagctggagggttccacagcacatttaggcttctcactagagagtgcttttcagggtactgtcaccagccagctctattgcaaaggccccgagtttagaaaaattgttaataaaaggaccaagcaggtttctgtacctggtcagtggtaaatctctctctctctctctctctctctctctctctctctctctctctcacacacacacacacatgtatacatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatgtatacatgtgtgtctatatactaggggtagatagttgacacctagagatatgtagtgataggtcacaggttctgactgagctggtagaagtagagagggggtgacagaggctggtattaagaaatcaagatatggtgtcttgatgtcagggtgctctcattgactgcctcccagtgtgcagaacgagagtgggggagggaggagccggttgtatgaacgagcacagacttaacctttctctgtctctttgtccatttaaaatacggagggtgacaaaacgcgctgaggcgagagtctacttagatgcaaacgtgggggtctgagtgtgttatcaatccctttaactttgacaggtggctcaacccgctgtttaaaactggtcataagcggagactgg aagaagatgacatgttctcagtgcttccagaagatcgctcaaagcacctcggagaggagcttcaacggtaagcagggggcggcgggaggaggtgaagcctgagtgtgcatgcgcagggtggccccttgctcgcagggtggcttggaggaggtctgacctcttcccacgtcccttgctctgtctggagctttaatgtccccacgttgcacgcctcccttcaggttgtcctgtcttggtctgacttagtgacttggggaaagaaaacctgggttggggctctataggtcttgatggctgagatctgtccctggggctggtctcaccagagcagtaacctgccctctctggctgccagggctgttgtaatccagcagagctcggcagtgacctgtccccggagatgtctccccttgaggtctctctttctggtgcaacagagtctgcctcacacatctttctgggtccctcgggacgagccagtgtttgtatagcatctgcggggtagccctgtcataaacacttacctgtccttctgcccttccgttttactcaacggcagtcctactgttcctgactccgcgtttgtctcactgcaggtactgggataa agaacttctgcgagccaagaaggactcgaggaagccctccttaacaaaggcaatcataaagtgttactggaagtct tacctgattttgggaatttttacgttaattgaggtaaatgttagtcatatcctgactgctgtctctgagtgggtttaccaggaggaggcgtggagtcctgacggtcgatcattgttggagtgagtggggaccagcaggcagaatgagctttgaaaatgtcttgggttcagggaggaagcgtggtgatgctcattatcacatctttggaatgggtgatgctttggtgactgaagctgaagacattgccgtttcttctaattaaagcgcagtgctatctcgcagctgttaagatcgcttactgctgttgcagacatctggagttcagtccccagcgcctacagggtggctcacagcccttcctgaggtccagttcacggagagtctccgccatcctctgacctgcatgggctccagcctatacgcagtacacacagattgcaggtacacacacacacacacacacacacacacacactacatacatcttaacaaatgtttgaagtccagtctcacaggaagctgacagcacctctgagtgctggtgttcggtgtgtaaattcaccttgtcctctttgatgctcatgaggaaatacatttggatcagagcccagaataaccagtgcccaataagtaattgttaactgaaccatgccagatctgataaatgccttcctgggagagtattgtcaactttcccttcccaggtaaacaagacatggtgtcatgtgcccgggcttgctgtggggccctggagtctctcaccctcctgctgaccgatcgttggctgggcactgagagctgaatctactctggccatggaccagcttcaactctcgtgacctcctgtaccattcatcctgggttcttgctgcggagcagattgctggtctgacgttgagttctggtggtatggacttagagagagctacagcgctcattccctgataaaccaaagctggaaggccatgagtccttgcagggTCACCCGTTAGTCAGATAGTCGGctgcccagtgggcagtgcctgtgctggtcaatggtctgctggaggggatcctggaagcctggctgtgtggcttgtccttctggtggcgtctttaagagaaaaatgttatttgtatatttatttacgtatgcgtgtgtttataccacatatatcaggatgtctaaggaggctgggagaggaggtctgagccattagaactggagtcacaaggcgggtgtgaatttcctgacagggatgctgggatctgaactcgggtcctctggaagagcagcaagccctcttcatcactggtaccctataatgggcttaagagtgttggaggtctcacaagtggctggacatcctatctatggaagttgcagctctgtggaagggtggtctctgatgcagtgaggtgaaagctgtcccaggtgtctttgatggggtgaggcaggtggctatccaagcaggagagtgcgctcttgctgagcacgccctgtgcatgttgaaaggcacagagttctgttctatggcctcggagaaaggtaatggaagtgcttggttccagagatcgaccttctggaggttgcaaaggtgccaccatccttgttccggacatgagtgctctctttgagagggcagtgcccactgcttgggtgctgtttgtagcttggagaaaggtaaccttgtgatgcagaccacatgctgccccatgttctcttctcccccagggtagtcaggcccactgtgcacctgggtggcctagacggagggaagcgtagtcaggcgggtgggtgcaggcaagaccttggtcggcagtgcactggccaagaacacacatggagacagggttggataattggttcagttgattgggggagcaagttgttttgttttatataaagataaggagagaaggaatgtgtttggtggaggtgtagtcaggtgtgggggaagggggtgcctctgcaggcccatgctgaggcatcccttccccctgagggaccagccacatgatggtatagtatagaatagagtttatctagggca
aiming at the technical scheme of the invention, other possible implementation modes are provided:
the CRISPR/Cas9 technology is a third-generation gene site-directed editing technology, and becomes a high-efficiency gene editing tool due to the precise targeted cutting function. However, other gene editing techniques, such as traditional zinc finger nucleases and transcriptional activator-like effector nuclease gene editing techniques, may also achieve ABCC4 gene knock-out.
2. The invention selects No.2 exon and No.3 exon to design gRNA for knockout. Since the ABCC4 gene contains 31 exons, gRNA can be designed at other exons to achieve the purpose of knocking out the ABCC4 gene.
Therefore, due to the diversity and the universality of the technology, different technologies or different specific implementation methods of the same technology may exist to construct ABCC4 knockout mice. However, based on the discovery of the early research result that the SNP site of ABCC4 mutation is related to the incidence risk of chronic obstructive pulmonary disease, the invention discloses a specific implementation method for constructing an ABCC4 knockout mouse by using a CRISPR/Cas9 technology for the first time. The mouse is used for making a slow obstructive pulmonary disease model, and lays a foundation for researching a mechanism of ABCC4 influencing ciliary structural dysfunction in the occurrence and development of slow obstructive pulmonary disease.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> inner Mongolia autonomous region people hospital
<120> method for constructing ABCC4 gene knockout mouse by CRISPR/Cas9 technology and application thereof
<130>20191209
<160>8
<170>PatentIn version 3.3
<210>1
<211>23
<212>DNA
<213> Artificial Synthesis
<400>1
tctactgggt ggacgaggca ggg 23
<210>2
<211>23
<212>DNA
<213> Artificial Synthesis
<400>2
tcacccgtta gtcagatagt cgg 23
<210>3
<211>25
<212>DNA
<213> Artificial Synthesis
<400>3
tagaagtacc ggatggaaaa taccc 25
<210>4
<211>25
<212>DNA
<213> Artificial Synthesis
<400>4
tgtgactcca gttctaatgg ctcag 25
<210>5
<211>26
<212>DNA
<213> Artificial Synthesis
<400>5
gggaatatac atgtgtctct cttggg 26
<210>6
<211>24
<212>DNA
<213> Artificial Synthesis
<400>6
catgccaatg gttcactcta aggt 24
<210>7
<211>25
<212>DNA
<213> Artificial Synthesis
<400>7
tctctatgtc ccaaagtgca gacac 25
<210>8
<211>4761
<212>DNA
<213> Artificial Synthesis
<400>8
catagatgat caaagatagc ttcttatcct agtgtgcttt gcttgacagc aaattaaacc 60
atgtgtctca gcctggggct ttttgagatt tgggtagtga gagagcctgg gagaagcaat 120
ctgtggtatc ttatgaaaaa aagtttgagt tgagcgtgtg ttgagttcat atattgaatt 180
taacactact ccctctttat aatgttgcta catacgatgc aaatgcagtt aactgtgcgg 240
tctcattgga cagggttacc agtgctttcc tacgtcatgg ggcttgggtc ttgagtttga 300
actcaggacc tgtatatgct aagcacttgt ttggctactt ttctatatac acccaaggcc 360
ctttcttaac tattatattt cttccccaag ctgattttac ctgctgattg aaaacaaagc 420
tcaccacaaa ccatattaag cctttgtaag cctaaaaaat gctaataaat tccacttaga 480
agtaccggat ggaaaatacc cgatgttcta ctggaagaga gggctcaggc gtgtaggtag 540
tgtctcagac atgtctggga gcccacctac attgatccaa ggcttgcaag gtgacaaatg 600
tcctgaaaac tcttggggag ggtgctaaga gagaggtgag ctggttggtt cctcagtgat 660
caactcactg atccctcaga atctactggg tggacgaggc aggggcattg gcacctcttg 720
ggacccccaa atttactttg gaagtccttg taatgtctgc tatggtcttg gggagctgtc 780
atagctctga gccaaaaagt ctgtgccttt ggagtgtcct gagcaagttg cgtggaggtt 840
gactgaacac cttgctccct ggagctgctg ggtgctctcc tctacagaaa cccctgagca 900
gcagcttctc caaggggaga gcttcccaga gtatgcagtc ccctgtaaag tgtgcactct 960
caagagagac aagaggaaag acatctgctt aattcagtaa tctcatctat caaggctttc 1020
tagaaggacc cacatttaat tgcccaagag agacacatgt atattccctg gccttctgaa 1080
gctttcgcac aaaagatgat agattgttcc tgtgagtggg ggtgggggtg ctgggtgggt 1140
cggtgggtgg gggacagaca ggagagggct gagcttccat ggacagtggc tgacatttgt 1200
catcccagag gatctgcagc atctttagat gtccttccat ttgcccaagg ctgagagaga 1260
gagagagaga cagacagaca gacagagaag gagacagaga gagacagaga ccactcactg 1320
gttagcagtc atatatccag aaagcagcca catctatgta gacattaaag ctggagggtt 1380
ccacagcaca tttaggcttc tcactagaga gtgcttttca gggtactgtc accagccagc 1440
tctattgcaa aggccccgag tttagaaaaa ttgttaataa aaggaccaag caggtttctg 1500
tacctggtca gtggtaaatc tctctctctc tctctctctc tctctctctc tctctctctc 1560
tctcacacac acacacacat gtatacatat atatatatat atatatatat atatatatat 1620
atatatatat atatatatat atatatgtat acatgtgtgt ctatatacta ggggtagata 1680
gttgacacct agagatatgt agtgataggt cacaggttct gactgagctg gtagaagtag 1740
agagggggtg acagaggctg gtattaagaa atcaagatat ggtgtcttga tgtcagggtg 1800
ctctcattga ctgcctccca gtgtgcagaa cgagagtggg ggagggagga gccggttgta 1860
tgaacgagca cagacttaac ctttctctgt ctctttgtcc atttaaaata cggagggtga 1920
caaaacgcgc tgaggcgaga gtctacttag atgcaaacgt gggggtctga gtgtgttatc 1980
aatcccttta actttgacag gtggctcaac ccgctgttta aaactggtca taagcggaga 2040
ctggaagaag atgacatgtt ctcagtgctt ccagaagatc gctcaaagca cctcggagag 2100
gagcttcaac ggtaagcagg gggcggcggg aggaggtgaa gcctgagtgt gcatgcgcag 2160
ggtggcccct tgctcgcagg gtggcttgga ggaggtctga cctcttccca cgtcccttgc 2220
tctgtctgga gctttaatgt ccccacgttg cacgcctccc ttcaggttgt cctgtcttgg 2280
tctgacttag tgacttgggg aaagaaaacc tgggttgggg ctctataggt cttgatggct 2340
gagatctgtc cctggggctg gtctcaccag agcagtaacc tgccctctct ggctgccagg 2400
gctgttgtaa tccagcagag ctcggcagtg acctgtcccc ggagatgtct ccccttgagg 2460
tctctctttc tggtgcaaca gagtctgcct cacacatctt tctgggtccc tcgggacgag 2520
ccagtgtttg tatagcatct gcggggtagc cctgtcataa acacttacct gtccttctgc 2580
ccttccgttt tactcaacgg cagtcctact gttcctgact ccgcgtttgt ctcactgcag 2640
gtactgggat aaagaacttc tgcgagccaa gaaggactcg aggaagccct ccttaacaaa 2700
ggcaatcata aagtgttact ggaagtctta cctgattttg ggaattttta cgttaattga 2760
ggtaaatgtt agtcatatcc tgactgctgt ctctgagtgg gtttaccagg aggaggcgtg 2820
gagtcctgac ggtcgatcat tgttggagtg agtggggacc agcaggcaga atgagctttg 2880
aaaatgtctt gggttcaggg aggaagcgtg gtgatgctca ttatcacatc tttggaatgg 2940
gtgatgcttt ggtgactgaa gctgaagaca ttgccgtttc ttctaattaa agcgcagtgc 3000
tatctcgcag ctgttaagat cgcttactgc tgttgcagac atctggagtt cagtccccag 3060
cgcctacagg gtggctcaca gcccttcctg aggtccagtt cacggagagt ctccgccatc 3120
ctctgacctg catgggctcc agcctatacg cagtacacac agattgcagg tacacacaca 3180
cacacacaca cacacacaca cactacatac atcttaacaa atgtttgaag tccagtctca 3240
caggaagctg acagcacctc tgagtgctgg tgttcggtgt gtaaattcac cttgtcctct 3300
ttgatgctca tgaggaaata catttggatc agagcccaga ataaccagtg cccaataagt 3360
aattgttaac tgaaccatgc cagatctgat aaatgccttc ctgggagagt attgtcaact 3420
ttcccttccc aggtaaacaa gacatggtgt catgtgcccg ggcttgctgt ggggccctgg 3480
agtctctcac cctcctgctg accgatcgtt ggctgggcac tgagagctga atctactctg 3540
gccatggacc agcttcaact ctcgtgacct cctgtaccat tcatcctggg ttcttgctgc 3600
ggagcagatt gctggtctga cgttgagttc tggtggtatg gacttagaga gagctacagc 3660
gctcattccc tgataaacca aagctggaag gccatgagtc cttgcagggt cacccgttag 3720
tcagatagtc ggctgcccag tgggcagtgc ctgtgctggt caatggtctg ctggagggga 3780
tcctggaagc ctggctgtgt ggcttgtcct tctggtggcg tctttaagag aaaaatgtta 3840
tttgtatatt tatttacgta tgcgtgtgtt tataccacat atatcaggat gtctaaggag 3900
gctgggagag gaggtctgag ccattagaac tggagtcaca aggcgggtgt gaatttcctg 3960
acagggatgc tgggatctga actcgggtcc tctggaagag cagcaagccc tcttcatcac 4020
tggtacccta taatgggctt aagagtgttg gaggtctcac aagtggctgg acatcctatc 4080
tatggaagtt gcagctctgt ggaagggtgg tctctgatgc agtgaggtga aagctgtccc 4140
aggtgtcttt gatggggtga ggcaggtggc tatccaagca ggagagtgcg ctcttgctga 4200
gcacgccctg tgcatgttga aaggcacaga gttctgttct atggcctcgg agaaaggtaa 4260
tggaagtgct tggttccaga gatcgacctt ctggaggttg caaaggtgcc accatccttg 4320
ttccggacat gagtgctctc tttgagaggg cagtgcccac tgcttgggtg ctgtttgtag 4380
cttggagaaa ggtaaccttg tgatgcagac cacatgctgc cccatgttct cttctccccc 4440
agggtagtca ggcccactgt gcacctgggt ggcctagacg gagggaagcg tagtcaggcg 4500
ggtgggtgca ggcaagacct tggtcggcag tgcactggcc aagaacacac atggagacag 4560
ggttggataa ttggttcagt tgattggggg agcaagttgt tttgttttat ataaagataa 4620
ggagagaagg aatgtgtttg gtggaggtgt agtcaggtgt gggggaaggg ggtgcctctg 4680
caggcccatg ctgaggcatc ccttccccct gagggaccag ccacatgatg gtatagtata 4740
gaatagagtt tatctagggc a 4761
Claims (9)
1. A method for constructing an ABCC4 gene knockout mouse by using CRISPR/Cas9 technology is characterized by comprising the following steps: the method comprises the following steps:
step one, selecting a gRNA target site: selecting gRNA target sites at the non-tandem repeat sequences within 2000bp of the upstream exon of the No.2 exon and within 2000bp of the downstream exon of the No.3 exon of the ABCC4 gene, and designing and synthesizing gRNAs;
step two, obtaining positive F0 mouse: cas9 protein and synthetic gRNA are directly injected into fertilized eggs of a C57BL/6N mouse, then the fertilized eggs are transplanted into the oviduct of a surrogate mother mouse, and after the mouse is born, the tail of the mouse is cut for PCR identification to obtain a positive F0-generation mouse;
step three, breeding and identifying the homozygote: breeding sexually mature positive F0 generation mice with wild mice for one generation respectively, obtaining offspring, clipping rat tail, extracting genomic DNA, performing PCR identification to obtain F1 generation heterozygotes, selfing the obtained sexually mature F1 generation heterozygotes, and breeding homozygotes.
2. The method for constructing ABCC4 knockout mice by using CRISPR/Cas9 technology according to claim 1, wherein the CRISPR/Cas9 technology comprises the following steps: the gRNA target site sequence in the first step is shown as SEQ ID NO.1 and SEQ ID NO. 2.
3. The method for constructing ABCC4 knockout mice by using CRISPR/Cas9 technology according to claim 1, wherein the CRISPR/Cas9 technology comprises the following steps: and step two, after the mouse is born, the mouse tail is cut to carry out PCR identification, and the PCR identification comprises the steps of extracting mouse tail genome DNA and carrying out PCR identification.
4. The method for constructing ABCC4 knockout mice by using CRISPR/Cas9 technology according to claim 3, wherein the CRISPR/Cas9 technology comprises the following steps: the method for extracting mouse tail genome DNA comprises the following steps:
(1) clipping 2-5mm rat tail tissue, placing in a 1.5mL centrifuge tube, adding 180. mu.L Buffer GL, 20. mu.L proteinase K and 10. mu.L RNase A;
(2) incubating overnight at 56 ℃;
(3) centrifuging at 12000rpm for 2min, and discarding impurities;
(4) adding 200 mu L of Buffer GB and 200 mu L of absolute ethyl alcohol, and fully and uniformly mixing;
(5) placing a centrifugal column matched with the kit in a collecting pipe, transferring a sample into the centrifugal column, centrifuging at 12000rpm for 2 minutes, and removing liquid in the collecting pipe;
(6) adding 500 μ L Buffer WA into the centrifugal column, centrifuging at 12000rpm for 1min, and discarding the liquid in the collecting tube;
(7) adding 700 mu L of Buffer WB into a centrifugal column, centrifuging at 12000rpm for 1min, and discarding liquid in a collecting pipe;
(8) repeating the step (7);
(9) placing the centrifugal column in a collecting pipe, and centrifuging at 12000rpm for 2 min;
(10) placing the centrifugal column in a new 1.5mL centrifuge tube, adding 50-200 μ L sterile water to the center of the centrifugal column membrane, and standing for 5 min;
(11) centrifuging at 12000rpm for 2min, and repeating the step (10) to increase DNA yield;
(12) and (5) detecting by agarose gel electrophoresis.
5. The method for constructing ABCC4 knockout mice by using CRISPR/Cas9 technology according to claim 3, wherein the CRISPR/Cas9 technology comprises the following steps: the primer sequence required by the PCR identification is shown as SEQ ID NO.3 to SEQ ID NO.7 in the sequence table.
6. The method for constructing ABCC4 knockout mice by using CRISPR/Cas9 technology according to claim 5, wherein the CRISPR/Cas9 technology comprises the following steps: the size of a band amplified by the primer F1R1 required by the PCR identification is 434bp, the size of a band amplified by the F1R2 is 592bp, the gene of endogenous mouse Rgs7 is an internal reference, the size of an electrophoresis band of a product obtained by the PCR reaction is 335bp, and 100bp DNA Ladder Marker (Thermo Scientific, SM0242) is used as a reference during agarose gel electrophoresis.
7. The method for constructing ABCC4 knockout mice by using CRISPR/Cas9 technology according to claim 5, wherein the CRISPR/Cas9 technology comprises the following steps: the PCR identification is carried out by configuring a 25 mu LPCR reaction system according to the table 3, taking the extracted rat tail genome DNA as a template, carrying out PCR amplification by using a primer F1R1 according to the table 4, carrying out agarose gel electrophoresis on the obtained product, and obtaining a band with the size of 434bp, namely, identifying the product as positive.
8. The method for constructing ABCC4 knockout mice by using CRISPR/Cas9 technology according to claim 1, wherein the CRISPR/Cas9 technology comprises the following steps: when the PCR identification in the third step is carried out, the electrophoretic bands with sizes of 434bp and 592bp are simultaneously contained, namely, heterozygotes are identified, and the electrophoretic band with the size of 434bp is only identified as homozygotes.
9. Use of an ABCC4 knockout mouse constructed according to the method of any one of claims 1 to 8, wherein: the gene is used for making a slow obstructive pulmonary disease model, and provides a reliable animal model for researching the function of ABCC4 gene in cilium development, the influence of maintaining airway homeostasis and the effect of the gene in the development of slow obstructive pulmonary disease.
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WO2022134014A1 (en) * | 2020-12-22 | 2022-06-30 | 百迈康生物医药科技(广州)有限公司 | Combination of sgrnas targeting ahrr gene and application thereof |
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