CN111996215A - Construction and identification method of systemic Plin1 gene knockout animal model - Google Patents
Construction and identification method of systemic Plin1 gene knockout animal model Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and provides a construction method and an identification method of a systemic Plin1 gene knockout animal model. The construction method comprises the following steps: designing sgRNA sequences aiming at sequences before and after the No. 2 exon of the Plin1 gene, constructing a Plin1 gene knockout vector, and carrying out in vitro transcription to obtain sgRNA; the sgRNA was mixed with Cas9 protein and microinjected into mouse zygotes to obtain systemic Plin1 knockout mice. The project has the beneficial effects that a pair of sgRNA sequences with the highest scores are screened out, so that the success rate of the project is ensured; the large fragment knockout of the No. 2 exon of the Plin1 gene is realized, and the knockout efficiency is improved; 3 primers are designed aiming at the knockout region, so that various genotypes of the mouse are successfully identified, and the condition that 2 primers can not distinguish the genotypes during large fragment knockout is avoided. Provides a convenient, reliable and economic animal model and a good foundation for researching the biological function of PLIN1 and the pathogenesis of obesity-related metabolic diseases.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to construction and an identification method of a systemic Plun 1 gene knockout animal model.
Background
With the rapid development of economy and the improvement of the living standard of people, obesity has become an increasingly serious global health problem. The incidence of overweight and obesity worldwide has increased dramatically over the last decades and has seen a rapid spread in both developed and developing countries. It is widely believed that changes in the living environment have been directly linked to the occurrence of obesity in recent years. Unhealthy eating habits, sedentary lifestyles, lack of exercise, sleeping habits, and changes in culture and population background may all induce obesity. However, in a recent study in foreign countries, it has been found that genetic factors may dominate the development of obesity under changes in the living environment. With the deepening of human whole gene plans and whole genome association researches, dozens of genes related to obesity occurrence are discovered, and the development of a series of researches aiming at the genes is helpful for comprehensively understanding the pathogenesis of obesity and can provide potential targets for the prevention and treatment of obesity.
The Plin1 gene is located in the q26 region of human chromosome 15, has a DNA length of more than 15 kb, comprises 9 exons and 8 introns, has multiple single nucleic acid polymorphism sites which increase the risk of obesity and is considered as a candidate gene related to obesity. In mammals, the Plin1 gene is highly expressed in white adipose tissue, and a single copy encodes perilipin (perilipin 1, PLIN 1) which is completely localized on the surface of a lipid droplet, and has an important role in biosynthesis of the lipid droplet. PLIN1 can enhance the synthesis of Triglyceride (TG) and promote the formation of fat droplets (diameter greater than or equal to 10 um) in single chamber, thereby improving the fat storage capacity of adipose tissue. Also, PLIN1 may be involved in the regulation of lipolysis by interacting with triglyceride Lipase (ATGL) and hormone-sensitive Lipase (HSL). Recent studies have also shown that PLIN1 may also be involved in inflammatory reactions in adipose tissue. At present, the biological function of PLIN1 is not completely clarified, but the down regulation of the gene expression level is closely related to the occurrence of various metabolic diseases such as type 2 diabetes, hypertension, fatty liver and the like, and further research on the function and the action of the PLIN1 gene has important significance for clarifying the pathogenesis of the diseases.
The gene editing technology is a technology for carrying out site-directed modification on a target gene, and is widely applied to the aspects of gene structure and function research, cell genetic engineering modification, new species culture of animals and plants and gene therapy of diseases. In recent years, due to the development of transgenic technology and the need of disease model construction, transgenic animals become a great hot spot in the field of gene editing research. By introducing exogenous genes into the genome of animals by various methods, the exogenous gene segments can realize the modification of animal cell chromosomes, thereby achieving the purpose of stable inheritance in transgenic animals. The construction of the transgenic animal model breaks through the inherent pattern of the natural genome sequence, so that people can deeply research the structure and the function of the gene, and a wider platform is created for the research of the gene and genetic information.
The CRISPR/Cas9 technology is a new gene editing technology in recent years, and compared with a Zinc Finger Nuclease (ZFN) technology and a transcription-like effector nuclease (TALEN) technology, the CRISPR/Cas9 technology is widely applied to the field of gene editing due to the advantages of simple operation, high efficiency and low cost.
The CRISPR/Cas9 system element comprises a Cas9 protein and sgRNA, wherein the Cas9 protein has the activity of two endonucleases, can be combined with a guide sequence sgRNA of a target sequence and guided to a target position by the sgRNA for cutting to cause Double Strand Break (DSB), and mutation is introduced under the action of a DNA damage repair mechanism to realize gene knockout, repair or insertion (figure 1). Since the CRISPR/Cas9 technology emerged, related researchers have made many improvements on the CRISPR/Cas9 technology, and an engineered system capable of achieving editing of a target sequence only through interaction of sgRNA and Cas9 protein is formed. The sgRNA and Cas9 protein in the system can act on a target gene through different vectors, and the gene editing can be conveniently realized no matter the target gene is a plasmid, a lentivirus or a mixture of the sgRNA and the Cas9 protein through direct transfection.
Chinese patent document CN107043787A discloses a construction method for obtaining a MARF1 site-directed mutagenesis mouse model based on CRISPR/Cas 9. Chinese patent document CN104293831A discloses a method for establishing a hypertension mouse model based on CRISPR/Cas9 gene knockout technology. Chinese patent document CN105950639A discloses a preparation method of a staphylococcus aureus CRISPR/Cas9 system and application thereof in constructing a gene modified mouse model. Chinese patent document CN106172238A discloses a method for establishing a miR-124 gene knockout mouse animal model by using CRISPR/Cas9 gene knockout technology and application thereof. Chinese patent document CN 107475300A discloses a method for establishing an Ifit3-eKO1 gene knockout mouse animal model by using CRISPR/Cas9 technology and application thereof. However, no report is found on the construction and identification method of systemic Plin1 gene knockout mouse animal model.
Disclosure of Invention
The invention aims to provide a method for constructing and identifying a systemic Plun 1 gene knockout mouse animal model aiming at the defects in the prior art.
The second purpose of the invention is to provide a pair of sgRNA sequences which target the No. 2 exon of the Plin1 gene based on CRISPR/Cas9 technology.
A third object of the present invention is to provide applications of the sgRNA described above.
In order to achieve the purpose, the invention adopts the technical scheme that: a construction method of a systemic Plin1 gene knockout mouse animal model based on CRISPR/Cas9 gene knockout technology comprises the following steps:
(1) designing 1 pair of sgRNA sequences aiming at sequences before and after the No. 2 exon of PLIN1 gene, and obtaining sgRNA by constructing a PLIN1 gene knockout vector and in vitro transcription;
(2) microinjection of mouse fertilized eggs of Cas9 sgRNA system formed by mixing sgRNA and Cas9 protein, F0Birth and identification of mouse generations, F0Sexual maturity and reproduction of mouse generation, F1Mouse generation identification, F1Male and female inbreeding of generation-positive mice to obtain F2The generation mouse is the systemic Plin1 gene knockout mouse.
Furthermore, the nucleotide sequence of sgRNA is shown in SEQ ID NO. 1 and SEQ ID NO. 2.
The sgRNA screening and obtaining method comprises the following steps:
(1) aiming at the exon sequence of the Plun 1 gene No. 2 of the mouse, the basic information of the Plun 1 gene of the mouse is inquired in NCBI, and a sgRNA sequence pair before and after the exon sequence of the Plun 1 gene No. 2 is designed and identified by applying a line tool http:// crispr.
(2) Evaluating and screening according to the off-target site, the gene factor and the possibility of mismatching, and selecting 1 pair of sgRNA sequences, wherein the nucleotide sequences are shown as SEQ ID NO 1 and SEQ ID NO 2;
(3) selecting a CRISPR gene knockout plasmid pX330 as a vector, and cutting a cutting site of the pX330 plasmid BBS by using a restriction endonuclease BBS to linearize the cutting site;
(4) after sticky ends are added to the 5 'ends of 2 Plin1 sgRNAs, the 5' ends are connected with a linearization vector by using T4 DNA ligase, sgRNA sequences are added at the position of pX330 gRNA scafford to construct Plin1 gene CRISPR/Cas9 knockout vectors Plin1-1 and Plin 1-2; the constructed plasmid vector is transformed into DH5 alpha flora, 2 d later, single colony sequencing and restriction endonuclease ApaLI + NcoI double enzyme digestion method identification are selected, and the vector sequence information is shown in SEQ ID NO 3 and 4;
(5) forward primers SG1-F, SG2-F and a universal downstream primer SG-R are designed according to an sgRNA in-vitro transcription kit, namely a PC1380 specification, the sequences are shown as SEQ ID NO:5,6 and 7, sgRNA transcription is carried out after a transcription template is amplified by PCR, and the size and the integrity of a sgRNA fragment are detected by an agarose gel electrophoresis experiment after the complete transcription and the purification.
The method for identifying the animal model of the Plun 1 gene knockout mouse comprises the following steps:
A. wait for F0Three weeks after the birth of the mouse, extracting tail DNA of the mouse, performing PCR amplification agarose gel electrophoresis by using primers F and R, sequencing and identifying the genotype, wherein the primer sequences are shown as SEQ ID NO. 8 and 9；
B. Selecting F0F obtained by mating generation-positive mouse and wild-type heteromouse1Replacing a mouse, and carrying out PCR amplification agarose gel electrophoresis sequencing by using a primer F and a Wt/He-R to identify the genotype, wherein the primer sequence is shown as SEQ ID NO. 8 and 10;
C. selecting F1Male and female inbreeding of generation-positive mice to obtain F2Mouse replacement according to F1The homozygous mouse is obtained by a mouse genotype identifying method, namely a mouse animal model.
The sgRNA is applied to the establishment of a gene-deficient mouse.
The invention has the beneficial effects that: according to the invention, a pair of sgRNA sequences with the highest scores are screened out by evaluating the front and back sequences of the No. 2 exon of the Plin1 gene through off-target sites, gene factors and the possibility of mismatching, so that the success rate of the project is ensured; meanwhile, the sgRNA is used as a targeting vector, so that large fragment knockout of the No. 2 exon of the Plin1 gene is realized, and the knockout efficiency is improved to a great extent. In addition, 3 primers are designed aiming at the knockout region, so that various genotypes of the mouse are successfully identified, and the condition that 2 primers can not distinguish the genotypes during large fragment knockout is avoided. According to the invention, a CRISPR/Cas9 gene knockout technology is used, large fragment knockout of No. 2 exon of the mouse Plin1 gene is realized for the first time, a mouse animal model of systemic Plin1 gene knockout is obtained, and successful implementation of the project provides a convenient, reliable and economic animal model for researching the biological function of PLIN1 and pathogenesis of obesity-related metabolic diseases.
The invention uses CRISPR/Cas9 gene knockout technology to realize large fragment knockout of No. 2 exon of mouse Plin1 gene for the first time, and carries out genotype identification through a plurality of primers to obtain a mouse animal model with systemic PLIN1 gene knockout. Successful implementation of the project provides a convenient, reliable and economic animal model and a good basis for studying the biological function of PLIN1 and the pathogenesis of obesity-related metabolic diseases.
Drawings
FIG. 1 is a technical scheme for establishing a systemic Plin1 gene knockout mouse model based on CRISPR/Cas9 technology;
FIG. 2 is a schematic diagram of a strategy for knocking out exon 2 of Plin1 gene based on CRISPR/Cas9 technology;
FIG. 3 is a map of pX330 plasmid vector;
FIG. 4 is the sequencing result and enzyme digestion identification electrophoresis chart of the Plun 1 gene knockout vector; in the figure: the upper diagram is a vector sequencing result diagram; the following figure is an enzyme cutting identification electrophoresis chart: m: DNA marker; 1: the enzyme digestion fragment of the Plun 1-1 vector; 2: plin1- -1 vector; 3: the enzyme digestion fragment of the Plun 1-2 vector; 4: plin1- -2 vector;
fig. 5 is an electropherogram of sgRNA in vitro transcription; in the figure: m: DNA marker; 1-3: sgRNA-Plin 1-1; 4-6: sgRNA-Plin 1-2;
FIG. 6 is a diagram of an identification strategy for knockout mice based on exon 2 of the Plin1 gene;
FIG. 7 is F0Generating an electrophoretogram for mouse PCR identification; in the figure: water: blank control; 1-23: numbering the mice; WT: a wild-type mouse; m: DNAmarker;
FIG. 8 is F0An electrophoretogram for comparing sequencing results before and after the generation 8 chimera and performing PCR identification; in the figure: left picture F0The generation 8 chimera is an electrophoretogram identified by the PCR of the Plin1 gene; the right picture is F0Comparing sequencing results of the No. 8 chimera Plun 1 gene before and after;
FIG. 9 is F1Comparing sequencing results before and after the generation of positive gene knockout mice and obtaining an electrophoretogram; in the figure: the upper figure is an electrophoretogram of the PCR identification of the Plun 1 gene of the No. 1,2,4,8,12,13 and 15 mouse, and the lower figure is an alignment of the sequencing results before and after the No. 1,2,4,8,12,13 and 15 Plun 1 gene;
FIG. 10 is F2Electrophorogram of the transgenic knockout mouse; in the figure: the upper diagram is an electrophoresis diagram for identifying the genotype of a mouse No. 1-17 by using a PCR of a primer F and a primer R, and the lower diagram is an electrophoresis diagram for identifying the genotype of the mouse No. 1-17 by using a PCR of a primer F and a Wt/He-R, wherein: white arrows indicate homozygous mouse genotypes.
Detailed Description
The following further describes embodiments of the present invention. So that those skilled in the art can understand the invention, it should be understood that the invention is not limited in scope to the specific embodiments, but that various changes may be apparent to those skilled in the art, which changes are within the spirit and scope of the invention as defined and defined in the claims, and that all inventive concepts utilizing the inventive concepts are protected.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The nomenclature used herein and the laboratory procedures are well known and commonly employed in the art. All operations performed using standard techniques are generally performed according to the product specifications and general technical requirements of the manufacturer of the instrument consumables and the references provided herein.
A method for establishing a systemic Plin1 gene knockout mouse model based on CRISPR/Cas9 gene knockout technology is shown in a technical route shown in figure 1, and comprises the following specific steps:
first, search the basic information of mouse Plun 1 gene in NCBI
(1) Knockout gene name (Ensembl/MGI/GenBank number): plin1 (ENSMUSG 00000030546/1890505/NM _ 001113471.1).
(2) Knockout gene website link (NCBI): https:// www.ncbi.nlm.nih.gov/gene/103968;
(3) knock out targeted exon: exon 2.
Strategy diagram of knocking out Plun 1 gene based on CRISPR/Cas9 technology, as shown in FIG. 2, realizes large fragment knocking out of No. 2 exon of Plun 1 gene.
Thirdly, confirming the upstream and downstream sequence information of the gene knockout site
A line tool (http:// criprp. mit. edu) is applied to design 2 pairs of sgRNA sequences which can recognize sequences before and after the No. 2 exon of the Plin1 gene, analysis is carried out according to off-target sites, gene factors and the possibility of mismatch, a pair of sgRNAs with the highest score are selected and complementary matched with corresponding antisense chains to synthesize, and the sgRNA sequences are shown as SEQ ID NO. 1 and SEQ ID NO. 2.
Fourthly, constructing sgRNA expression vector
CRISPR plasmid pX330 (fig. 3) was selected as the backbone vector. Cutting a pX330 plasmid BBS restriction enzyme site (positions 245 and 267) by using a restriction enzyme BBS to linearize the plasmid BBS restriction enzyme site, incubating for 30 min at 37 ℃, performing 1% agarose gel electrophoresis, and recovering a linearized vector by using a recovery kit; adding sticky ends to the 5' ends of the sgRNAs of 2 Plin1 genes to adjust the concentration to 100 mu M, incubating at 37 ℃ for 30 min in a system containing T4 PNK, then slowly reducing the temperature to 25 ℃ after incubating at 95 ℃ for 5 min, and annealing and phosphorylating the sgRNAs; diluting annealed and phosphorylated sgRNA to 1 mu M, then connecting the sgRNA with a recovered linearized vector by using T4 DNA ligase, incubating at 37 ℃ for 1 h, and then staying overnight at 16 ℃ to construct Plun 1 gene CRISPR/Cas9 knockout vectors Plun 1-1 and Plun-2, wherein the vector information is shown as SEQ ID NO:3, 4;
pX330 vector linearization reaction system
sgRNA and pX330 vector connection reaction system
Fifthly, the constructed sgRNA vector is transformed into a DH5 alpha escherichia coli flora, a single colony is picked after 2 d of routine culture, plasmid DNA is extracted for sequencing, the vector is identified through a restriction endonuclease ApaLI + NcoI double-enzyme cutting method, the sequencing and enzyme cutting results are shown in figure 4, the sgRNA sequence is successfully added to the position of pX330 gRNA scafford, and 5 fragments (497, 527, 959, 1246 and 5195 bp) with expected sizes are obtained.
Six, sgRNA in vitro transcription
Designing a forward primer SG1-F, SG2-F and a universal downstream primer SG-R of an in-vitro transcription template according to a sequence of the sgRNA, wherein the sequence is shown as SEQ ID NO:5,6 and 7, carrying out PCR amplification on the sgRNA transcription template, recovering the sgRNA transcription template, carrying out sgRNA transcription according to the instruction of an in-vitro transcription kit PC1380 (purchased from Honghong biotech GmbH, Suzhou) and carrying out a 2% agarose gel electrophoresis experiment to detect the size and the integrity of a sgRNA fragment after the complete transcription and purification; as shown in FIG. 5, the sgRNA in vitro transcript fragment was intact, with a size between 100 and 200 bp, identical to the expected result.
Seven, superovulation and fertilized egg acquisition
Injecting 5-10 UI pregnant mare serum gonadotropin into abdominal cavity of C57BL/6J female mouse of about 8 weeks old, injecting 5-10 UI human chorionic gonadotropin after 46-48 hours to promote superovulation of the mouse, mating with male mouse in cage, picking the female mouse with thrombus on the next day, sterilizing abdomen with 75% ethanol after decapitation, opening abdominal cavity, cutting two lateral oviducts, transferring into M2 culture medium preheated to 37 ℃ and containing 3% hyaluronidase, puncturing the enlarged ampulla of oviduct with fine forceps under an inverted microscope to allow fertilized egg to flow out, standing and digesting for 1-3 minutes to allow fertilized egg to disperse, then washing with preheated M2 culture medium for 3 times, transferring 30-50 fertilized eggs into another culture dish containing fresh M2 culture medium liquid drop, covering with mineral oil, placing on the stage of micromanipulation system, a total of 113 fertilized eggs were obtained.
Eighthly, construction of ligation male mouse and pseudopregnant female mouse
A C57BL/6J male mouse aged about 8 weeks is intraperitoneally injected with 0.15 ml of 5% chloral hydrate, after the mouse is completely anesthetized, the middle position of two hind limbs of the mouse is sterilized by 75% ethanol, an opening of about 1 cm is transversely cut by an ophthalmological scissors, fat pads around the testis of the mouse are clamped by blunt forceps to pull out the vas deferens, epididymis, testis and the like, the vas deferens is gently lifted, and the vas deferens on the other side is cut by scissors (one section is cut off), and the vas deferens on the other side is cut by the same operation. The completion of the occlusion of the testis, etc. back into the mouse and the replacement, suturing the mouse and transferring it to a new mouse cage. After the mice revive, the mice are fed normally for 2 weeks to completely recover, and ligation male mice are obtained. The ligated male mice and normal 8-week-old female mice are combined into a cage, and the next day, the tied female mice are picked to be pseudopregnant female mice.
Ninth, microinjection and embryo transplantation of fertilized egg
Mixing the sgRNA obtained in the sixth step with the purchased Cas9 protein, adjusting the concentration to a proper concentration, injecting 1-2 pl into a germ cell pronucleus by a microscopic injection needle, after all germ cells are injected, picking the germ cells surviving injection under an inverted microscope, transferring the germ cells into a preheating chamber to preheatPlacing in M16 culture medium at 37 deg.C, placing in cell culture box at 37 deg.C and 5% CO2Culturing for 2 h under the condition, and keeping 63 fertilized eggs alive. And C, anaesthetizing the pseudopregnant female mouse obtained in the step eight by using 5% chloral hydrate, cutting a 1-2 cm small opening from the last rib along the back midline after disinfecting the back by using 75% ethanol, slowly pulling out the ovary, the oviduct and the uterus after finding a fat pad by using tweezers, tearing off the thin wall coating the ovary and the oviduct by using fine tweezers under a body microscope, exposing the umbrella end of the oviduct, blowing the cultured fertilized egg into the oviduct by using an egg moving needle through the umbrella end of the oviduct, resetting tissues such as the ovary and the fat pad of the mouse after the same operation is carried out on the lateral oviduct, suturing the opening, completing embryo transplantation, and transplanting 4 pseudopregnant female mice together. F is the mouse delivered after 19-21 days after the female pseudopregnant mouse recovers0Mouse generation.
Based on the strategy diagram of mouse genotype identification of exon 2 of knockout Plun 1 gene, F, R and Wt/He-R3 primers are designed to be used for identifying mutant type and wild type mice respectively, as shown in FIG. 6. The primer sequence is shown as SEQ ID NO:8,9 and 10.
Eleven treat F0Three weeks after birth, extracting tail DNA of the mouse, performing PCR amplification agarose gel electrophoresis by using primers F and R to identify the genotype, and determining the genotype by using the electrophoresis result as shown in figure 7, wherein 24 mice are born in total, and 12 mice are positive (4 and 8). The size of the knockout fragment is about 700 bp.
Twelve selecting F0The generation 8 is subjected to electrophoresis and sequencing identification, and the result is shown in FIG. 8, and 741bp containing the No. 2 exon sequence of the Plin1 gene is knocked out; f is to be0Male mice No. 8 and No. 11 were mated with wild-type female mice, respectively, to obtain F1Mouse generation, extracting mouse tail DNA, performing PCR amplification agarose gel electrophoresis by using primers F, R and Wt/He-R, and sequencing to identify genotype, wherein the electrophoresis and sequencing results are shown in figure 9; the offspring of No. 11 mice No. 1,2 and 4 (both male and 8 mouse offspring No. 8), and the offspring of No. 12,13 and 15 (both female) mice are all F1A positive heterozygous mouse; mouse No. 11F1Generation Plin1 gene knockout fragment size and number 8 mouse and F thereof1The generation is slightly different, and 747 bp is deleted.
Thirteen, selecting F1The positive heterozygote mouse No. 8 is inbred with the male and female mice No. 12,13 and 15, and the inbreeding is F after the mouse is born2Substituting a mouse, extracting tail DNA of the mouse, amplifying agarose gel electrophoresis by using primers F and R and Wt/He-RPCR to identify the genotype, wherein the electrophoresis result is shown in figure 10, and 17 mice are born together, wherein the No. 12 and No. 13 (1 male and 1 female) are all homozygotic mice; no. 11 and No. 17 (2) are all wild type mice; no. 1-10, No. 14-16 (6 male and 7 female) mice are all heterozygote mice, and basically accord with Mendelian inheritance law.
PCR reaction system
PCR reaction conditions
Fourteen, F2The mouse with genotype homozygote in the generation is the mouse animal model.
Sequence listing
<110> university of Shanxi medical science
<120> construction and identification method of systemic Plun 1 gene knockout animal model
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ttggagggcg aatgttacat 20
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gaacttcagg cgtatgaccc 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca 60
aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa 120
atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta 180
aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata 240
tcttgtggaa aggacgaaac accgttggag ggcgaatgtt acatgtttta gagctagaaa 300
tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 360
tttttttcta gacgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 420
cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt 480
ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 540
gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac 600
cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 660
cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat 720
tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg 780
gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc 840
ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg 900
gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg 960
ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt 1020
actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa 1080
gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca 1140
cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac 1200
gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca 1260
aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc 1320
ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg 1380
cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg 1440
atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc 1500
gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc 1560
aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg 1620
gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc 1680
taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac 1740
aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac 1800
ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag 1860
ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac 1920
gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc 1980
cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc 2040
ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc 2100
aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc 2160
gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga 2220
gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag 2280
caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac 2340
aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc 2400
agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag 2460
gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac 2520
ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa 2580
gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc 2640
atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga 2700
aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc 2760
gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg 2820
ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg 2880
aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc 2940
atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac 3000
tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc 3060
aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg 3120
gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag 3180
gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg 3240
atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc 3300
aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc 3360
ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac 3420
atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg 3480
gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc 3540
gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac 3600
cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc 3660
atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag 3720
aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa 3780
ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg 3840
aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc 3900
gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg 3960
aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc 4020
ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc 4080
acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac 4140
aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg 4200
aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc 4260
tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag 4320
ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag 4380
gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag 4440
accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc 4500
gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg 4560
agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa 4620
gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac 4680
cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc 4740
aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc 4800
atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac 4860
aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa 4920
aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc 4980
ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc 5040
tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac 5100
gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg 5160
gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag 5220
aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt 5280
gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg 5340
atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc 5400
gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc 5460
ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 5520
cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 5580
atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 5640
ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc 5700
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 5760
ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 5820
gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 5880
gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 5940
cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc 6000
cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 6060
tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 6120
aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 6180
ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 6240
actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta 6300
ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 6360
gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 6420
gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 6480
cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 6540
atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 6600
catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 6660
ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 6720
ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 6780
cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 6840
ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 6900
tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 6960
cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 7020
actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 7080
aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 7140
tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 7200
ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 7260
tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 7320
gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 7380
gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 7440
tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg 7500
gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 7560
ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 7620
gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 7680
aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 7740
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 7800
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 7860
tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 7920
gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 7980
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 8040
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 8100
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 8160
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 8220
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 8280
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 8340
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 8400
acggttcctg gccttttgct ggcc 8424
<210> 4
<211> 8424
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca 60
aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa 120
atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta 180
aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata 240
tcttgtggaa aggacgaaac accggaactt caggcgtatg acccgtttta gagctagaaa 300
tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 360
tttttttcta gacgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 420
cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt 480
ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 540
gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac 600
cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 660
cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat 720
tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg 780
gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc 840
ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg 900
gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg 960
ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt 1020
actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa 1080
gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca 1140
cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac 1200
gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca 1260
aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc 1320
ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg 1380
cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg 1440
atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc 1500
gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc 1560
aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg 1620
gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc 1680
taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac 1740
aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac 1800
ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag 1860
ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac 1920
gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc 1980
cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc 2040
ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc 2100
aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc 2160
gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga 2220
gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag 2280
caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac 2340
aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc 2400
agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag 2460
gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac 2520
ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa 2580
gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc 2640
atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga 2700
aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc 2760
gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg 2820
ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg 2880
aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc 2940
atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac 3000
tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc 3060
aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg 3120
gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag 3180
gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg 3240
atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc 3300
aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc 3360
ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac 3420
atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg 3480
gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc 3540
gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac 3600
cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc 3660
atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag 3720
aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa 3780
ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg 3840
aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc 3900
gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg 3960
aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc 4020
ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc 4080
acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac 4140
aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg 4200
aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc 4260
tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag 4320
ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag 4380
gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag 4440
accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc 4500
gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg 4560
agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa 4620
gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac 4680
cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc 4740
aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc 4800
atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac 4860
aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa 4920
aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc 4980
ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc 5040
tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac 5100
gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg 5160
gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag 5220
aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt 5280
gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg 5340
atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc 5400
gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc 5460
ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 5520
cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 5580
atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 5640
ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc 5700
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 5760
ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 5820
gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 5880
gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 5940
cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc 6000
cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 6060
tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 6120
aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 6180
ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 6240
actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta 6300
ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 6360
gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 6420
gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 6480
cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 6540
atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 6600
catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 6660
ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 6720
ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 6780
cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 6840
ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 6900
tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 6960
cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 7020
actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 7080
aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 7140
tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 7200
ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 7260
tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 7320
gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 7380
gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 7440
tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg 7500
gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 7560
ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 7620
gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 7680
aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 7740
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 7800
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 7860
tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 7920
gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 7980
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 8040
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 8100
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 8160
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 8220
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 8280
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 8340
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 8400
acggttcctg gccttttgct ggcc 8424
<210> 5
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ttaatacgac tcactatagg gttggagggc gaatgttaca tgttttagag ctagaaatag 60
<210> 6
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttaatacgac tcactatagg ggaacttcag gcgtatgacc cgttttagag ctagaaatag 60
<210> 7
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aaaaaaagca ccgactcggt gccacttttt c 31
<210> 8
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ctgagagaag gcttaacctt gctgg 25
<210> 9
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
agctttccat cctgcaagtg agtcag 26
<210> 10
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aggtggcaag gacagagaca gtgag 25
Claims (6)
1. A method for constructing a systemic Plin1 gene knockout mouse animal model is characterized in that: the method is based on CRISPR/Cas9 gene knockout technology and comprises the following steps:
(1) designing 1 pair of sgRNA sequences aiming at sequences before and after the No. 2 exon of the Plin1 gene, and obtaining sgRNA by constructing a Plin1 gene knockout vector and in vitro transcription;
(2) microinjection of mouse fertilized eggs of Cas9 sgRNA system formed by mixing sgRNA and Cas9 protein, F0Birth and identification of mouse generations, F0Sexual maturity and reproduction of mouse generation, F1Mouse generation identification, F1Male and female inbreeding of generation-positive mice to obtain F2The generation mouse is the systemic Plin1 gene knockout mouse.
2. The method of constructing a systemic Plin1 gene knockout mouse animal model according to claim 1, characterized in that: the nucleotide sequence of the sgRNA is shown in SEQ ID NO 1 and SEQ ID NO 2.
3. The method of constructing a systemic Plin1 gene knockout mouse animal model according to claim 1 or 2, characterized in that: the sgRNA screening and obtaining method comprises the following steps:
(1) aiming at the exon sequence of the Plun 1 gene No. 2 of the mouse, the basic information of the Plun 1 gene of the mouse is inquired in NCBI, and a sgRNA sequence pair before and after the exon sequence of the Plun 1 gene No. 2 is designed and identified by applying a line tool http:// crispr.
(2) Evaluating and screening according to the off-target site, the gene factor and the possibility of mismatching, and selecting 1 pair of sgRNA sequences, wherein the nucleotide sequences are shown as SEQ ID NO 1 and SEQ ID NO 2;
(3) selecting a CRISPR gene knockout plasmid pX330 as a vector, and cutting the enzyme cutting sites of the pX330 plasmid BBS, namely 245 th and 267 th positions by using a restriction enzyme BBS to linearize the plasmid;
(4) after sticky ends are added to the 5 'ends of 2 Plin1 sgRNAs, the 5' ends are connected with a linearization vector by using T4 DNA ligase, sgRNA sequences are added at the position of pX330 gRNA scafford to construct Plin1 gene CRISPR/Cas9 knockout vectors Plin1-1 and Plin 1-2; the constructed plasmid vector is transformed into DH5 alpha flora, 2 d later, single colony sequencing and restriction endonuclease ApaLI + NcoI double enzyme digestion method identification are selected, and the vector sequence information is shown in SEQ ID NO 3 and 4;
(5) forward primers SG1-F, SG2-F and a universal downstream primer SG-R are designed according to an sgRNA in-vitro transcription kit, namely a PC1380 instruction, and the sequences are shown as SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: and 7, carrying out sgRNA transcription after the transcription template is amplified by PCR, and carrying out an agarose gel electrophoresis experiment to detect the size and the integrity of the sgRNA fragment after the sgRNA is completely transcribed and purified.
4. The method of constructing a systemic Plin1 gene knockout mouse animal model according to claim 1, wherein the method comprises the steps of: the method also includes identifying a Plin1 gene knockout mouse animal model.
5. The method of constructing a systemic Plin1 gene knockout mouse animal model according to claim 4, wherein: the method for identifying the animal model of the Plun 1 gene knockout mouse comprises the following steps:
A. wait for F0Three weeks after the birth of the mouse, extracting tail DNA of the mouse, performing PCR amplification by using primers F and R, performing agarose gel electrophoresis, sequencing and identifying the genotype, wherein the primer sequence is shown as SEQ ID NO:8, SEQ ID NO: 9 is shown in the figure;
B. selecting F0F obtained by mating generation-positive mouse and wild-type heteromouse1Mouse generation, PCR amplification by using a primer F, Wt/He-R, agarose gel electrophoresis, sequencing and genotype identification, wherein the primer sequence is shown as SEQ ID NO:8, SEQ ID NO: 10 is shown in the figure;
C. selecting F1Male and female inbreeding of generation-positive mice to obtain F2Mouse replacement according to F0The homozygous mouse is obtained by a mouse genotype identifying method, namely a mouse animal model.
6. The method of claim 2 for constructing a systemic Plin1 knockout mouse animal model, the sgRNA is used for establishing knockout mice.
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| CN112553199A (en) * | 2020-11-30 | 2021-03-26 | 深圳市人民医院 | Construction method and application of snhg17-KO gene knockout mouse model |
| CN112553254A (en) * | 2020-12-23 | 2021-03-26 | 成都药康生物科技有限公司 | IL10 gene knockout mouse model and construction method and application thereof |
| CN112852803A (en) * | 2021-02-05 | 2021-05-28 | 上海市第六人民医院 | Construction method and application of systemic EEPD1 knockout animal model |
| CN113913425A (en) * | 2020-12-22 | 2022-01-11 | 百迈康生物医药科技(广州)有限公司 | sgRNA combination of targeted AHRR gene and application thereof |
| CN115058456A (en) * | 2022-06-23 | 2022-09-16 | 五邑大学 | Construction method and application of HPRT gene knockout animal model |
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| CN112852803A (en) * | 2021-02-05 | 2021-05-28 | 上海市第六人民医院 | Construction method and application of systemic EEPD1 knockout animal model |
| CN112852803B (en) * | 2021-02-05 | 2023-10-31 | 上海市第六人民医院 | Method for constructing systemic EEPD1 knockout animal model and application thereof |
| CN115058456A (en) * | 2022-06-23 | 2022-09-16 | 五邑大学 | Construction method and application of HPRT gene knockout animal model |
| CN115058456B (en) * | 2022-06-23 | 2023-09-19 | 五邑大学 | Construction method and application of animal model with HPRT gene knocked out |
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