CN108251456A - A kind of preparation method of the atherosclerosis mouse model of NOD genetic backgrounds - Google Patents

A kind of preparation method of the atherosclerosis mouse model of NOD genetic backgrounds Download PDF

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CN108251456A
CN108251456A CN201810065368.4A CN201810065368A CN108251456A CN 108251456 A CN108251456 A CN 108251456A CN 201810065368 A CN201810065368 A CN 201810065368A CN 108251456 A CN108251456 A CN 108251456A
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apoe
ldlr
ivt
mouse
atherosclerosis
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CN108251456B (en
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梁银明
张黎琛
卢燎勋
王旭刚
黄蓉
晁天柱
郑前前
罗静
谷妍蓉
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Xinxiang Medical University
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    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

The present invention relates to a kind of preparation methods of the atherosclerosis mouse model of NOD genetic backgrounds, belong to genetic engineering and genetic modification technique field.The present invention for the first time knocks out ApoE the and LDLR genes progress in NOD genetic background mouse simultaneously, obtains the gene knock-out mice model that can generate atherosclerosis of NOD genetic background mouse.The aorta that the present invention obtains the atherosclerosis mouse model of NOD genetic backgrounds has apparent atherosclerotic plaque to be formed, compared with the Atherosclerosis Model C57B/L6 background ApoE knock out mice of traditional classical, its occurring degree is consistent, the method of the present invention provides a kind of gene knock-out mice model of completely new NOD genetic background atherosclerosis for researcher, and the fundamental research and clinical conditions to atherosclerosis have highly important application value.

Description

A kind of preparation method of the atherosclerosis mouse model of NOD genetic backgrounds
Technical field
The present invention relates to a kind of preparation methods of the atherosclerosis mouse model of NOD genetic backgrounds, belong to gene work Journey and genetic modification technique field.
Background technology
Atherosclerosis is one of the main reason for cardiovascular and cerebrovascular disease occurs, its mechanism, which will be furtherd investigate, to be had Help solve the problems, such as that cardiovascular and cerebrovascular disease healing is difficult, the death rate is high.NOD mouse be non-obese diabetes strain, 30 week old When diabetes cumulative life-incidence male and female be respectively 60~80% and 20~30%, female mice has insulin-dependent diabetes mellitus, clinical Symptom and human Type I diabetes quite it is similar, NOD mouse are type-1 diabetes mellitus research and humanized model establish in using the most Extensive mouse species, but NOD genetic background mice gene knockouts models still extremely lack, and are particularly ground in atherosclerosis Study carefully field.
There is result of study to show that NOD genetic backgrounds mouse has very strong resistance to atherosclerosis disease (Keren P,George J,Keren G,Harats D:Non-obese diabetic(NOD)mice exhibit an increased cellular immune response to glycated-LDL but are resistant to high fat diet induced atherosclerosis.Atherosclerosis 2001,157:285-292.), and this reality Test room have early period the result shows that, even if individually knocking out ApoE in NOD genetic background mouse either after LDLR genes, warp Forage feed high in fat is crossed, single-gene knock-out mice is not can induce yet and atherosclerosis disease occurs, so being carried on the back in NOD heredity The gene knockout model of atherosclerosis can be generated by being obtained in scape mouse, will be had a very important significance.
Invention content
The object of the present invention is to provide a kind of preparation methods of the atherosclerosis mouse model of NOD genetic backgrounds, should Method operability is strong, and structure success rate is high, is provided very well for studying atherosclerotic and type-1 diabetes mellitus and its complication Mouse genetic model.
To achieve these goals, the technical solution adopted in the present invention is：
A kind of preparation method of the atherosclerosis mouse model of NOD genetic backgrounds, including：By ApoE in NOD mouse Gene and LDLR genes knock out jointly after to get.
Since NOD genetic backgrounds mouse has very strong resistance to atherosclerosis disease, the present invention in NOD in order to lose The gene knock-out mice model for being obtained in background mice and generating atherosclerosis is passed, it for the first time will be in NOD genetic background mouse ApoE and LDLR genes progress knock out simultaneously.Pass through further experimental study, the results showed that ApoE and LDLR is dual-gene to be lacked The mouse of mistake, after induction is fed by high lipid food, the aorta of mouse has apparent atherosclerotic plaque to be formed, with The Atherosclerosis Model C57B/L6 background ApoE knock out mice of traditional classical is compared, and occurring degree is consistent, shows The gene knock-out mice model of NOD genetic background atherosclerosis is built successfully.The present invention realizes in NOD mouse for the first time The dual-gene knockouts of ApoE and LDLR, and obtain the stabilization genetic model that can realize atherosclerosis-inducing disease high in fat.
When knocking out the ApoE genes using CRISPR/Cas9 systems, 3 target sequences are selected, it is as follows respectively：
ApoE-sgRNA1：5‘-CCTAGCCGAGGGAGAGCCGG AGG-3’；
ApoE-sgRNA2：5‘-GTAATCCCAGAAGCGGTTCA GGG-3’；
ApoE-sgRNA3：5‘-CTTCTGGGATTACCTGCGCT GGG-3’.
When knocking out the LDLR genes using CRISPR/Cas9 systems, 3 target sequences are selected, it is as follows respectively：
Llr-sgRNA1：5‘-TCCATCACACACAAACTGCG GGG-3’；
LDLR-sgRNA2：5‘-AGTTGCAGCGGAAGTGGGCG GGG-3’；
LDLR-sgRNA3：5‘-CAGTCTTTGGGCCTGCGACG GGG-3’.
The ApoE genes selected in the present invention and the specific target practice site of LDLR genes have very high shear efficiency.
When knocking out ApoE genes and LDLR genes jointly, 7 primers are synthesized, 6 double-stranded DNAs are obtained by PCR amplification； 6 double-stranded DNAs are transcribed into 6 sgRNAmRNA, 6 sgRNA mRNA and Cas9mRNA co-injections are entered small In mouse fertilized egg cell, dual-gene knockout fertilized egg cell is obtained；The dual-gene knockout fertilized oocyte is entered into vacation Pregnant female mice fallopian tubal, can obtain F0 mouse after birth；
The primer difference is as follows：
ApoE-IVT-1：TTAATACGAC TCACTATAGG GCCTAGCCGA GGGAGAGCCG GGTTTTAGAG CTAGAAATAG CAAG；
ApoE-IVT-2：TTAATACGAC TCACTATAGG GGTAATCCCA GAAGCGGTTC AGTTTTAGAG CTAGAAATAG CAAG；
ApoE-IVT-3：TTAATACGAC TCACTATAGG GCTTCTGGGA TTACCTGCGC TGTTTTAGAG CTAGAAATAG CAAG；
LDLR-IVT-1：TTAATACGAC TCACTATAGG GTCCATCACA CACAAACTGC GGTTTTAGAG CTAGAAATAG CAAG；
LDLR-IVT-2：TTAATACGAC TCACTATAGG GAGTTGCAGC GGAAGTGGGC GGTTTTAGAG CTAGAAATAG CAAG；
LDLR-IVT-3：TTAATACGAC TCACTATAGG GCAGTCTTTG GGCCTGCGAC GGTTTTAGAG CTAGAAATAG CAAG；IVT-R：AAAAAAGCACCGACTCGGTGCC；
By the IVT-R respectively with ApoE-IVT-1, ApoE-IVT-2, ApoE-IVT-3, LDLR-IVT-1, LDLR- For PCR amplification after IVT-2 and LDLR-IVT-3 combinations, 6 double-stranded DNAs are obtained.
The present invention is practiced shooting, and obtain biradical simultaneously using 6 sgRNA for NOD mouse ApoE and LDLR genes Because of the knock out mice of large fragment deletion, this six sgRNA genes that can ensure to be practiced shooting can thoroughly lose biology work( Energy.
The Cas9 mRNA be by pST1374-cas9 carriers through Agel enzymes linearisation after, then in-vitro transcription obtain.
During the PCR amplification, the template used is pX330 plasmid.T7 promoter sequences are added into synthesis in the present invention Primer sequence 5 ' end, pass through PCR amplification, obtain with T7 promoters double-stranded DNA.
The final concentration of 6 sgRNA mRNA and Cas9 mRNA is 50ng/ μ L.
The dual-gene knockout fertilized oocyte is entered into false pregnancy female mice fallopian tubal, F0 mouse are obtained, by identification Selection ApoE and LDLR is dual-gene while the F0 mouse that knock out mate to obtain with NOD mouse F1 generation hybrid mice, then by F1 generation Hybrid mice is selfed, filter out ApoE and LDLR it is dual-gene and meanwhile knock out homozygotic individual be NOD genetic backgrounds Atherosclerosis mouse model.
The identification of the ApoE genes uses following primer：
ApoE-F PCR-F：5‘-CGAGGGTGAAAGAGCTGGAC-3’；
ApoE-F PCR-R：5‘-GCCTCCAGACCCACTTCAAA-3’.Preferably, the ApoE-F PCR-F primers 5 ' are held and are marked using fluorescence, is specifically marked using HEX fluorescence.
The identification of the LDLR genes uses following primer：
LDLR-F PCR-F：5‘-AGACGTGCTCCCAGGATG-3’；
LDLR-F PCR-R：5‘-GTTCTGTGGCCACTCATCG-3.Preferably, the 5 of the LDLR-F PCR-F primers It ' holds and is marked using fluorescence, is specifically marked using FAM fluorescence.
The present invention utilizes the glimmering of double fluorescing systems when carrying out genotype detection to ApoE and LDLR Double knockout mices Light round pcr, can be by a PCR process, while obtains the genotype of 2 target genes, be greatly saved reagent and when Between cost.
The atherosclerosis mouse model of above-mentioned NOD genetic backgrounds is in the fundamental research of atherosclerosis Application.Or the atherosclerosis mouse model of the NOD genetic backgrounds is controlled in studying atherosclerotic clinical diagnosis Application in treatment.
Using CRISPR/Cas9 systems ApoE and LDLR to be knocked out simultaneously dual-gene at home and abroad in NOD genetic background mouse It still belongs to the first time, there is very high originality and very important basic research and actual application value, it is also athero- for research artery Hardening and type-1 diabetes mellitus and its complication provide good mouse genetic model.The preparation method of the present invention is carried for researcher For a kind of gene knock-out mice model of completely new NOD genetic background atherosclerosis, the basis of atherosclerosis is managed There is highly important application value by research and clinical conditions such as diabetic complication etc..
Description of the drawings
Fig. 1 builds flow chart for NOD genetic background atherosclerosis mouse model；
Fig. 2 is the result figure for carrying out genotype detection to obtained Founder mouse using double fluorescent PCR systems；
Fig. 3 is the result figure for carrying out genotype detection to obtained #1 mouse using Sanger sequencings；
Fig. 4 is the result figure for carrying out genotype detection to obtained #2 mouse using Sanger sequencings；
Fig. 5 is the western blotting expressing quantities of NOD genetic background atherosclerosis mouse models Testing result figure；
Fig. 6 is that genotype mice feeds the later aorta oil red O stain comparison diagram of high lipid food different time；
Fig. 7 is that genotype mice feeds the later aorta oil red O stain result statistics of high lipid food different time Comparison diagram；
Fig. 8 feeds 20 weeks later heart frozen section oil red O stain results pair of high lipid food for genotype mice Than figure；
Fig. 9 feeds high lipid food 20 weeks later heart frozen sections and H＆E coloration results pair for genotype mice Than figure；
Figure 10 is the area oil red O stain system that genotype mice feeds patch in 20 weeks later hearts of high lipid food Count comparison diagram.
Specific embodiment
With reference to specific embodiment, the present invention is described in further detail.NOD in following embodiment and test example Mouse is provided by Nanjing University-Nanjing biological medicine research institute, and B6 mouse tie up the limited public affairs of tonneau China's experimental animal technology by Beijing Department provides, and ApoE single-genes knock out B6 mouse, and by the purchase of U.S. JAX laboratories, (it can suffer from artery congee after high lipid food nursing Sample hardens, and is the tool mouse of common classics in laboratory research atherosclerosis), used high lipid food is purchased from Research Diets companies, number D12108C.PX330 Plasmid DNA is purchased from Addgene；PST1374-cas9 plasmids are by yellow army Professor is presented.Other experimental article unexplained references, are commercial goods.
Embodiment 1
The preparation method (flow is as shown in Figure 1) of the atherosclerosis mouse model of NOD genetic backgrounds in the present embodiment, Include the following steps：
First, according to CRISPR/Cas9 system principles, using CRISPOR Photographing On-line softwares, determine that NOD mouse wait to knock out Gene A poE (Gene ID：And LDLR (Gene ID 88057)：96765) specific target sites ApoE-sgRNA1, ApoE- SgRNA2, ApoE-sgRNA3 and LDLR-sgRNA1, LDLR-sgRNA2, LDLR-sgRNA3.
In mouse genome database ensembl (http://asia.ensembl.org) in find mouse ApoE (Transcript ID：) and LDLR (Transcript ID ENSMUST00000174064.8： ENSMUST00000034713.8) gene DNA sequence, then using Photographing On-line software CRISPOR (http:// Crispor.tefor.net/crispor.cgi), exon3 (the exon ID in mouse ApoE genes are determined： ) and the exon4 of LDLR genes (exon ID ENSMUSE00000231320：ENSMUSE00000217581 3 spies are respectively selected in) Target sequence of the different in nature site as sgRNA, these three target sequences are respectively：
ApoE-sgRNA1：CCTAGCCGAGGGAGAGCCGG AGG (as shown in SEQ ID NO.1)；
ApoE-sgRNA2：GTAATCCCAGAAGCGGTTCA GGG (as shown in SEQ ID NO.2)；
ApoE-sgRNA3：CTTCTGGGATTACCTGCGCT GGG (as shown in SEQ ID NO.3)；
LDLR-sgRNA1：TCCATCACACACAAACTGCG GGG (as shown in SEQ ID NO.4)；
LDLR-sgRNA2：AGTTGCAGCGGAAGTGGGCG GGG (as shown in SEQ ID NO.5)；
LDLR-sgRNA3：CAGTCTTTGGGCCTGCGACG GGG (as shown in SEQ ID NO.6).
2nd, 6 sgRNA mRNA are obtained.
1) 7 primers (hundred Li Ge Bioisystech Co., Ltd of Shanghai) are ordered：
ApoE-IVT-1：TTAATACGAC TCACTATAGG GCCTAGCCGA GGGAGAGCCG GGTTTTAGAG CTAGAAATAG CAAG (as shown in SEQ ID NO.7)；
ApoE-IVT-2：TTAATACGAC TCACTATAGG GGTAATCCCA GAAGCGGTTC AGTTTTAGAG CTAGAAATAG CAAG (as shown in SEQ ID NO.8)；
ApoE-IVT-3：TTAATACGAC TCACTATAGG GCTTCTGGGA TTACCTGCGC TGTTTTAGAG CTAGAAATAG CAAG (as shown in SEQ ID NO.9)；
LDLR-IVT-1：TTAATACGAC TCACTATAGG GTCCATCACA CACAAACTGC GGTTTTAGAG CTAGAAATAG CAAG (as shown in SEQ ID NO.10)；
LDLR-IVT-2：TTAATACGAC TCACTATAGG GAGTTGCAGC GGAAGTGGGC GGTTTTAGAG CTAGAAATAG CAAG (as shown in SEQ ID NO.11)；
LDLR-IVT-3：TTAATACGAC TCACTATAGG GCAGTCTTTG GGCCTGCGAC GGTTTTAGAG CTAGAAATAG CAAG (as shown in SEQ ID NO.12)；
IVT-R：AAAAAAGCACCGACTCGGTGCC (as shown in SEQ ID NO.13).
2) it ' holds, by PCR amplification, obtains and start with T7 in 5 that T7 promoter sequences are added into the primer sequence of synthesis The double-stranded DNA of son, specially：
PCR reaction systems are：Template is pX330 Plasmid DNA, adds in 10ng；(10 μ of sense primer ApoE-IVT-1,2,3 M) and LDLR-IVT-1,2,3 (10 μM) are separately added into 2 μ L；Downstream universal primer IVT-R (10 μM) is separately added into 2 μ L；2×Taq Master Mix (being purchased from vazyme, P111-01) add in 25 μ L；Supplement H2O to 50 μ L of total volume.
PCR response procedures are：94 DEG C, 5min；94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C, 30s；72 DEG C, 10min；Recurring number It is 30 times.After PCR EPs (end of program), using invitrogen PCR QIAquick Gel Extraction Kits (invitrogen, K220001), PCR is purified Product.
3) in-vitro transcription obtains sgRNA mRNA：In-vitro transcription kit uses NEB kits (NEB, E2050S), in detail Reaction step is as follows：It is required according to kit, prepares reaction system, sequentially add 10 μ L of NTP Buffer Mix；Template DNA 1μg；T7RNA Polymerase Mix 2μL；Supplement H2O to 30 μ L of total volume；It is placed in 37 DEG C of reactions to be incubated 4 hours, obtains body Outer transcription product；Recycling band is required to obtain to specifications using Ambion RNA purification kits (Ambion, AM1909) sgRNA mRNA。
3rd, Cas9 mRNA are obtained.
1) Cas9 is extracted using high-purity plasmid extraction kit (TIANGEN Biotech (Beijing) Co., Ltd., DP116) Then vector plasmid DNA uses AgeI restriction enzymes (NEB, R3552S) digested plasmid DNA using following system：Plasmid DNA 10μg；CutSmart Buffer 20μL；2 μ L of AgeI restriction enzymes；Supplement H2O to 200 μ L of total volume.Use envelope Chewing-gum seals nozzle, is subsequently placed in 37 DEG C of incubator reaction overnights.
Digestion products are detected by agarose gel electrophoresis, single band is presented after digestion products electrophoresis, shows carrier matter Grain DNA linearisations are completed.It is produced using DNA QIAquick Gel Extraction Kits (TIANGEN Biotech (Beijing) Co., Ltd., DP205) purifying digestion Object obtains Cas9 in-vitro transcription template DNAs.
2) in-vitro transcription obtains Cas9 mRNA：In-vitro transcription kit uses invitrogen kits (invitrogen, AMB1345-5), detailed reaction step are as follows：It is required according to kit, prepares reaction system, sequentially add T72*NTP/ARCA 10μL；10*T7Reaction Buffer 2μL；1 μ g of Cas9 in-vitro transcriptions template DNA；T7 Enzyme Mix 2μL；Supplement H2O (invitrogen, 10977015) to 20 μ L of total volume；It is placed in 37 DEG C of reactions to be incubated 2 hours, Ran Houjia Enter 1 μ L TURBO DNase, after abundant mixing, be placed in 37 DEG C of reactions and be incubated 15min；Next again into more than reaction product Tailings reactions system, 20 μ L of mMESSAGEmMACHINE T7 Ultra reaction products are prepared in the following order；H2O (invitrogen, 10977015) 36 μ L；5*E-PAP Buffer 20μL；25mM MnCl210μL；ATP Solution 10 μL；4 μ L of E-PAP after abundant mixing, are placed in 37 DEG C of reactions and are incubated 45min.After obtaining in-vitro transcription product, use Ambion RNA purification kits (Ambion, AM1909) recycling Cas9 mRNA.
4th, by 6 sgRNA mRNA and Cas9 mRNA of acquisition by micro-injection system inject NOD background mices by Spermiovum.
By injecting hormone to 8 week old NOD background mices female mices, acquisition fertilized eggs are thin after super ovulation and mating Born of the same parents.6 sgRNA mRNA and Cas9 mRNA are diluted to end using the ultra-pure water (invitrogen, 10977015) of no RNA enzyme Concentration is 50ng/ μ L, is injected into NOD background mice fertilized egg cells by micro-injection system after mixing, is placed in culture Case (37 DEG C, CO2After a concentration of 5%) culture 1 hour, the cell of survival is selected, it is defeated to be implanted into false pregnancy ICR mouse female mices In oviduct.27 fertilized egg cells of co-injection in the present embodiment are survived 12, are transplanted 1 false pregnancy female mice, are finally obtained 2 Founder mouse.
5th, Founder murine genes type is identified.
Murine genes type is detected by double fluorescent PCRs：Obtain 2 Founder mouse are cut into rat-tail, extract DNA, then Template DNA is utilized it as, while fluorescent PCR amplification is carried out using 2 pairs of primers, is using primer sequence：
ApoE-F PCR-F：5 '-CGAGGGTGAAAGAGCTGGAC-3 ' (as shown in SEQ ID NO.14,5 ' end uses HEX fluorescence is marked)；
ApoE-F PCR-R：5 '-GCCTCCAGACCCACTTCAAA-3 ' (as shown in SEQ ID NO.15)；
LDLR-F PCR-F：(as shown in SEQ ID NO.16,5 ' hold and use FAM 5 '-AGACGTGCTCCCAGGATG-3 ' Fluorescence is marked)；
LDLR-F PCR--R：5 '-GTTCTGTGGCCACTCATCG-3 ' (as shown in SEQ ID NO.17)；Wherein ApoE Wild type DNA profiling expanding fragment length is 268bp, and LDLR wild type DNA profilings expanding fragment length is 230bp.
PCR reaction systems are：Template is rat-tail DNA, 20ng；Each primer respectively adds in 2 μ L；2×Taq Master Mix (being purchased from vazyme, P111-01) adds in 25 μ L；Supplement H2O to 50 μ L of total volume.
PCR response procedures are：94 DEG C, 5min；94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C, 30s；72 DEG C, 10min；Recurring number It is 30 times.
After obtaining PCR product, 2 μ L products is taken if there is band, then PCR product to be diluted 200 times for electrophoresis detection After, PCR product after formamide, the dilution and standard molecular weight internal reference DNA are mixed, it is enterprising that denaturation is placed on sequenator As a result row Capillary Electrophoresis carries out data analysis using Genemapper softwares, directly obtain the size of PCR product, and wild After the PCR product size of type control is compared, the specific number that base is inserted into or is lacked is obtained.
The results are shown in Figure 2, compares and is compared with wild-type amplification product, it is found that 2 Founder mouse (are ordered respectively Entitled #1 and #2) amplified fragments compared with wild type (such as Fig. 2-A), lacked.#1 mouse Apeo gene locis two Chromosome lacks 78 bases, and two chromosomes of LDLR gene locis lack 129 bases (such as Fig. 2-B), #2 mouse APOE gene locis item chromosome lacks 7 bases, 79 bases of another chromosome deficiency；One dye of LDLR gene locis Colour solid lacks 14 bases, 127 bases of another chromosome deficiency (such as Fig. 2-C).
Detection murine genes type is cloned by TA.It is using primer sequence：
ApoE-S PCR-F：5 '-AAGGCAGGAGGATTCAAGGC-3 ' (as shown in SEQ ID NO.18)；
ApoE-S PCR-R：5 '-GCCTAGTCTCGGCTCTGAAC-3 ' (as shown in SEQ ID NO.19)；
LDLR-S PCR-F：5 '-CCACCCATGGCTCTGCTAAA-3 ' (as shown in SEQ ID NO.20)；
LDLR-S PCR-R：5 '-CCTTTCTGGGCAGTCTGGTT-3 ' (as shown in SEQ ID NO.21).
Wherein ApoE wild types DNA profiling expanding fragment length is 447bp (as shown in SEQ ID NO.22), and LDLR is wild Type DNA profiling expanding fragment length is 593bp (as shown in SEQ ID NO.23), and PCR reaction systems are same as above with program.Take 2 μ L# The pcr amplification product of 1 and #2 mouse clones coupled reaction (TIANGEN Biotech (Beijing) Co., Ltd., DP205) for TA, so Connection product is converted into DH5 α competent cells afterwards, is incubated overnight in 37 DEG C of incubators, after it grows monoclonal, picking list Clone is cultivated, and sample presentation carries out sequencing identification.2 Founder rats carry out sequencing identification, #1 sequencing results such as Fig. 3 Shown, A is the genotype after ApoE gene knockouts, and B is the genotype after LDLR gene knockouts；#2 sequencing results as shown in figure 4, A and B is the genotype after ApoE gene knockouts, and C and D are the genotype after LDLR gene knockouts；It is identical with Fig. 2 results, Show that double fluorescent PCRs have very high accuracy.
6th, ApoE and LDLR expression of gene protein amounts are detected by western blotting：Using liquid nitrogen by Mouse Liver After dirty tissue grinder, cracked using RIPA, lysate using Bio-rad protein electrophoresis systems be separated by electrophoresis with Afterwards, hybridization check is carried out using APOE specific antibodies (ab5281) and LDLR specific antibodies (ab183597), as a result such as Fig. 5 It is shown, it can be seen from the figure that APOE and LDLR protein expressions have completely disappeared.
7th, 2 Founder mouse are returned and are selfed respectively and obtain the dual-gene knockout NOD mouse of ApoE and LDLR Homozygote.Specially：Founder mouse with wild-type NOD mouse are hybridized, select heterozygote selfing in generation after hybridization, from Offspring is handed over to obtain the dual-gene knockout homozygotes of ApoE and LDLR after testing, this mouse is the dual-gene knockout arteries of ApoE and LDLR Atherosis mouse genetic model.
8th, knockout diabetic mice genetic model dual-gene to ApoE and LDLR carries out phenotypic evaluation：To ApoE and LDLR The NOD mouse of dual-gene knockout, the B6 mouse of ApoE single-genes knockout, wild type NOD and B6 control mice continuously feed high in fat Feed 20 weeks.
Aorta is obtained, is dyed (Fig. 6) using oil red O after dissecting and being unfolded, obtains plaque area (Fig. 7)；It obtains Heart carries out frozen section and carries out H＆E dyeing (Fig. 8) and oil red O stain (Fig. 9), and count oil red O stain plaque area (Figure 10).As a result as illustrated in figures 6-10, analyze result above, it can be seen that continuous feeding high lipid food is after 20 weeks, ApoE and The area of patch and ApoE single-genes knockout B6 mouse are identical and aobvious in LDLR Double knockout mices aorta and heart It writes and is more than wild type NOD and B6 control mice, show that it has serious Atheromatosis after high lipid food is fed Disease.
Data above show the present invention successfully pass CRISPR/Cas9 gene Knockouts obtain NOD backgrounds ApoE and LDLR is dual-gene to knock out atherosclerosis mouse genetic model.
<110>Xinxiang College of Medical Science
<120>A kind of preparation method of the atherosclerosis mouse model of NOD genetic backgrounds
<160> 23
<170> SIPOSequenceListing 1.0
<211> 23
<212> DNA
<213>Artificial sequence
<221> ApoE-sgRNA1
<400> 1
cctagccgag ggagagccgg agg 23
<211> 23
<212> DNA
<213>Artificial sequence
<221> ApoE-sgRNA2
<400> 2
gtaatcccag aagcggttca ggg 23
<211> 23
<212> DNA
<213>Artificial sequence
<221> ApoE-sgRNA3
<400> 3
cttctgggat tacctgcgct ggg 23
<211> 23
<212> DNA
<213>Artificial sequence
<221> LDLR-sgRNA1
<400> 4
tccatcacac acaaactgcg ggg 23
<211> 23
<212> DNA
<213>Artificial sequence
<221> LDLR-sgRNA2
<400> 5
agttgcagcg gaagtgggcg ggg 23
<211> 23
<212> DNA
<213>Artificial sequence
<221> LDLR-sgRNA3
<400> 6
cagtctttgg gcctgcgacg ggg 23
<211> 64
<212> DNA
<213>Artificial sequence
<221> ApoE-IVT-1
<400> 7
ttaatacgac tcactatagg gcctagccga gggagagccg ggttttagag ctagaaatag 60
caag 64
<211> 64
<212> DNA
<213>Artificial sequence
<221> ApoE-IVT-2
<400> 8
ttaatacgac tcactatagg ggtaatccca gaagcggttc agttttagag ctagaaatag 60
caag 64
<211> 64
<212> DNA
<213>Artificial sequence
<221> ApoE-IVT-3
<400> 9
ttaatacgac tcactatagg gcttctggga ttacctgcgc tgttttagag ctagaaatag 60
caag 64
<211> 64
<212> DNA
<213>Artificial sequence
<221> LDLR-IVT-1
<400> 10
ttaatacgac tcactatagg gtccatcaca cacaaactgc ggttttagag ctagaaatag 60
caag 64
<211> 64
<212> DNA
<213>Artificial sequence
<221> LDLR-IVT-2
<400> 11
ttaatacgac tcactatagg gagttgcagc ggaagtgggc ggttttagag ctagaaatag 60
caag 64
<211> 64
<212> DNA
<213>Artificial sequence
<221> LDLR-IVT-3
<400> 12
ttaatacgac tcactatagg gcagtctttg ggcctgcgac ggttttagag ctagaaatag 60
caag 64
<211> 22
<212> DNA
<213>Artificial sequence
<221> IVT-R
<400> 13
aaaaaagcac cgactcggtg cc 22
<211> 20
<212> DNA
<213>Artificial sequence
<221> ApoE-F PCR-F
<400> 14
cgagggtgaa agagctggac 20
<211> 20
<212> DNA
<213>Artificial sequence
<221> ApoE-F PCR-R
<400> 15
gcctccagac ccacttcaaa 20
<211> 18
<212> DNA
<213>Artificial sequence
<221> LDLR-F PCR-F
<400> 16
agacgtgctc ccaggatg 18
<211> 19
<212> DNA
<213>Artificial sequence
<221> LDLR-F PCR-R
<400> 17
gttctgtggc cactcatcg 19
<211> 20
<212> DNA
<213>Artificial sequence
<221> ApoE-S PCR-F
<400> 18
aaggcaggag gattcaaggc 20
<211> 20
<212> DNA
<213>Artificial sequence
<221> ApoE-S PCR-R
<400> 19
gcctagtctc ggctctgaac 20
<211> 20
<212> DNA
<213>Artificial sequence
<221> LDLR-S PCR-F
<400> 20
ccacccatgg ctctgctaaa 20
<211> 20
<212> DNA
<213>Artificial sequence
<221> LDLR-S PCR-R
<400> 21
cctttctggg cagtctggtt 20
<211> 447
<212> DNA
<213>Mouse
<221>ApoE wild type DNA profiling amplified fragments
<400> 22
aaggcaggag gattcaaggc caacctgggc tacacactaa ttgagaaagg aaacaagaag 60
gggctgggga cccagatagg aggcaaccct ggatcagctg gtgcctgccg agggtgaaag 120
agctggacac tcacgtcagt tcttgtgtga cttgggagct ctgcagctct tcctggacct 180
ggtcagacag cgtctgcacc cagcgcaggt aatcccagaa gcggttcagg gcctgctccc 240
agggttggtt gctttgccac tcgagctgat ctgtcacctc cggctctccc tcggctaggc 300
atcctgtgta gagtaagttt aaggctgggt caggacctac agtctttttg ggtctctttg 360
aagtgggtct ggaggctgtg gagtctgttt agatactaca agcatgcact gtcttgtatc 420
ctatgtagtt cagagccgag actaggc 447
<211> 593
<212> DNA
<213>Mouse
<221>LDL wild type DNA profiling amplified fragments
<400> 23
ccacccatgg ctctgctaaa tgggttgtgt gtttttgaag cacacggtcc tgtctgcgaa 60
taggctggtg taagccatag cctgacaggg ccctctcctc cctgtgcacc cctgcagccc 120
ccaagacgtg ctcccaggat gacttccgat gccaggatgg caagtgcatc tccccgcagt 180
ttgtgtgtga tggagaccga gattgcctag atggctctga tgaggcccac tgccaggcca 240
ccacttgtgg ccccgcccac ttccgctgca actcatccat atgcatcccc agtctttggg 300
cctgcgacgg ggatgtcgac tgtgttgacg gctccgatga gtggccacag aactgccagg 360
gccgagacac ggcctccaaa ggcgttagca gcccctgctc ctccctggag ttccactgtg 420
gtagcagtga gtgtatccat cgcagctggg tctgtgacgg cgaggcagac tgcaaggaca 480
agtcagatga ggagcactgc ggtaaggccc tggggaggag ggatggatga ttggctcccc 540
aggccctgct ggggagcttt ggggcttagc aataaccaga ctgcccagaa agg 593

Claims (10)

1. a kind of preparation method of the atherosclerosis mouse model of NOD genetic backgrounds, it is characterised in that：Including：NOD is small After ApoE genes and LDLR genes knock out simultaneously in mouse to get.
2. preparation method according to claim 1, it is characterised in that：CRISPR/ is used when knocking out the ApoE genes Cas9 systems select 3 target sequences, as follows respectively：
ApoE-sgRNA1：5‘-CCTAGCCGAGGGAGAGCCGG AGG-3’；
ApoE-sgRNA2：5‘-GTAATCCCAGAAGCGGTTCAGGG-3’；
ApoE-sgRNA3：5‘-CTTCTGGGATTACCTGCGCT GGG-3’.
3. preparation method according to claim 1 or 2, it is characterised in that：It is used when knocking out the LDLR genes CRISPR/Cas9 systems select 3 target sequences, as follows respectively：
LDLR-sgRNA1：5‘-TCCATCACACACAAACTGCG GGG-3’；
LDLR-sgRNA2：5‘-AGTTGCAGCGGAAGTGGGCG GGG-3’；
LDLR-sgRNA3：5‘-CAGTCTTTGGGCCTGCGACG GGG-3’.
4. preparation method according to claim 3, it is characterised in that：When knocking out ApoE genes and LDLR genes jointly, 7 primers are synthesized, 6 double-stranded DNAs are obtained by PCR amplification；6 double-stranded DNAs are transcribed into 6 sgRNAmRNA, by institute It states 6 sgRNAmRNA with Cas9mRNA co-injections to enter in mouse fertilized egg cell, obtains dual-gene knockout fertilized egg cell； The dual-gene knockout fertilized oocyte is entered into false pregnancy female mice fallopian tubal, F0 mouse are obtained after birth；
The primer difference is as follows：
ApoE-IVT-1：TTAATACGAC TCACTATAGG GCCTAGCCGA GGGAGAGCCG GGTTTTAGAG CTAGAAATAG CAAG；
ApoE-IVT-2：TTAATACGAC TCACTATAGG GGTAATCCCA GAAGCGGTTC AGTTTTAGAG CTAGAAATAG CAAG；
ApoE-IVT-3：TTAATACGAC TCACTATAGG GCTTCTGGGA TTACCTGCGC TGTTTTAGAG CTAGAAATAG CAAG；
LDLR-IVT-1：TTAATACGAC TCACTATAGG GTCCATCACA CACAAACTGC GGTTTTAGAG CTAGAAATAG CAAG；
LDLR-IVT-2：TTAATACGAC TCACTATAGG GAGTTGCAGC GGAAGTGGGC GGTTTTAGAG CTAGAAATAG CAAG；
LDLR-IVT-3：TTAATACGAC TCACTATAGG GCAGTCTTTG GGCCTGCGAC GGTTTTAGAG CTAGAAATAG CAAG；IVT-R：AAAAAAGCACCGACTCGGTGCC；
By the IVT-R respectively with ApoE-IVT-1, ApoE-IVT-2, ApoE-IVT-3, LDLR-IVT-1, LDLR-IVT-2 or For PCR amplification after LDLR-IVT-3 combinations, 6 double-stranded DNAs are obtained.
5. preparation method according to claim 4, it is characterised in that：The Cas9mRNA is by pST1374-cas9 carriers After the linearisation of Agel enzymes, transcription in vitro obtains.
6. preparation method according to claim 4, it is characterised in that：During the PCR amplification, the template used is pX330 Plasmid.
7. preparation method according to claim 4, it is characterised in that：By the dual-gene knockout fertilized oocyte extremely False pregnancy female mice, after obtaining F0 mouse, the F0 mouse that identification selects ApoE and LDLR dual-gene while knocks out mate with NOD mouse It is selfed to F1 generation hybrid mice, then by F1 generation hybrid mice, it is dual-gene while knock out to filter out ApoE and LDLR Homozygotic individual be NOD genetic backgrounds atherosclerosis mouse model.
8. preparation method according to claim 6, it is characterised in that：The identification of the ApoE genes uses following primer：
ApoE-F PCR-F：5‘-CGAGGGTGAAAGAGCTGGAC-3’；
ApoE-F PCR-R：5‘-GCCTCCAGACCCACTTCAAA-3’.
9. preparation method according to claim 6, it is characterised in that：The identification of the LDLR genes uses following primer：
LDLR-F PCR-F：5‘-AGACGTGCTCCCAGGATG-3’；
LDLR-F PCR-R：5‘-GTTCTGTGGCCACTCATCG-3.
10. the atherosclerosis mouse model for the NOD genetic backgrounds that preparation method as described in claim 1 obtains is in artery Application in the fundamental research of atherosis.
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