CN110331172A - Method based on CRISPR-Cas9 system building albefaction Hamster model - Google Patents

Method based on CRISPR-Cas9 system building albefaction Hamster model Download PDF

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CN110331172A
CN110331172A CN201910632733.XA CN201910632733A CN110331172A CN 110331172 A CN110331172 A CN 110331172A CN 201910632733 A CN201910632733 A CN 201910632733A CN 110331172 A CN110331172 A CN 110331172A
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hamster
albefaction
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crispr
primer
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CN110331172B (en
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王全新
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Liaoning Changsheng Biotechnology Co Ltd
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Abstract

The invention belongs to genetic engineering fields, and in particular to the method based on CRISPR-Cas9 system building albefaction Hamster model.Method based on CRISPR-Cas9 system building albefaction Hamster model model of the invention, comprising the following steps: determine hamster TRY gene targeting site sequence；Prepare gRNA；External microinjection: collecting hamster fertilized eggs, gRNA and Cas9 RNA be injected into the fertilized eggs into hamster, carries out transplant to the fertilized eggs of injection.The sgRNA of selectively targeted hamster TRY gene prepared by the present invention can accurately target hamster TYR gene and realize gene knockout, generate the albefaction hamster of TYR gene delection.

Description

Method based on CRISPR-Cas9 system building albefaction Hamster model
Technical field
The invention belongs to genetic engineering fields, and in particular to based on CRISPR-Cas9 system building albefaction Hamster model Method.
Background technique
Golden Hamster (hamster) is a kind of important minitype animal experiment, it be mainly used in most tumour, cardiovascular diseases and In immune Research, annual usage amount is huge.Then, due to the technical restriction of Hamster embryos operation, domestic and international large scale preparation turns Gene or gene knockout mould Hamster model are made slow progress.It is different from the standardization embryo transfer technology of mouse, hamster fertilized eggs pair Temperature, PH are sensitive, in vitro operating difficulties, so far, not yet effectively break through 2 cell transplantation of fertilized eggs retardance, false pregnancy hamster It is low to transplant success rate.Therefore, preparation hamster passes through transplanting fertilized eggs mostly and causes to pass through shifting in the hamster body of natural conception in 0.5 day Fertilized eggs and naturally fertilized egg competition development are planted, engineering hamster is lost in preparation, but cannot be distinguished after homograft offspring birth.
Summary of the invention
The purpose of the present invention is to provide a kind of methods based on CRISPR-Cas9 system building albefaction Hamster model.
The method based on CRISPR-Cas9 system building albefaction Hamster model of specific embodiment according to the present invention, Method the following steps are included:
(1) hamster TRY gene targeting sequence is determined；
(2) sgRNA is prepared；
(3) cas9mRNA is prepared；
(4) external microinjection: hamster fertilized eggs are collected, gRNA and Cas9RNA are injected into the fertilized eggs into hamster；
(5) transplant is carried out to the fertilized eggs of injection.
The method based on CRISPR-Cas9 system building albefaction Hamster model of specific embodiment according to the present invention, step Suddenly in (1), TRY gene targeting sequence is as shown in SEQ ID No.1.
SEQ ID No.1:GGGTGGATGACCGTGAGTCCTGG。
Wild 1593 bases of hamster Try full length gene, nucleotide sequence is as shown in SEQ ID NO.2, Try gene 530 amino acid are encoded, amino acid sequence is as shown in SEQ ID NO.3.The hamster of mutation generates from the 66th amino acid Frameshift mutation forms the short of only 103 amino acid in+353-455bp formation terminator codon TAA (SEQ ID NO.4) Peptide (SEQ ID NO.5) cannot generate normal TYR albumen, generate albefaction.
SEQ ID NO.2:
ATGTTCCTGGCTGTTTTGTATTGCCTTCTGTGGAGTTTCCAGATCTCTTCTTCCCATTTCCCTCGAGCT TGTGTCTCCTCAAAAAACTTGATGGCAAAAGAATGCTGTCCACCATGGACAGGTGACGGGAGTCCCTGCGGCCAACT TTCAGGCAGAGGTTCTTGCCAGGACATCCTTCTTTCCAATGCACCCACTGGACCTCAATTTCCCTTCAAAGGGGTGG ATGACCGTGAGTCCTGGCCCTCTGTATTTTACAATAGAACCTGCCAGTGCTCTGGCAACTTCATGGGATTCAACTGC GGAAACTGTAAATTTGGATTTGGGGGATCAAATTGCACAGAGAAGCGACTCTTAATTAGAAGAAACATCTATGATTT GAGTGTCCCAGAAAAGAATAAGTTCTTTGCTTACCTAAATTTAGCAAAGCATACTATCAGCCCAGTCTATGTTATCC CCATAGGTACATATGGTCAAATGAACAATGGGTCAACACCCATGTTTAATGATATAAGCGTCTATGATCTCTTTGTA TGGATGCATTACTATGTGTCAAGAGACACTCTGCTTGGAGGTTCTGAAATCTGGAGGGACGTTGATTTTGCCCACGA AGCACCAGGGTTTCTGCCTTGGCACAGACTTTTCTTGCTACTGTGGGAACAAGAAATACGAGAGCTAACAGGCGATG AGAACTTCACCATTCCATACTGGGACTGGCGGGATGCCGAAAATTGTGATATTTGCACAGATGAGTACTTGGGAGGT CGCCACCCTGAAAATCCTAATCTACTCAGTCCAGCATCCTTCTTCTCCTCATGGCAGATCATCTGCAGCAGATCAGA AGAGTACAATAGCCGTCAGGCTTTATGCGATGGGACACCTGAGGGACCACTGATACGCAATCCTGGAAACCATGACA AAACCAAAACCCCGAGGCTCCCATCTTCGGCAGATGTGGAATTTTGTCTGACTTTAACCCAGTATGAAGCTGGCTCC ATGGATAAAACTGCCAATTTCAGCTTTAGAAACACACTGGAAGGATTTGCAAATCCACTCACAGGCATAGCGGATTC TTCTCGGAGCACAATGCACAATGCTTTGCACATCTATATGAATGGAACAATGTCCCAAGTCCAGGGATCGGCCAATG ATCCCATTTTTCTTCTTCATCATGCTTTTGTGGACAGTGTTTTTGAACAATGGCTTCGAAGACACCGTCCTCTTCTG GAAGTTTACCCAGAAGCGAATGCACCTATTGGTCATAACAGAGAATCCTACATGGTCCCTTTCCTACCACTGTATAG AAATGGTGATTTCTTCATTTCATCTAAGGATCTGGGATATGACTACAGCTACCTACAGGATCCAGATCCAGGCTTTT ACAGAAACTATATTGAGCCCTACTTGGAGCAAGCCTCTAGGATCTGGCCATGGCTACTTGGGGCAGCACTAGTGGGA GCTGTTGTTGCTGCAGCTCTGGCAGGGCTCAGCAGTCATCTGTGCTGTCACAAGAGGAGGCATCTCCAGGAAGAGAG ACAACCACTCCTCATGGAAAAAGATGATTACCACAACTTGCTGTATCAGAGCCATCTTTGA
SEQ ID NO.3:
MFLAVLYCLLWSFQISSSHFPRACVSSKNLMAKECCPPWTGDGSPCGQLSGRGSCQDILLSNAPTGPQ FPFKGVDDRESWPSVFYNRTCQCSGNFMGFNCGNCKFGFGGSNCTEKRLLIRRNIYDLSVPEKNKFFAYLNLAKHT ISPVYVIPIGTYGQMNNGSTPMFNDISVYDLFVWMHYYVSRDTLLGGSEIWRDVDFAHEAPGFLPWHRLFLLLWEQ EIRELTGDENFTIPYWDWRDAENCDICTDEYLGGRHPENPNLLSPASFFSSWQIICSRSEEYNSRQALCDGTPEGP LIRNPGNHDKTKTPRLPSSADVEFCLTLTQYEAGSMDKTANFSFRNTLEGFANPLTGIADSSRSTMHNALHIYMNG TMSQVQGSANDPIFLLHHAFVDSVFEQWLRRHRPLLEVYPEANAPIGHNRESYMVPFLPLYRNGDFFISSKDLGYD YSYLQDPDPGFYRNYIEPYLEQASRIWPWLLGAALVGAVVAAALAGLSSHLCCHKRRHLQEERQPLLMEKDDYHNL LYQSHL&
SEQ ID NO.4
ATGTTCCTGGCTGTTTTGTATTGCCTTCTGTGGAGTTTCCAGATCTCTTCTTCCCATTTCCCTCGAGCT TGTGTCTCCTCAAAAAACTTGATGGCAAAAGAATGCTGTCCACCATGGACAGGTGACGGGAGTCCCTGCGGCCAACT TTCAGGCAGAGGTTCTTGCCAGGACATCCTTCTTTCCAATGCACCCACTGGCCCTCTGTATTTTACAATAGAACCTG CCAGTGCTCTGGCAACTTCATGGGATTCAACTGCGGAAACTGTAAATTTGGATTTGGGGGATCAAATTGCACAGAGA AGCGACTCTTAATTAGAAGAAACATCTATGATTTGAGTGTCCCAGAAAAGAATAAGTTCTTTGCTTACCTAAATTTA GCAAAGCATACTATCAGCCCAGTCTATGTTATCCCCATAGGTACATATGGTCAAATGAACAATGGGTCAACACCCAT GTTTAATGATATAAGCGTCTATGATCTCTTTGTATGGATGCATTACTATGTGTCAAGAGACACTCTGCTTGGAGGTT CTGAAATCTGGAGGGACGTTGATTTTGCCCACGAAGCACCAGGGTTTCTGCCTTGGCACAGACTTTTCTTGCTACTG TGGGAACAAGAAATACGAGAGCTAACAGGCGATGAGAACTTCACCATTCCATACTGGGACTGGCGGGATGCCGAAAA TTGTGATATTTGCACAGATGAGTACTTGGGAGGTCGCCACCCTGAAAATCCTAATCTACTCAGTCCAGCATCCTTCT TCTCCTCATGGCAGATCATCTGCAGCAGATCAGAAGAGTACAATAGCCGTCAGGCTTTATGCGATGGGACACCTGAG GGACCACTGATACGCAATCCTGGAAACCATGACAAAACCAAAACCCCGAGGCTCCCATCTTCGGCAGATGTGGAATT TTGTCTGACTTTAACCCAGTATGAAGCTGGCTCCATGGATAAAACTGCCAATTTCAGCTTTAGAAACACACTGGAAG GATTTGCAAATCCACTCACAGGCATAGCGGATTCTTCTCGGAGCACAATGCACAATGCTTTGCACATCTATATGAAT GGAACAATGTCCCAAGTCCAGGGATCGGCCAATGATCCCATTTTTCTTCTTCATCATGCTTTTGTGGACAGTGTTTT TGAACAATGGCTTCGAAGACACCGTCCTCTTCTGGAAGTTTACCCAGAAGCGAATGCACCTATTGGTCATAACAGAG AATCCTACATGGTCCCTTTCCTACCACTGTATAGAAATGGTGATTTCTTCATTTCATCTAAGGATCTGGGATATGAC TACAGCTACCTACAGGATCCAGATCCAGGCTTTTACAGAAACTATATTGAGCCCTACTTGGAGCAAGCCTCTAGGAT CTGGCCATGGCTACTTGGGGCAGCACTAGTGGGAGCTGTTGTTGCTGCAGCTCTGGCAGGGCTCAGCAGTCATCTGT GCTGTCACAAGAGGAGGCATCTCCAGGAAGAGAGACAACCACTCCTCATGGAAAAAGATGATTACCACAACTTGCTG TATCAGAGCCATCTTTGA
SEQ ID NO.5
MFLAVLYCLLWSFQISSSHFPRACVSSKNLMAKECCPPWTGDGSPCGQLSGRGSCQDILLSNAPTG P LYFTIEPASALATSWDSTAETVNLDLGDQIAQRSDS
The method based on CRISPR-Cas9 system building albefaction Hamster model of specific embodiment according to the present invention, step Suddenly in (2), TRY sgRNA1 and PX330TRAC R primer are designed, primer sequence difference is as follows:
Piwil1sgRNA1 primer:
ATAATACGACTCACTATAGg GGGTGGATGACCGTGAGTCC GTTTTAGAGCTAGAAATAG；
PX330TRAC R primer:
AAAAGCACCGACTCGGTGCC。
The method based on CRISPR-Cas9 system building albefaction Hamster model of specific embodiment according to the present invention, step Suddenly in (2), using TRY sgRNA1 and PX330TRAC R primer amplification PX330 plasmid, template DNA is obtained, then polymerize by T7 Enzyme synthesizes TRY sgRNA.
The method based on CRISPR-Cas9 system building albefaction Hamster model of specific embodiment according to the present invention, step Suddenly in (3), upstream primer CAS9-T7F and downstream primer CAS9-T7R is designed, primer sequence difference is as follows:
CAS9-T7F:
GAAATTAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGAC；
CAS9-T7R:
GCGAGCTCTAGGAATTCTTAC。
Beneficial effects of the present invention:
The present invention utilizes CRISPR/CAS9 gene editing technology, designs hamster TYR sgRNA (single-guide RNA) Target sequence constructs the hamster of TRY gene knockout by pronuclear microinjection, and display offspring is that albefaction gold hamster is (referred to as white Change hamster), using albefaction hamster as transplant recipient, gold hamster (as donor) provides fertilized eggs and is used for gene modification, will infuse Fertilized eggs after penetrating are migrated to immediately in the albefaction hamster body to mate, and the fertilized eggs of transplanting and the fertilized eggs of albefaction hamster compete It develops, in the offspring given birth to, albefaction hamster and non-albefaction gold hamster will occurs, non-albefaction gold hamster is to need to identify Gene modification hamster, convenient for transplanting the screening and Quick-type identification of offspring.Therefore, constructed TRY gene knockout albefaction hamster It can be used as transplant recipient, the preparation for hamster genetic engineering mouse.
Detailed description of the invention
Fig. 1 shows hamster TYR gene knockout the sequencing results；
Fig. 2 shows the observation of TYR gene knockout hamster hair color.
Specific embodiment
The type and raising of experimental animal:
The experiment hamster that the present invention uses ties up experimental animal technology company, tonneau China purchased from Beijing, cures in Nanjing Medical University The raising of medicine Experimental Animal Center, rearing conditions barrier environment, using 22-26 DEG C of temperature, humidity 40-60%, light application time 10: 14.Experimental implementation obtains experimental animal Ethics Committee, Nanjing Medical University and examines and supervise.
The building of embodiment 1:CRISPR/Cas9 system
1.1 design hamster TYR gene specific target practice sequences
In the exon 2 of Syrian holden hamsters TYR gene, designs gene specific target practice DNA and identify sequence.
Hamster TYR gene targeting sequence is GGGTGGATGACCGTGAGTCCTGG.
1.2 preparation TRY sgRNA
TYR sgRNA1 primer and PX330TRAC R primer are designed, PX330 plasmid is expanded, obtains template DNA, then pass through T7 polymerase synthesizes TYR sgRNA.It is specific as follows:
Primer component: 1) specific oligonucleotide sequence CRISPRF, the sequence include T7 promoter, sgRNA sequence and portion Divide sgRNA skeleton part；
TYR sgRNA1 primer:
ATAATACGACTCACTATAGg GGGTGGATGACCGTGAGTCC GTTTTAGAGCTAGAAATAG；
PX330TRAC R primer:
AAAAGCACCGACTCGGTGCC。
Above-mentioned primer makees 100 μ l PCR reaction systems respectively, size 127bp, reaction condition be (94 DEG C of 30s, 30cycles(94℃ 20s,58℃ 30s,72℃ 20s),72℃ 10min,4℃∞).PCR product directly uses PCR product pure Change kits DNA, the i.e. template DNA of sgRNA.
Transcript reagent box MEGAshortscriptTM(No.1345) it is transcribed in vitro.SgRNA is carried out pure with ethyl alcohol after reaction Change.No RNase water dissolution makes 1 μ g/ μ l of its concentration.- 80 DEG C are stored in, for injecting.
1.3 preparation Cas9mRNA
The Cas9DNA template source of use in PCR method expand PX330 plasmid.Design the upstream primer of the sequence containing T7 CAS9-T7F and downstream primer CAS9-T7R, makees 100 μ l PCR reaction systems, size 4269bp, and reaction condition is (94 DEG C 30s,30cycles(94℃ 30s,58℃ 30s,72℃ 5min),72℃10min,4℃∞).PCR product agarose electricity Swimming, cuts glue purification.The transcription of transcript reagent box, transcription product purifies mRNA. with LiCl and is stored in -80 DEG C, for injecting.
CAS9-T7F:
GAAATTAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGAC；
CAS9-T7R:
GCGAGCTCTAGGAATTCTTAC。
Embodiment 2: building TRY knocks out hamster
2.1 obtain hamster fertilized eggs
6-8 week old gold female mice is chosen, in the 9 points one morning of feelings phase, pregnant mare serum gonadotrop(h)in (PMSG) is injected intraperitoneally (PMSG) the super row of (15IU/100g) induction.It 4th day 6 pm, mates and mates with male mouse.2nd day 9 points of the morning, genital tract Whether microscopy sperm mates.
Fallopian tubal is taken, fertilized eggs are collected in M2 culture solution, HEMC-9 cultivates fertilized eggs.Fertilized eggs are directly used in micro- note It penetrates.In vitro operation is completed in 30 minutes.
2.2 protokaryons (PN) injection
20ng/ μ l sgRNA1 and 50ng/ μ l cas9mRNA PN injection, fertilized eggs directly transplanting enters replace-conceive storehouse after injection In mouse rat body.
The 2.3 fertilization implantation of ovums
Replace-conceive mouse mates with male mouse on the same day for ovum mouse.2nd day, the fertilized eggs injected are entered into replace-conceive from common oviduct transplantation Mouse.40 fertilized eggs migrate in 2 hamster replace-conceive rat bodies after procaryotic injection, and every hamster is implanted into 14-20 pieces of fertilization Ovum.
Be born 6 hamsters altogether, wherein 2 knock out albefaction hamster for homozygote, one is variegated hamster, and 3 are wild type.
2.4 genotyping
After selecting hamster wean, identified for genes is carried out with toe tissue.15 days hamsters after birth are cut into toe and extract DNA, are used PCR amplification is identified.
(1) PCR amplification genomic DNA, 20 μ L systems of reaction, size 183bp, primer are as follows:
HAM-TYR-F:CCAACTTTCAGGCAGAGGTTC；
HAM-TYR--R:TCTCTGTGCAATTTGATCCCC。
Glue purification is cut after PCR primer agarose electrophoresis identification, is sequenced and is identified with primer HAM-TYR-F.
Wild 1593 bases of hamster Try full length gene, nucleotide sequence is as shown in SEQ ID NO.2, Try gene 530 amino acid are encoded, amino acid sequence is as shown in SEQ ID NO.3.The hamster of mutation generates from the 66th amino acid Frameshift mutation forms the short of only 103 amino acid in+353-455bp formation terminator codon TAA (SEQ ID NO.4) Peptide (SEQ ID NO.5) cannot generate normal TYR albumen, generate albefaction.
As shown in Figure 1, identified for genes shows 4#14/17 base FOUNDER1 albefactions (male mouse) of missing respectively, 5# is lacked 22/43 base FOUNDER2 albefactions (male mouse) are lost, it is variegated (female mice) that 6# lacks 17 base FOUNDER3.
It chooses albefaction hamster 5# to mate with wild type, obtains F1 generation, be golden yellow raw mouse, identified for genes takes the missing storehouse 43bp Mouse mutually mates, and offspring is albefaction and gold hamster, and it is TYR mut43 that albefaction hamster, which is determined lane type, and homozygous is fertile.
The phenotypic analysis of 2.5TYR gene knockout hamster
It is albefaction hamster that TYR gene pure, which knocks out TYR (-/-) hamster, and TYR (+/-) hamster is that white is chimeric with golden yellow, As shown in Fig. 2, TYR gene pure knockout is fertile, fertility-rate does not influence.
Embodiment 3:TYR albefaction hamster prepares as receptor mouse for genetic engineering hamster
It is albefaction hamster that TYR gene pure, which knocks out hamster, replaces replace-conceive with the albefaction hamster of 6-8 weeks TYR gene knockout Hamster, so that offspring can be distinguished by color after being born.Albefaction replace-conceive mouse, gold hamster mate with male mouse on the same day for ovum mouse. 2nd day, the fertilized eggs injected are entered into replace-conceive mouse from common oviduct transplantation.Every hamster is implanted into 15 pieces of fertilized eggs.Offspring goes out within 16 days It is raw, it can be distinguished by color after 3 days, identify albefaction hamster, it is 100% that CAS9, which knocks out efficiency,.
Although the embodiments of the invention are described in conjunction with the attached drawings, but patent owner can be in appended claims Within the scope of make various deformations or amendments, as long as it does not exceed the scope of protection described in the claims to the invention, all should Within protection scope of the present invention.
Sequence table
<110>long-living Biotechnology Ltd. in Liaoning
<120>method based on CRISPR-Cas9 system building albefaction Hamster model
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>hamster (Cricetinae)
<400> 1
gggtggatga ccgtgagtcc tgg 23
<210> 2
<211> 1593
<212> DNA
<213>hamster (Cricetinae)
<400> 2
atgttcctgg ctgttttgta ttgccttctg tggagtttcc agatctcttc ttcccatttc 60
cctcgagctt gtgtctcctc aaaaaacttg atggcaaaag aatgctgtcc accatggaca 120
ggtgacggga gtccctgcgg ccaactttca ggcagaggtt cttgccagga catccttctt 180
tccaatgcac ccactggacc tcaatttccc ttcaaagggg tggatgaccg tgagtcctgg 240
ccctctgtat tttacaatag aacctgccag tgctctggca acttcatggg attcaactgc 300
ggaaactgta aatttggatt tgggggatca aattgcacag agaagcgact cttaattaga 360
agaaacatct atgatttgag tgtcccagaa aagaataagt tctttgctta cctaaattta 420
gcaaagcata ctatcagccc agtctatgtt atccccatag gtacatatgg tcaaatgaac 480
aatgggtcaa cacccatgtt taatgatata agcgtctatg atctctttgt atggatgcat 540
tactatgtgt caagagacac tctgcttgga ggttctgaaa tctggaggga cgttgatttt 600
gcccacgaag caccagggtt tctgccttgg cacagacttt tcttgctact gtgggaacaa 660
gaaatacgag agctaacagg cgatgagaac ttcaccattc catactggga ctggcgggat 720
gccgaaaatt gtgatatttg cacagatgag tacttgggag gtcgccaccc tgaaaatcct 780
aatctactca gtccagcatc cttcttctcc tcatggcaga tcatctgcag cagatcagaa 840
gagtacaata gccgtcaggc tttatgcgat gggacacctg agggaccact gatacgcaat 900
cctggaaacc atgacaaaac caaaaccccg aggctcccat cttcggcaga tgtggaattt 960
tgtctgactt taacccagta tgaagctggc tccatggata aaactgccaa tttcagcttt 1020
agaaacacac tggaaggatt tgcaaatcca ctcacaggca tagcggattc ttctcggagc 1080
acaatgcaca atgctttgca catctatatg aatggaacaa tgtcccaagt ccagggatcg 1140
gccaatgatc ccatttttct tcttcatcat gcttttgtgg acagtgtttt tgaacaatgg 1200
cttcgaagac accgtcctct tctggaagtt tacccagaag cgaatgcacc tattggtcat 1260
aacagagaat cctacatggt ccctttccta ccactgtata gaaatggtga tttcttcatt 1320
tcatctaagg atctgggata tgactacagc tacctacagg atccagatcc aggcttttac 1380
agaaactata ttgagcccta cttggagcaa gcctctagga tctggccatg gctacttggg 1440
gcagcactag tgggagctgt tgttgctgca gctctggcag ggctcagcag tcatctgtgc 1500
tgtcacaaga ggaggcatct ccaggaagag agacaaccac tcctcatgga aaaagatgat 1560
taccacaact tgctgtatca gagccatctt tga 1593
<210> 3
<211> 530
<212> PRT
<213>hamster (Cricetinae)
<400> 3
Met Phe Leu Ala Val Leu Tyr Cys Leu Leu Trp Ser Phe Gln Ile Ser
1 5 10 15
Ser Ser His Phe Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Ala
20 25 30
Lys Glu Cys Cys Pro Pro Trp Thr Gly Asp Gly Ser Pro Cys Gly Gln
35 40 45
Leu Ser Gly Arg Gly Ser Cys Gln Asp Ile Leu Leu Ser Asn Ala Pro
50 55 60
Thr Gly Pro Gln Phe Pro Phe Lys Gly Val Asp Asp Arg Glu Ser Trp
65 70 75 80
Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met
85 90 95
Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Gly Gly Ser Asn Cys
100 105 110
Thr Glu Lys Arg Leu Leu Ile Arg Arg Asn Ile Tyr Asp Leu Ser Val
115 120 125
Pro Glu Lys Asn Lys Phe Phe Ala Tyr Leu Asn Leu Ala Lys His Thr
130 135 140
Ile Ser Pro Val Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Asn
145 150 155 160
Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Ser Val Tyr Asp Leu Phe
165 170 175
Val Trp Met His Tyr Tyr Val Ser Arg Asp Thr Leu Leu Gly Gly Ser
180 185 190
Glu Ile Trp Arg Asp Val Asp Phe Ala His Glu Ala Pro Gly Phe Leu
195 200 205
Pro Trp His Arg Leu Phe Leu Leu Leu Trp Glu Gln Glu Ile Arg Glu
210 215 220
Leu Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr Trp Asp Trp Arg Asp
225 230 235 240
Ala Glu Asn Cys Asp Ile Cys Thr Asp Glu Tyr Leu Gly Gly Arg His
245 250 255
Pro Glu Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp
260 265 270
Gln Ile Ile Cys Ser Arg Ser Glu Glu Tyr Asn Ser Arg Gln Ala Leu
275 280 285
Cys Asp Gly Thr Pro Glu Gly Pro Leu Ile Arg Asn Pro Gly Asn His
290 295 300
Asp Lys Thr Lys Thr Pro Arg Leu Pro Ser Ser Ala Asp Val Glu Phe
305 310 315 320
Cys Leu Thr Leu Thr Gln Tyr Glu Ala Gly Ser Met Asp Lys Thr Ala
325 330 335
Asn Phe Ser Phe Arg Asn Thr Leu Glu Gly Phe Ala Asn Pro Leu Thr
340 345 350
Gly Ile Ala Asp Ser Ser Arg Ser Thr Met His Asn Ala Leu His Ile
355 360 365
Tyr Met Asn Gly Thr Met Ser Gln Val Gln Gly Ser Ala Asn Asp Pro
370 375 380
Ile Phe Leu Leu His His Ala Phe Val Asp Ser Val Phe Glu Gln Trp
385 390 395 400
Leu Arg Arg His Arg Pro Leu Leu Glu Val Tyr Pro Glu Ala Asn Ala
405 410 415
Pro Ile Gly His Asn Arg Glu Ser Tyr Met Val Pro Phe Leu Pro Leu
420 425 430
Tyr Arg Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp
435 440 445
Tyr Ser Tyr Leu Gln Asp Pro Asp Pro Gly Phe Tyr Arg Asn Tyr Ile
450 455 460
Glu Pro Tyr Leu Glu Gln Ala Ser Arg Ile Trp Pro Trp Leu Leu Gly
465 470 475 480
Ala Ala Leu Val Gly Ala Val Val Ala Ala Ala Leu Ala Gly Leu Ser
485 490 495
Ser His Leu Cys Cys His Lys Arg Arg His Leu Gln Glu Glu Arg Gln
500 505 510
Pro Leu Leu Met Glu Lys Asp Asp Tyr His Asn Leu Leu Tyr Gln Ser
515 520 525
His Leu
530
<210> 4
<211> 1550
<212> DNA
<213>hamster (Cricetinae)
<400> 4
atgttcctgg ctgttttgta ttgccttctg tggagtttcc agatctcttc ttcccatttc 60
cctcgagctt gtgtctcctc aaaaaacttg atggcaaaag aatgctgtcc accatggaca 120
ggtgacggga gtccctgcgg ccaactttca ggcagaggtt cttgccagga catccttctt 180
tccaatgcac ccactggccc tctgtatttt acaatagaac ctgccagtgc tctggcaact 240
tcatgggatt caactgcgga aactgtaaat ttggatttgg gggatcaaat tgcacagaga 300
agcgactctt aattagaaga aacatctatg atttgagtgt cccagaaaag aataagttct 360
ttgcttacct aaatttagca aagcatacta tcagcccagt ctatgttatc cccataggta 420
catatggtca aatgaacaat gggtcaacac ccatgtttaa tgatataagc gtctatgatc 480
tctttgtatg gatgcattac tatgtgtcaa gagacactct gcttggaggt tctgaaatct 540
ggagggacgt tgattttgcc cacgaagcac cagggtttct gccttggcac agacttttct 600
tgctactgtg ggaacaagaa atacgagagc taacaggcga tgagaacttc accattccat 660
actgggactg gcgggatgcc gaaaattgtg atatttgcac agatgagtac ttgggaggtc 720
gccaccctga aaatcctaat ctactcagtc cagcatcctt cttctcctca tggcagatca 780
tctgcagcag atcagaagag tacaatagcc gtcaggcttt atgcgatggg acacctgagg 840
gaccactgat acgcaatcct ggaaaccatg acaaaaccaa aaccccgagg ctcccatctt 900
cggcagatgt ggaattttgt ctgactttaa cccagtatga agctggctcc atggataaaa 960
ctgccaattt cagctttaga aacacactgg aaggatttgc aaatccactc acaggcatag 1020
cggattcttc tcggagcaca atgcacaatg ctttgcacat ctatatgaat ggaacaatgt 1080
cccaagtcca gggatcggcc aatgatccca tttttcttct tcatcatgct tttgtggaca 1140
gtgtttttga acaatggctt cgaagacacc gtcctcttct ggaagtttac ccagaagcga 1200
atgcacctat tggtcataac agagaatcct acatggtccc tttcctacca ctgtatagaa 1260
atggtgattt cttcatttca tctaaggatc tgggatatga ctacagctac ctacaggatc 1320
cagatccagg cttttacaga aactatattg agccctactt ggagcaagcc tctaggatct 1380
ggccatggct acttggggca gcactagtgg gagctgttgt tgctgcagct ctggcagggc 1440
tcagcagtca tctgtgctgt cacaagagga ggcatctcca ggaagagaga caaccactcc 1500
tcatggaaaa agatgattac cacaacttgc tgtatcagag ccatctttga 1550
<210> 5
<211> 103
<212> PRT
<213>hamster (Cricetinae)
<400> 5
Met Phe Leu Ala Val Leu Tyr Cys Leu Leu Trp Ser Phe Gln Ile Ser
1 5 10 15
Ser Ser His Phe Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Ala
20 25 30
Lys Glu Cys Cys Pro Pro Trp Thr Gly Asp Gly Ser Pro Cys Gly Gln
35 40 45
Leu Ser Gly Arg Gly Ser Cys Gln Asp Ile Leu Leu Ser Asn Ala Pro
50 55 60
Thr Gly Pro Leu Tyr Phe Thr Ile Glu Pro Ala Ser Ala Leu Ala Thr
65 70 75 80
Ser Trp Asp Ser Thr Ala Glu Thr Val Asn Leu Asp Leu Gly Asp Gln
85 90 95
Ile Ala Gln Arg Ser Asp Ser
100
<210> 6
<211> 59
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ataatacgac tcactatagg gggtggatga ccgtgagtcc gttttagagc tagaaatag 59
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gcgagctcta ggaattctta c 21

Claims (5)

1. the method based on CRISPR-Cas9 system building albefaction Hamster model, which is characterized in that the method includes following steps It is rapid:
(1) hamster TRY gene targeting sequence is determined；
(2) sgRNA is prepared；
(3) cas9 mRNA is prepared；
(4) external microinjection: hamster fertilized eggs are collected, gRNA and Cas9 RNA is injected into the fertilized eggs into hamster；
(5) transplant is carried out to the fertilized eggs of injection.
2. the method according to claim 1 based on CRISPR-Cas9 system building albefaction Hamster model, feature exist In in step (1), TRY gene targeting sequence is as shown in SEQ ID No.1.
3. the method according to claim 1 based on CRISPR-Cas9 system building albefaction Hamster model, feature exist In in step (2), design TRY sgRNA1 and PX330 TRAC R primer, primer sequence is distinguished as follows:
Piwil1 sgRNA1 primer:
ATAATACGACTCACTATAGg GGGTGGATGACCGTGAGTCC GTTTTAGAGCTAGAAATAG；
PX330 TRAC R primer:
AAAAGCACCGACTCGGTGCC。
4. the method according to claim 3 based on CRISPR-Cas9 system building albefaction Hamster model, feature exist In using TRY sgRNA1 and PX330 TRAC R primer amplification PX330 plasmid, obtaining template DNA, then pass through in step (2) T7 polymerase synthesizes TRY sgRNA.
5. the method according to claim 1 based on CRISPR-Cas9 system building albefaction Hamster model, feature exist In in step (3), design upstream primer CAS9-T7F and downstream primer CAS9-T7R, primer sequence is distinguished as follows:
CAS9-T7F:
GAAATTAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGAC；
CAS9-T7R:
GCGAGCTCTAGGAATTCTTAC。
CN201910632733.XA 2019-07-14 2019-07-14 Method for constructing whitened hamster model based on CRISPR-Cas9 system Active CN110331172B (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
赵彦斌等: "黑线仓鼠及其白化突变系TYR、TYRP1的基因表达水平比较分析", 《中国比较医学杂志》 *
陈傍柱等: "利用crispr/cas9技术制备白化小鼠", 《第十三届中国实验动物科学年会论文集》 *

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