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Method for constructing albino hamster model based on CRISPR-Cas9 system

An albino hamster, CAS9-T7F technology, applied in the field of genetic engineering, can solve problems such as the block of fertilized egg 2-cell transplantation, the low success rate of pseudo-pregnant hamster transplantation, and the difficulty in operation.

Active Publication Date: 2019-10-15
辽宁长生生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the technical limitations of hamster embryo manipulation, the large-scale production of transgenic or gene knockout hamster models at home and abroad has been slow.
Different from the standardized embryo transfer technology in mice, hamster fertilized eggs are sensitive to temperature and pH, and it is difficult to operate in vitro. So far, it has not effectively broken through the blockage of fertilized egg 2 cell transplantation, and the success rate of pseudopregnant hamster transplantation is low.
Therefore, most hamsters are prepared by transplanting fertilized eggs to naturally conceive hamsters for 0.5 days, and the transplanted fertilized eggs compete with natural fertilized eggs to prepare genetically engineered hamsters, but the allografted offspring cannot be distinguished after birth.

Method used

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  • Method for constructing albino hamster model based on CRISPR-Cas9 system
  • Method for constructing albino hamster model based on CRISPR-Cas9 system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: CRISPR / Cas9 system construction

[0039] 1.1 Design of hamster TYR gene specific targeting sequence

[0040] In the second exon of the Syrian golden hamster TYR gene, a gene-specific targeting DNA recognition sequence was designed.

[0041] The hamster TYR gene targeting sequence is GGGTGGATGACCGTGAGTCCTGG.

[0042] 1.2 Preparation of TRY sgRNA

[0043] Design TYR sgRNA1 primer and PX330TRAC R primer, amplify PX330 plasmid, obtain template DNA, and then synthesize TYR sgRNA by T7 polymerase. details as follows:

[0044] Primer components: 1) specific oligonucleotide sequence CRISPRF, which sequence includes T7 promoter, sgRNA sequence and part of sgRNA backbone;

[0045] TYR sgRNA1 primer:

[0046] ATAATACGACTCACTATAGgGGGTGGATGACCGTGAGTCCGTTTTAGAGCTAGAAATAG;

[0047] PX330TRAC R Primer:

[0048] AAAAGCACCGACTCGGTGCC.

[0049] The above primers were used as 100 μl PCR reaction system with a size of 127 bp, and the reaction conditions were (94°C 30s,...

Embodiment 2

[0057] Embodiment 2: Construction TRY knocks out hamster

[0058] 2.1 Obtaining hamster fertilized eggs

[0059] Golden female mice aged 6-8 weeks were selected, and at 9 am on the first day of estrus, pregnant horse serum gonadotropin (PMSG) (15IU / 100g) was intraperitoneally injected to induce superovulation. At 6:00 p.m. on the fourth day, the male mice were caged together for mating. At 9 o'clock in the morning of the second day, check whether the sperm have mated with the reproductive tract microscope.

[0060] The fallopian tubes were taken, fertilized eggs were collected in M2 culture medium, and fertilized eggs were cultured in HEMC-9. Fertilized eggs were used directly for microinjection. The in vitro operation is completed within 30 minutes.

[0061] 2.2 Pronuclear (PN) injection

[0062] 20ng / μl sgRNA1 and 50ng / μl cas9mRNA PN were injected, and fertilized eggs were directly transplanted into surrogate hamster mothers after injection.

[0063] 2.3 Implantation o...

Embodiment 3

[0077] Embodiment 3: TYR albino hamster is used as recipient mouse for the preparation of genetically engineered hamster

[0078] TYR gene homozygous knockout hamsters are albino hamsters, and 6-8 weeks old TYR gene knockout albino hamsters are used to replace pregnant hamsters so that offspring can be distinguished by color after birth. Albino surrogate mice and golden hamster egg donor mice were mated with male mice on the same day. On the second day, the injected fertilized eggs were transplanted into surrogate mice from the oviduct. Fifteen fertilized eggs were implanted into each hamster. The offspring were born on the 16th day and could be distinguished by color after 3 days to identify the albino hamster, and the knockout efficiency of CAS9 was 100%.

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to a method for constructing an albino hamster model based on a CRISPR-Cas9 system. The method for constructing the albino hamster model based on the CRISPR-Cas9 system comprises the following steps: determining a hamster TRY gene targeting site sequence; preparing gRNA (guide ribonucleic acid); performing in-vitro microinjection: collecting fertilized eggs of the hamster, injecting the gRNA and Cas9 RNA into the fertilized eggs of the hamster, and transplanting and cultivating the injected fertilized eggs. The sgRNA of the specific targeting hamster TRY gene prepared by the invention can accurately target a hamster TYR gene and realize gene knockout, and an albino hamster with TYR gene deletion is produced.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for constructing an albino hamster model based on a CRISPR-Cas9 system. Background technique [0002] The golden hamster (hamster) is an important small experimental animal, which is mainly used in tumor, cardiovascular disease and immune research, and is used in a huge amount every year. However, due to the technical limitations of hamster embryo manipulation, the large-scale production of transgenic or gene knockout hamster models at home and abroad has been slow. Different from the standardized embryo transfer technology in mice, hamster fertilized eggs are sensitive to temperature and pH, and it is difficult to operate in vitro. So far, there has been no effective breakthrough in the blockage of fertilized egg 2 cell transplantation, and the success rate of pseudopregnant hamster transplantation is low. Therefore, hamsters are mostly prepared by transp...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N15/89A01K67/027
CPCC12N15/907C12N9/22C12N15/89A01K67/0275A01K2217/075A01K2227/105A01K2267/0331A01K2267/0387A01K2267/0375
Inventor 王全新
Owner 辽宁长生生物技术股份有限公司
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