Targeted sgRNA for editing pig APN gene and modified carrier as well as preparation method and application thereof

A technology of gene editing and gene modification, applied in the field of genetic engineering, to achieve strong specificity, clean and clear genetic background, and reduce the work of transgenic safety assessment

Inactive Publication Date: 2017-08-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although more and more genes are currently being used as biomarkers and therapeutic targets for livestock diseases, the key genes and their editing methods for piglet diarrhea have not yet been applied in my country

Method used

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  • Targeted sgRNA for editing pig APN gene and modified carrier as well as preparation method and application thereof
  • Targeted sgRNA for editing pig APN gene and modified carrier as well as preparation method and application thereof
  • Targeted sgRNA for editing pig APN gene and modified carrier as well as preparation method and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0049] The present invention also provides the preparation method of above-mentioned porcine APN gene modification carrier, comprises the following steps:

[0050] Step (a): Complementary pairing of the sense strand and the antisense strand of sgRNA-A213 to form an A213 double-stranded DNA molecule;

[0051] Step (b): Complementary pairing of the sense strand and the antisense strand of sgRNA-A258 to form an A258 double-stranded DNA molecule;

[0052] Step (c): connecting the A213 double-stranded DNA molecule to an expression vector to construct a vector expressing sgRNA-A213;

[0053] Step (d): connecting the A258 double-stranded DNA molecule to an expression vector to construct a vector expressing sgRNA-A258;

[0054] Step (e): Using the vector expressing sgRNA-A213 as a template, amplify the sgRNA-A213 sequence containing the U6 promoter by PCR, connect it to the cloning vector, and obtain the U6-sgRNA-A213 sequence after enzyme digestion;

[0055] Step (f): link the U6-s...

Embodiment 1

[0070] The design of embodiment 1sgRNA and the construction of carrier

[0071] According to the DNA sequence (as shown in SEQ ID NO.5) and mRNA sequence (as shown in SEQ ID NO.6) of the porcine APN gene in NCBI, two sgRNAs are designed in the coding frame region of the APN gene, which are sgRNA-A213 and sgRNA -A258, adding cohesive linker sequences to its two ends, respectively. Add the adapter sequence CACC to the 5' end of the F strand of each sgRNA sequence, and add the adapter sequence AAAC to the 5' end of the R strand of the reverse complementary sequence. If the first base at the 5' end of the sgRNA sequence is not G, then it should be First add a G to the 5' end of the F chain of the sgRNA sequence, and then add the linker sequence CACC. Correspondingly, add a C to the 3' end of the R chain of its reverse complementary sequence, so that it can be combined with the PX461 vector digested by BbsI The cohesive ends are complementary.

[0072] The sense and antisense str...

Embodiment 2

[0097] Example 2 Transfection of pig somatic cells and screening of APN gene edited cell clones

[0098] Cell culture and transient transfection:

[0099] Inoculate pig fetal fibroblasts in a 6-well cell culture plate, and when cultured to 50-70% confluence in DMEM medium containing 15% fetal bovine serum, according to the requirements of the instructions, use Lipofectamine 2000 liposomes to inoculate Example 1 The provided vector PX461-A258+A213 was transferred into the cells.

[0100] Flow cytometric sorting and monoclonal culture of transfected cells:

[0101] The transfected cells were cultured in a 37°C incubator for 48 hours, digested with 0.1% trypsin, sorted the positive cells expressing GFP by flow cytometry, and seeded the cells at a density of 50-100 cells / 100mm culture dish , cultured in DMEM medium containing 15% fetal bovine serum and 2.5ng / mL fibroblast growth factor (bFGF) for 9 to 12 days until obvious single-cell colonies appeared.

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Abstract

The invention provides a targeted sgRNA for editing a pig APN gene and a modified carrier as well as a preparation method and application thereof, and relates to the technical field of gene engineering. The sgRNA and the gene modified carrier provided by the invention are strong in speciality, and effectively edit the pig APN gene on cell level by a CRISPR/Cas9n system, and positive cells are edited by the obtained APN gene into donor cells to carry out somatic cell cloning and embryo transferring, so that the obtained APN gene edited cloning pig has ability of resisting piglet diarrhea. According to the preparation method of the piglet diarrhea resistant pig provided by the invention, a receptor of a virus causing the piglet diarrhea is destroyed, any other exogenous genes cannot be introduced except edition of a targeted gene APN, non-speciality edition on a non-APN gene area on a gene group cannot be carried out, a genetic background is clean and clear, and a late safety assessment work of transgenesis is greatly reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a targeting sgRNA for porcine APN gene editing, a modified carrier and a preparation method and application thereof. Background technique [0002] Piglet diarrhea is a highly infectious disease in newborn piglets and weaned piglets, which brings huge economic losses and harm to the pig industry in my country. Currently, porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) and transmissible gastroenteritis virus (Transmissible gastroenteritis virus, TGEV) are considered to be the main and most common viruses that cause diarrhea in piglets. [0003] Many studies at the cell level have shown that porcine aminopeptidase (pAPN) is the main receptor for PEDV and TGEV infecting pigs. In the study of producing pigs resistant to PRRS, it was found that pigs whose receptors for PRRS virus have been destroyed by gene editing technology will not be infected by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N15/877C12N15/66A01K67/027
CPCC12N15/1137A01K67/0276A01K2217/075A01K2227/108A01K2267/02C12N15/66C12N15/8509C12N15/8778C12N15/907
Inventor 张坤王少华
Owner ZHEJIANG UNIV
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