Single guide ribonucleic acid (sgRNA) capable of effectively editing pig ROSA26 gene, and application of sgRNA

An editing and gene technology, applied in DNA/RNA fragments, recombinant DNA technology, animal husbandry, etc., can solve problems such as limiting the breeding and application prospects of transgenic pigs

Active Publication Date: 2017-07-04
重庆吉渝科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These factors limit the breeding and application prospects of transgenic pigs

Method used

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  • Single guide ribonucleic acid (sgRNA) capable of effectively editing pig ROSA26 gene, and application of sgRNA
  • Single guide ribonucleic acid (sgRNA) capable of effectively editing pig ROSA26 gene, and application of sgRNA
  • Single guide ribonucleic acid (sgRNA) capable of effectively editing pig ROSA26 gene, and application of sgRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1-1. Design of sgRNA sequence and construction of PX330 expression vector

[0025] Three sgRNA sequences targeting porcine ROSA26 locus were designed and synthesized. The shRNA sequence designed above was synthesized; the DNA sequences of the six single-stranded sgRNAs were annealed respectively to form three oligonucleotide chains targeting sgRNAs at different positions in the first intron of porcine ROSA26; and then the oligonucleotides were polymerized Nucleotides were ligated into the PX330 plasmid vector.

[0026] The sequences of the three sgRNAs and the sequences of their action sites are:

[0027] SgRNA-86 sequence: 5-ATCGCAGTGGTAGTCAAGAT-3;

[0028] The sequence of the sgRNA-86 action site: 5-ATCTTGACTACCACTGCGAT-3;

[0029] SgRNA-89 sequence: 5-ATCTTGAGCATAGGCCCAAC-3;

[0030] The sequence of the sgRNA-89 action site: 5-GTTGGGCCTATGCTCAAGAT-3;

[0031] SgRNA-91 sequence: 5-TACGGTCAGATAACTCTCAC-3;

[0032] The sequence of the sgRNA-91 action site: 5-GTGAG...

Embodiment 2

[0042] Construction of targeting vector for site-specific integration of EGFP

[0043] According to the screened high-efficiency sgRNA, design and construct the EGFP gene targeting vector (PUC57-EGFP-KI-Donor) matching the sgRNA. The main components of the targeting vector are: upstream homology arm, SA site, EGFP gene, PolyA sites, downstream homology arms, and backbone vectors for prokaryotic expression. The EGFP site-specific integration targeting plasmid and the screened sgRNA can specifically genetically modify the ROSA26 site of the pig genome, and then combine the method of fluorescence microscopy and PCR to easily analyze the foreign gene at the sgRNA recognition site Feasibility of integration and expression. figure 2 It is a schematic diagram of the EGFP targeted targeting plasmid vector of the present invention.

Embodiment 3

[0045] (1) Co-transfection of PX330 plasmid and PUC57-EGFP-KI-Donor plasmid

[0046] Resuscitate the fetal fibroblasts of the primary pigs, and when they are transferred to the F3 generation, digest the fetal fibroblasts of the F3 pigs, wash with DPBS 2 to 3 times, discard the supernatant, add electrotransfection buffer, and then PX330 Add the plasmid and the PUC57-EGFP-KI-Donor plasmid to the cells and the buffer in proportion, mix gently with a pipette, then gently transfer the mixture into the electrode cup, and place the electrode cup on the electroporation instrument Perform electric shock operation. After the electric shock was completed, the electrode cup was left to stand at 4° C. for 10 minutes, and then the mixture in the electrode cup was transferred to a cell culture dish. Finally, the cell culture dish was placed in a 39°C carbon dioxide incubator for cultivation. After 12 hours of incubation, the medium was changed.

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Abstract

The invention discloses single guide ribonucleic acid (sgRNA) capable of effectively editing a pig ROSA26 gene, and application of the sgRNA. Based on the sgRNA capable of specifically identifying the pig ROSA26 gene in a pig genome, a cell line of enhanced green fluorescent protein (EGFP) site-specific integrated porcine fetal fibroblasts is successfully established by using a CRISPR/Cas9-mediated gene knock-in technique; the results show that the cell line can stably and efficiently express an EGFP gene; furthermore, in the preparation process of the cell line, exogenous promoter genes and positive and negative screening marker genes are not introduced into the cell line. Therefore, the sgRNA greatly improves the safety of transgenic pigs, and has important significance for eliminating the biological and food potential safety hazards of agricultural products of the transgenic pigs.

Description

technical field [0001] The invention discloses a sgRNA capable of effectively editing pig ROSA26 gene and its application, and also discloses a site-specific strategy and feasibility analysis of a safer exogenous gene related to the sgRNA, belonging to the field of biotechnology. [0002] technical background [0003] CRISPR / Cas9 is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight against invading viruses and foreign DNA. The CRISPR / Cas9 system confers immunity by incorporating fragments of invading phage and plasmid DNA into CRISPR and utilizing corresponding CRISPR RNAs (crRNAs) to direct the degradation of homologous sequences. Later, researchers discovered that it is a precise and versatile gene editing tool that can be used to delete, add, activate or inhibit target genes in other organisms, including humans, mice, pigs, cattle, sheep, zebrafish, bacteria , Drosophila, yeast, nematodes, and crops. Usin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11A01K67/027
Inventor 逄大欣谢子聪欧阳红生
Owner 重庆吉渝科技有限公司
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