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sgRNA for specifically recognizing pig AP2 site and application of sgRNA

A specific recognition and species-specific technology, applied in the field of genetic engineering, can solve the problems of inaccurate repair and loss of gene function, and achieve the effect of strong specificity and reducing off-target phenomena.

Pending Publication Date: 2020-09-11
烟台佰博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CRISPR system can generate double-strand breaks at the target sequence, and the body can then use non-homologous end link repair mechanisms to repair itself, but this repair is often imprecise and can lead to loss of gene function

Method used

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  • sgRNA for specifically recognizing pig AP2 site and application of sgRNA
  • sgRNA for specifically recognizing pig AP2 site and application of sgRNA
  • sgRNA for specifically recognizing pig AP2 site and application of sgRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Construction of sgRNA expression plasmids.

[0040] A sequence encoding sgRNA upstream of the stop codon of the porcine AP2 gene (GI: 399533) was selected, as shown in SEQ ID NO.2, specifically: ATGTATCATGAAAGGTGTCA, named AP2-sgR.

[0041] Extract the coding region of the AP2 gene, and the target position is as follows (the underline is the guide sequence AP2-sgR):

[0042]

[0043] Add G to the 5' end of the above-mentioned guide sequence AP2-sgR to obtain a forward oligonucleotide, and add C to the 3' end of its complementary sequence to obtain a reverse oligonucleotide, and the oligonucleotide chains to be synthesized were respectively Named AP2-sgR-F (shown in SEQ ID NO.3) and AP2-sgR-R (shown in SEQ ID NO.4), the sequences are shown in the table below, and the underlined part is the restriction site:

[0044] sequence name Sequence (5'-3') AP2-sgR-F accg GATGTATCATGAAAGGTGTCA

AP2-sgR-R aaac TGACACCTTTCATGATACATC

[0045...

Embodiment 2

[0049] Efficiency verification of sgRNA expression plasmids.

[0050] (1) Nucleofection

[0051] The recombinant plasmid pgl3-AP2-sgR obtained in Example 1 was transfected into 3D4-Cas9 cell line by liposome transfection, the specific operation process is as follows:

[0052] The day before the transfection, the 3D4-Cas9 cell line was inoculated into a 6-well culture dish, and the transfection was performed when the cell density reached about 80% on the second day, and the operation was performed according to the operation manual of lipo2000 (Invitrogen). After 24 hours of transfection, the fluorescence can be observed. After 48 hours of transfection, cells with GFP fluorescence were sorted by flow cytometry.

[0053] (2) Cell Genomic DNA Extraction

[0054] Collect the cells obtained by the above sorting into EP tubes, and extract the genomic DNA of the cells. The specific operation process is as follows:

[0055] ① Add 200uL buffer GA to the EP tube containing the cell pe...

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Abstract

The invention discloses sgRNA for specifically recognizing a pig AP2 site. The sgRNA is authenticated at the upstream of a termination codon of a pig AP2 gene, is strong in specificity and is capableof effectively reducing off-target phenomena in a CRISPR / Cas9 system; and the sgRNA is capable of guiding Cas9 nuclease to realize accurate and efficient targeted modification on the pig AP2 site, soas to analyze the functions of the AP2 gene, so that a foundation is laid for authentication of a novel friendly locus; and besides, the AP2 gene is a gene capable of realizing specific expression infat cells, and specific expression of exogenous gene tissues is expected to be achieved through the successful authentication of the friendly locus.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a sgRNA for specifically recognizing porcine AP2 sites and applications thereof. Background technique [0002] In the process of constructing transgenic animals and gene gain-of-function research, whether foreign genes can be expressed correctly, efficiently and stably in the animal genome is the key to the success of transgenic technology. The random integration of exogenous genes in the animal genome will cause expression instability or epigenetic modification and silence expression due to the position effect and dosage effect of the gene. However, site-specific integration of exogenous genes into specific sites will avoid the occurrence of the above situation, and enable the continuous and stable expression of exogenous genes in the genome. In recent years, with the emergence of ZFN and TALEN technologies, especially CRISPR / cas9 technology, new tools have been p...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N15/12
CPCC07K14/47C12N15/113C12N15/85C12N15/907C12N2800/107C12N2310/20
Inventor 阮进学庄荣志李奎吴添文王超
Owner 烟台佰博生物科技有限公司