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Porcine somatostatin gene editing site and application thereof

A technology of somatostatin and gene editing, applied in the fields of somatostatin, application, genetic engineering, etc., which can solve the problems of complex operation process, few applications, and high off-target rate

Pending Publication Date: 2018-02-16
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the technologies represented by ZFN and TALEN can achieve targeted genome modification, but they have certain cytotoxicity, complicated operation process, and high off-target rate, so they are rarely used in mammalian genome editing.

Method used

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  • Porcine somatostatin gene editing site and application thereof
  • Porcine somatostatin gene editing site and application thereof
  • Porcine somatostatin gene editing site and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Design and synthesis of sgRNA for specifically targeting SST gene in CRISPR / Cas9 specific knockout pig SST gene

[0031] 1. Design of sgRNA targeting porcine SST gene

[0032] Using the website to design SST-sgRNA, the specific steps are: ① Log in to the GenBank website, search for the sequence of the porcine SST gene and download it, the nucleotide sequence of which is shown in SEQ ID NO:2. ② Open the target site prediction website (http: / / crispr.mit.edu / ), use the target site online design tool "CRISPR design tool", enter the gene sequence in the text box, select the species, and the gene sequence can be obtained For all target sites, several groups of target sites are selected after comprehensive consideration of various parameters. ③ The 20nt oligonucleotide sgRNA core sequence was designed according to the GG(N)19NGG sequence.

[0033] 2. Construction of sgRNA oligonucleotides

[0034] According to the selected sgRNA, add AAACACCG to the 5' end of the ...

Embodiment 2

[0038] The preparation of embodiment 2sgRNA, Cas9mRNA

[0039] 1. Main reagents:

[0040] pmLM3613 (Addgene No.42251), BsaI, and PmeI enzymes were purchased from NEB Company; MEGA shortscript Kit (AM1354) sgRNA in vitro transcription kit, mMESSAGE mMACHINE Kit (AM1344) Cas9 in vitro transcription kit, Poly(A) Tailing Kit were purchased from Ambion Company; pDR274 (Addgene No.42250) plasmid was provided by Hubei Provincial Key Laboratory of Animal Embryo Engineering and Molecular Breeding; other commonly used reagents were domestic analytically pure; primer synthesis and DNA sequencing were completed by Shenzhen Huada Gene Company.

[0041] 2. Operation steps:

[0042] (1) In vitro transcription to obtain sgRNA:

[0043] 1) Construction of plasmid pDR274 expressing sgRNA (such as figure 2 shown).

[0044] ①Synthesis of double-strand oligonucleotide strands: sgRNA-g1 sites with high target activity add TAGG to the 5' end of the sense strand template, which is complementary to...

Embodiment 3

[0058] The collection of embodiment 3 oocytes and in vitro maturation culture

[0059] 1. Main solution preparation

[0060] (1) Preparation of oocyte maturation medium in vitro: the maturation medium is composed of improved TCM-199,

[0061] That is to add 3.05mmol / L glucose, 1mmol / L L-glutamine, 0.91mmol / L sodium pyruvate, 0.57mmol / L L-cysteine, 75ug / mL penicillin, 50ug / mL streptomycin sulfate. After mixing according to the above components, adjust the pH to 7.2-7.4, filter on the ultra-clean workbench, and store at 4°C for later use. 2 hours before the experiment, 10 IU / mL hCG, 10 IU / mL PMSG, and 10% pFF were added to the culture medium, and it was preheated in a constant temperature incubator for use.

[0062] (2) Preparation of embryo culture medium: composed of NCSU-23 basal medium + 4mg / mL BSA. NCSU-23 components are: 108.73mmol / L NaCl, 1.19mmol / L MgSO 4 , 25.07mmol / L NaHCO 3 , 4.78mmol / L KCl, 1.70mmol / L CaCl 2 , 5.55mmol / L glucose, 1.19mmol / L KH 2 PO 4 , 1.00...

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Abstract

The invention discloses a porcine somatostatin gene editing site and application thereof. The nucleotide sequence is as shown in SEQ ID NO:1. By injecting sgRNA and Cas9mRNA containing the coding sequence into porcine embryos, site-directed mutagenesis of porcine SST gene is realized. Through the CRISPR-Cas9 technique, porcine endogenous gene SST is edited at the embryo level. Statistical resultsshow that the in-vitro digestion activity is 65% after SSA luciferase detection at the site, porcine SST gene can be knocked out at the embryo level, and point mutation of SST is formed in cells. Theporcine somatostatin gene editing site is of great significance for promoting application of the endogenous gene knockout or exogenous gene site-specific integration technology in the research and production of transgenic pigs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a porcine somatostatin gene editing site and application thereof, in particular to a porcine somatostatin gene SST editing site (30-52) and application thereof. Background technique [0002] It has always been extremely difficult to accurately knock foreign genes into the pig genome. Common methods such as microinjection, electroporation, calcium phosphate precipitation, and retroviral vector infection cannot achieve targeted integration of exogenous genes at specific locations in the genome due to their randomness; although gene targeting technology that relies on homologous recombination can The source gene is introduced into the specific position of the target cell, but the gene targeting efficiency is low, the operation is difficult, and the marker gene introduced in the cell screening process will also cause potential biological safety hazards in the later stage. These prob...

Claims

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Application Information

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IPC IPC(8): C12N15/16C12N15/113C12N15/90
CPCC07K14/655C12N15/113C12N15/907
Inventor 任红艳毕延震李晓敏李莉华再东郑新民刘西梅肖红卫华文君张立苹
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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