Zinc finger nuclease knockout specific target site of myostatin gene

A DNA molecule and encoding technology, applied in genetic engineering, plant genetic improvement, and microbial-based methods, can solve problems such as heavy experimental workload, low success rate, and long cycle time, and achieve improved research efficiency and gene knockout high efficiency effect

Active Publication Date: 2011-12-28
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, although a variety of gene knockout animal models have been successfully prepared using this technology, the shortcomings of large experimental workload, long cycle time, and low success rate have always restricted the application of traditional gene knockout technology.
The efficiency of traditional gene knockout is about 1 / 10,000, or even lower

Method used

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  • Zinc finger nuclease knockout specific target site of myostatin gene
  • Zinc finger nuclease knockout specific target site of myostatin gene
  • Zinc finger nuclease knockout specific target site of myostatin gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Design and Construction of ZFN Plasmid for Specific Target Site Sequence of Porcine MSTN Gene

[0028] 1. Design of specific target site sequence for porcine MSTN gene

[0029] Construct a zinc liponuclease (ZFN) plasmid pZFN plasmid that knocks out specific sites in the target sequence of the Meishan pig MSTN gene. The ZFN is composed of a DNA binding domain and a DNA cutting domain. The ZFN pair specifically recognizes and binds ≥ 24 bp bases in the coding region , to ensure the high precision and high efficiency of ZFN cutting MSTN gene.

[0030] Zinc lipid nuclease (ZFN) specific knockout target site sequence is located in the second exon of MSTN gene (the nucleotide sequence of MSTN is sequence 4), and the specific target site sequence is CCTTCCCAGGAC cagga GAAGATGGGCTGGTA (artificially synthesized, sequence 1, Sequence 1 is the 3770-3801 nucleotides from the 5' end of sequence 4), see for details figure 1 As shown, the partial sequences at both ends (...

Embodiment 2

[0034] Example 2, zinc finger nuclease site-directed knockout of MSTN gene

[0035] 1. Isolation and culture of pig fetal fibroblast primary cells:

[0036] (1) Select purebred Meishan pregnant sows (Taicang Breeding Pig Farm) who have been pregnant for 35 days as experimental animals, take out the uterus containing the embryos through surgery, place them in a sterile beaker, seal with a parafilm and bring them into the laboratory;

[0037] (2) Remove the capsule around the embryo in a sterile ultra-clean bench, and place the embryo in a sterile beaker with PBS added;

[0038] (3) Transfer the embryos one by one to a small sterile petri dish, remove the head, tail and limbs of the embryo with sterile scissors and forceps, and cut the tissue into 1mm 3 sized pieces, and add a little serum to moisten the tissue;

[0039] (4) Spread the small pieces evenly on the wall of the bottle, and the distance between each small piece is about 0.2cm-0.5cm. After the small pieces are coate...

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Abstract

The invention discloses a specific target site for site-directed knockout of gene Myostatin (MSTN) by zinc finger nuclease (ZFN). The invention provides a DNA molecule, which can be as shown in the following (1) or (2) or (3): (1) is the DNA molecule shown in sequence 1 of a sequence table; (2) is the DNA molecule which hybridizes with a DNA sequence restricted by (1) under a strict condition and encodes an associated protein with the same function. Experiments of the invention prove that, the invention positions a specific target sequence site DNA fragment in the MSTN gene and constructs ZFN plasmids to the specific site, and nucleofaction of the plasmids to porcine embryonic fibroblasts can realize site-directed knockout or mutation of the specific target sequence in the MSTN gene, and can thoroughly destroy the expression of the MSTN gene.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a zinc finger nuclease to knock out the specific target site of Myostatin gene. Background technique [0002] The traditional gene knockout technology is based on the Cre / LoxP system. Through homologous recombination, the exogenous gene is integrated into a certain site on the genome of the target cell, so as to achieve the purpose of site-specific modification and transformation of a certain gene on the chromosome. a technique. So far, although a variety of gene knockout animal models have been successfully prepared using this technology, the shortcomings of large experimental workload, long cycle time, and low success rate have always restricted the application of traditional gene knockout technology. The efficiency of traditional gene knockout is about 1 in 10,000, or even lower. [0003] Zinc Finger Nuclease (ZFN) is a chimeric protein formed by the recombination of the cleavag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/63C12N5/10C12N1/21C12N15/85C12R1/19
Inventor 崔文涛汤茂学钱丽丽李奎
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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