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Monopterus albus gene editing method

A gene editing, eel technology, applied in the field of eel gene editing, can solve the problem of not establishing gene editing technology and so on

Active Publication Date: 2016-12-14
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, no gene editing techniques have been established in eels so far

Method used

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  • Monopterus albus gene editing method
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Obtaining fertilized embryos at the 1-cell stage of rice field eel:

[0032] Artificial oxytocin breeding of rice field eels was carried out in an indoor breeding system, and the oxytocin hormone was produced by Ningbo Sansheng Pharmaceutical Co., Ltd. The injection dose of female eel (50-100g) was injected with 3mg of carp pituitary gland (PG), 10μg of luteinizing hormone releasing hormone A2 (LHRH-A2) and 500IU of chorionic gonadotropin (HCG) per 100g of female eel body weight. ). 24 hours after female eel injection, male eel (>200g) was injected with LHRH-A2 at 5μg / 100g body weight. After 40 hours of female eel injection, the eggs were squeezed out; at the same time, male eel sperm were obtained, and the sperm were placed in Danielal buffer (1.74mol / L NaCl, 0.02mol / L KCl, 0.01mol / L MgSO 4 ·7H 2 O,0.02mol / LCa(NO 3 ) 2 4H 2 O, 0.15mol / L Hepes, pH 7.2) immediately poured into the eggs, shake gently to mix, and let stand for 5min to complete the fertilization proce...

Embodiment 2

[0034] Construct the TALENs vector of the eel target gene (Dmrt1):

[0035] Taking the target gene Dmrt1 of rice field eel as an example, the TALENs target site design website (https: / / tale-nt.cac.cornell.edu / ) was used to select the "AGGACAAGCAGCGCAA" sequence of the Dmrt1 gene as the gene editing target. The recognition sequence of the arm is TTACTTCCACTATGAACA, and the recognition sequence of the right arm of TALENs is GCAGGTGCTGGACTGC( figure 1 ). According to the optimized "Golden Gate" cloning method reported in the literature, the TALENs expression vector targeting the rice field eel Dmrt1 gene was assembled and constructed (Liu et al., 2014). Plasmids for each repeat fragment (pNI1-pNI10 corresponds to bases A1-A10, pNN1-pNN10 corresponds to bases G1-G10, pHD1-pHD10 corresponds to bases C1-C10, pNG1-pNG10 corresponds to bases T1-T10) , and the plasmids corresponding to the last base (pLRNI corresponds to base A, pLRNN corresponds to base G, pLRHD corresponds to base ...

Embodiment 3

[0046] Example 3, microinjection of rice field eel 1-cell stage embryo

[0047]A layer of 1% agar was poured on the bottom of a 9 cm petri dish, and then 40 fertilized eggs at the 1-cell stage were placed. Under a stereomicroscope (Olympus, Japan), adjust the eel embryo animal pole upward; mix the left and right arms of TALENs mRNA of Dmrt1, so that the final concentration after mixing is 100 ng / μL; use a nitrogen-pressurized quantitative microinjection system (Warner PLI-100A, USA) were injected one by one into the animal poles of fertilized eggs at the 1-cell stage of rice field eel, and the injection volume of each fertilized egg was 2nL.

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Abstract

The invention discloses a monopterus albus gene editing method. The monopterus albus gene editing method comprises the following steps: 1, acquiring an artificially inseminated monopterus albus 1-cell stage embryo by establishing an indoor eel whole artificial propagation technology; 2, establishing a transcription activator-like effector nuclease gene editing vector of a target gene, performing in-vitro transcription to synthesize mRNA, and transferring the mRNA into the monopterus albus 1-cell stage fertilization embryo by adopting a microinjection method; 3, screening to obtain a monopterus albus with a mutant target gene through a three-primer detection method, and determining the mutation type of the target gene through sequencing. According to the method, a micromanipulation technology for a monopterus albus embryo is established for the first time, a precise monopterus albus endogenous gene editing technology is established for the first time, and powerful technological means is provided for performing function research on a monopterus albus gene and genetic improvement of monopterus albus breeding variety.

Description

technical field [0001] The invention relates to the technical field of aquatic animal genetic breeding and functional gene verification research, and more specifically relates to a method for gene editing of rice field eels. Background technique [0002] Field eel (Monopterus albus), commonly known as eel, belongs to the order Synbranchidae, the family Synbranchidae, and the genus Monopterus albus. Rice field eel has delicious meat and high nutritional value. It is a famous and excellent object of aquaculture in my country and has important economic value. Statistics show that the demand for rice field eels at home and abroad is more than 3 million tons. However, in 2014, the national rice field eel farming output was only 357,000 tons (2015 China Fishery Statistical Yearbook). With the improvement of people's living standards, the market demand for rice field eels is still increasing year by year. However, rice field eel has the biological characteristics of hermaphroditi...

Claims

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Application Information

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IPC IPC(8): C12N15/89A01K67/027
CPCA01K67/0276A01K2227/40A01K2267/02C12N15/89
Inventor 胡炜冯科陈戟朱作言
Owner INST OF AQUATIC LIFE ACAD SINICA
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