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Method for producing genetically modified water buffalos by utilizing lentiviral vector

A lentiviral vector and transgenic technology, applied in biochemical equipment and methods, genetic engineering, plant genetic improvement, etc., can solve the difficulty of transgenic buffalo, low gene transfection efficiency, difficult stable integration and long-term expression of exogenous target genes And other issues

Inactive Publication Date: 2011-09-07
GUANGXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Compared with other species, buffalo somatic cells and embryos are not easy to be infected by exogenous genes, and the efficiency of gene transfection is low. At the same time, the exogenous target genes are not easy to be stably integrated and expressed for a long time; increased difficulty

Method used

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  • Method for producing genetically modified water buffalos by utilizing lentiviral vector
  • Method for producing genetically modified water buffalos by utilizing lentiviral vector
  • Method for producing genetically modified water buffalos by utilizing lentiviral vector

Examples

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Embodiment 1

[0031] From 2009 to 2010, buffalo IVF fertilized eggs were infected with CMV-promoted lentivirus, and 582 fertilized eggs were injected, 154 of which developed to the blastocyst stage, accounting for 26.5% of the injected embryos, of which 66 blastocysts Express GFP, accounting for 42.9% of the total number of blastocysts. Take 8 positive blastocysts and transplant them into the uterus of 4 surrogate cows. Two months later, B-ultrasound checks the pregnancy of 2 surrogate cows. Among them, a calf was born on December 17, 2010. The genomic DNA samples of the umbilical cord were detected by PCR, and the expression The exogenous gene GFP and lentivirus expression element gene prove that injection of lentivirus into the perivitelline space of buffalo fertilized eggs can effectively produce transgenic buffalo.

Embodiment 2

[0033]From 2009 to 2010, two lentiviruses with promoters of CMV and Ubc were used to infect buffalo IVF fertilized eggs at different developmental stages respectively. 466 fertilized eggs of different stages were injected respectively, and 113 fertilized eggs developed to the blastocyst stage, accounting for 24.2% of the injected embryos, including 61 blastocysts expressed GFP, accounting for 54.0% of the total blastocysts. This example proves that injection of lentivirus into the perivitelline space of buffalo fertilized eggs at different developmental stages can effectively produce buffalo transgenic embryos.

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Abstract

The invention relates to a method for producing genetically modified water buffalos by utilizing a lentiviral vector, comprising the following steps: carrying out package and concentration on high-titer lentiviral, infecting and screening fetal fibroblast of a water buffalo; using a microinjection method to prepare a genetically modified DNAchimeric embryo of the water buffalo; transplating a positive embryo to the receptor water buffalo. By utilizing the method provided by the invention, exogenous-gene water buffalo fibroblast, DNAchimeric blastaea of the genetically modified water buffalo, more than 30% of somatic cell expression exogenous genes and more than 40% of blastaea expression exogenous genes are obtained and a genetically modified water buffalo with green-fluorescent-protein modified genes is obtained.

Description

technical field [0001] The invention relates to animal biotechnology, in particular to a method for producing transgenic buffaloes using lentiviral vectors. Background technique [0002] Buffalo is one of the main large livestock in southern my country. It has biological characteristics such as strong adaptability, high temperature and high humidity resistance, strong disease resistance, resistance to rough feeding, and long service life. It is very suitable for breeding in rural areas in southern my country. It is the development trend of the buffalo animal husbandry industry to develop and utilize the buffalo breeding resources and transform the existing draft buffalo into commercial buffalo for milk or milk and meat. However, the reproductive rate of buffalo is very low at present, and the production performance of the population is poor. It is urgent to develop and improve a group of high agricultural technologies closely related to livestock genetic improvement to impro...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/873A61D19/04A01K67/027
Inventor 李湘萍石德顺孟凡丽刘庆友陆凤花罗婵蒋建荣
Owner GUANGXI UNIV