Method for breeding genetically modified animal by using recombinant adenovirus vector

A technology for recombinant adenovirus and transgenic animals, applied in the fields of molecular biology, genetic engineering, and cell biology, can solve the problems of low overall efficiency, simple and efficient transgenic methods, early embryo damage, etc.

Inactive Publication Date: 2009-09-02
NORTHWEST A & F UNIV
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Problems solved by technology

However, this method has unavoidable defects: it is necessary to use a microscopic operating system to precisely inject exogenous DNA into the male pronucleus of fertilized eggs, which will inevitably

Method used

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  • Method for breeding genetically modified animal by using recombinant adenovirus vector
  • Method for breeding genetically modified animal by using recombinant adenovirus vector
  • Method for breeding genetically modified animal by using recombinant adenovirus vector

Examples

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Embodiment 1

[0020] Example 1 Preparation method of human thrombin III transgenic animal

[0021] (1) Synthesis, cloning and sequence determination of target protein (such as human thrombin III) cDNA:

[0022] 1. Extraction of total RNA from human liver tissue

[0023] (1) Cut a small piece of frozen tissue, quickly weigh it, and then transfer it into the freshly prepared lysis buffer, and add 1 ml of lysis buffer for every 50 mg of tissue.

[0024] (2) Homogenize the liver tissue to break the cells, pre-filter and centrifuge once, and collect the filtrate.

[0025] (3) Add an equal volume of chloroform, shake and mix, centrifuge and aspirate the supernatant.

[0026] (4) Add an equal volume of 70% ethanol, mix gently, and centrifuge to obtain total RNA.

[0027] (5) Dissolve the precipitate in Rnase-free water treated with 0.01% DEPC, and perform content determination by conventional methods.

[0028] 2. Synthesis of total cDNA by reverse transcription

[0029] (1) Take 1~2μg of total breast ...

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Abstract

The invention relates to a method for breeding a genetically modified animal by using a recombinant adenovirus vector. The invention adopts a technical proposal that: a replication-defective adenoviral framework plasmid vector carries a foreign target gene sequence to produce a recombinant adenovirus vector in a packaging cell (293, 911 and other cells) containing a sequence of an Ela region; the recombinant adenovirus vector is combined with a donor egg or fertilized egg cell; and the donor egg or fertilized egg cell is first cultured and developed in vitro and then transferred into the body of a receptor animal for normally breeding so as to produce the genetically modified animal which is genetically stable and has functions of different target genes. The method for breeding the genetically modified animal is high in success rate. The genetically modified animal can be used in the confirmation of functional genes, the expression of pharmaceutical proteins (including organs, blood and mammary glands), and the building of models of genetically modified animal having functions of different target genes and animal genetic diseases.

Description

Technical field [0001] The invention belongs to the technical fields of cell biology, molecular biology and genetic engineering, in particular to a method for preparing transgenic animals by using adenovirus as a carrier. Background technique [0002] With the advent of the post-genomic era of life sciences, the research and application of genes and their products has attracted more and more attention from researchers. Transgenic animal technology is one of the most effective technical methods. At present, internationally The strategy is to process fertilized eggs, eggs and sperm to obtain transgenic animals from three aspects. In view of the instability of egg and sperm processing methods, in the preparation of transgenic animals, the fertilized egg processing method is still the most effective and classic method. Now, the existing method of preparing transgenic animals is processed for fertilized eggs. The summary is as follows. [0003] Prokaryotic phase microinjection of fore...

Claims

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Application Information

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IPC IPC(8): C12N15/861A01K67/027C12R1/93
Inventor 李青旺韩增胜赵红卫胡建宏宁国柱
Owner NORTHWEST A & F UNIV
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