Methods and compositions for generating a genetically modified animal using lentiviral vectors

A technology of lentiviral vectors and compositions, applied in the field of methods and compositions, capable of solving problems such as low embryo survival rate

Inactive Publication Date: 2005-06-29
OZGENE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

While nuclear transfer ensures that all animals born are transgenic, embryo survival rates are low and the technique itself has its own problems

Method used

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  • Methods and compositions for generating a genetically modified animal using lentiviral vectors
  • Methods and compositions for generating a genetically modified animal using lentiviral vectors
  • Methods and compositions for generating a genetically modified animal using lentiviral vectors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0175] Example 1: Generation of Genetically Modified Mice by Exposure of Lentiviral Vectors to the Plasma Membrane

[0176] A. Lentiviral transfer vector

[0177] Plasmid:

[0178] To construct the pPCW-eGFP gene transfer vector, a PCR-amplified DNA fragment containing EGFP cDNA (derived from pEGFP-C1, Clontech) was ligated into the BamHI / XhoI site of the pHR-CMV-LacZ plasmid (Naldini et al., Science 272: 263-7, 1996), produce pHR-CMV-eGFP; then PCR amplify the 150bp DNA sequence (4327-4483) containing central polypurine sequence (PPT) and central termination site (CTS) of HIV-1 pSG3 molecular clone ( Ghosh et al., Virology 194:858-864, 1993), and ligated into the unique ClaI site of pHR-CMV-eGFP. To reduce eGFP expression (Zufferey et al., J. Virol. 7:2886, 1999), a post-transcriptional regulatory element derived from duck hepatitis virus (WPRE) was inserted downstream of eGFP, generating the pPCW-eGFP gene transfer vector. This construct was used in the cell experiments i...

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Abstract

The present invention provides methods and compositions for integrating a nucleotide sequence of interest into a genome. Specifically, the method of the present invention comprises: contacting the lentiviral vector with oocytes, early embryos or blastocysts under conditions that allow entry of the viral vector and subsequent integration of the nucleotide sequence of interest into the genome. The method also includes culturing the oocyte or embryo contacted with the lentiviral vector under conditions that allow the formation of a pre-implantation embryo. The pre-implantation embryo can be conceived to be transferred into a recipient vertebrate, allowing it to develop into at least one genetically modified animal. The methods and compositions of the invention thus enable the production of genetically modified animals, in particular vertebrates, especially mammals.

Description

field of invention [0001] The present invention provides methods and compositions for integrating a nucleotide sequence of interest into the genome of an animal. Background of the invention [0002] The ability to manipulate the genetic makeup of animals in model organisms is widely exploited to functionally characterize genes and dissect complex networks of cooperating genes. Genetically modified model organisms have been shown to provide promising avenues for understanding gene function. For example, genetic modification of livestock allows the search for improved disease resistance, growth rate, husbandry efficiency, carcass composition, and ways to increase the nutritional quality and health benefits of meat products. However, current methods of producing such genetically modified animals remain cost-effective and inefficient. [0003] Pronuclear microinjection has been favored for some time to generate transgenic animals. In this method, microinjection of pronuclei c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A01K67/027C07HC12N5/00C12N15/00C12N15/867
CPCA01K67/0275A01K2217/05A01K2227/105C12N2740/15043A01K67/027C12N5/10C12N15/867
Inventor R·拉马哈德兰F·科恩特金J·维基菲尔德
Owner OZGENE
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