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Compositions and methods for modifying the content of polyunsaturated fatty acids in biological cells

A fatty acid and substance technology, applied in the field of compositions for changing the content of polyunsaturated fatty acids in animal cells, can solve problems such as lack of 12- and 15-dehydrogenase activities

Inactive Publication Date: 2007-04-18
THE GENERAL HOSPITAL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although for the most part animals contain enzymes that convert LA (18:2n-6) and ALA (18:3n-3) to long-chain PUFAs (where conversion rates are limited), they lack synthetic 12- and 15-dehydrogenase activities necessary for precursors (precursors) PUFA, LA, ALA (please refer to Knutzon et al., J.Biol.Chem. 273 : 29360-29366, 1998)

Method used

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  • Compositions and methods for modifying the content of polyunsaturated fatty acids in biological cells
  • Compositions and methods for modifying the content of polyunsaturated fatty acids in biological cells
  • Compositions and methods for modifying the content of polyunsaturated fatty acids in biological cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Construction of recombinant adenovirus

[0081] The recombinant adenovirus carrying the fat-1 gene was constructed following the steps similar to those described by He et al. (please refer to Proc. Natl. Acad. Sci. USA95: 2509-2514, 1998). The n-3 fatty acid dehydrogenase cDNA (fat-1 gene) in pCE8 was kindly provided by Dr. J. Browse (Washington State University) (but can also be synthesized or cloned using common techniques and information in the art; please refer to Spychalla et al. al., Proc. Natl. Acad. Sci. USA 94:1142-1147, 1997; US Pat. No. 6,194,167; and refer to FIGS. 17A and 17B). The cDNA insert gene of pCE8 was excised from the plasmid by restriction enzyme EcoRI / Kpnl double shearing reaction and inserted into the transfer vector (shutter vector), and then the adenovirus backbone was recombined according to the method of He et al. (supra). Two first-generation recombinant adenoviruses of type 5 were formed: Ad.GFP carrying green fluorescent prote...

Embodiment 2

[0083] Example 2: Adenovirus culture and infection of cardiomyocytes

[0084] Cardiomyocytes were isolated from one day old mice using the National Cardiomyocyte Isolation System (provided by Worthington Biochemical Corp., Freehold, NJ). Then place it in a 6-well plate, place it in a tissue culture machine (containing 5% CO) in F-10 culture medium (containing 5% fetal bovine serum, 30% horse serum, 2 and 98% relative humidity) for cultivation. Cells used in experiments need to be cultured for 2-3 days. Add different concentrations (5*10 9 -10 10 pfu) of virus particles. After culturing for 24 hours, 10 μM of 18:2n-6 and 20:4n-6 were added to the culture medium. Forty-eight hours after infection, the cells can be used (eg, for analysis of gene expression, fatty acid composition, viability, or growth capacity (eg, proliferation or division rate)).

Embodiment 3

[0085] Example 3: Detection of fat-1 expression and RNA analysis by fluorescence microscopy

[0086] There are many well-known methods in the field of molecular biology to detect gene expression. Here, the expression of fat-1 in cardiomyocytes after the above-mentioned infection steps was measured by visual detection of infected cells and RNase enzyme protection assay.

[0087] In detail, GFP co-expression was used to identify infected cells as well as cells expressing the transgene. Approximately 48 hours after infection, almost all cells (>90%) showed bright fluorescence, indicating a high gene transfer rate and a high expression level of the transgene (see Figure 1). Expression of Fat-1 gene transcripts can also be performed using RPA III TM The equipment (provided by Ambion) was determined by the RNase enzyme protection test method. Briefly, total RNA was extracted from cultured cells using RNA extraction equipment (provided by Qiagen) according to the manufacturer's pr...

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Abstract

The present invention features compositions and methods for effectively altering PUFA content in animal cells, i.e., cells other than C. elegans, such as cells of birds or fish, such as muscle cells, nerve cells ( in the peripheral or central nervous system), adipocytes, endothelial cells, and cancer cells, the composition includes a nucleic acid encoding fat-1, which is optionally and operably linked to a persistently active or tissue-specific promoter or Other regulatory sequences, and pharmaceutically acceptable formulations with nucleic acids or biologically active variants thereof. The compositions and methods include a modified fat-1 gene, wherein the gene contains at least one optimized codon. The modified cells, whether in vivo or in vitro (i.e., in tissue culture), transgenic animals (especially fish and birds) comprising these modified cells, and food obtained from those animals (also That is, meat or other edible parts of animals (ie, liver, kidney or pancreas) are also included within the scope of the present invention.

Description

[0001] related application [0002] This application claims priority to US Provisional Application No. 60 / 542098, filed February 4, 2004, and US Provisional Application No. 60 / 555422, filed March 22, 2004. Both applications are also incorporated herein by reference in their entirety. [0003] governmental support [0004] Portions of this work were supported by grants from the National Institutes of Health (CA79553). Accordingly, the US Government has certain rights in this invention. [0005] The technical field to which the invention belongs [0006] The invention relates to a composition and a method for changing the polyunsaturated fatty acid content in animal cells. 【Background technique】 [0007] Polyunsaturated fatty acids (Polyunsaturated Fatty Acids, PUFA) refer to fatty acids with 18 or more carbon atoms and two or more double bonds in the carbon chain. They can be divided into two groups according to the position of the double bond closest to the methyl termina...

Claims

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Application Information

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IPC IPC(8): A01N63/00C07H21/02C07H21/04C12N15/00C12N15/63
Inventor J·X·康
Owner THE GENERAL HOSPITAL CORP
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