Tissue products derived from animals lacking any expression of functional alpha 1,3 galactosyltransferase

A galactosyl and transferase technology, which can be used in bone implants, skin transplantation, medical science, etc., and can solve problems such as allele effects and phenotype changes.

Inactive Publication Date: 2007-04-25
REVIVICOR INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0027] Despite predictions and predictions, no one knows whether disrupting both alleles of the α-1,3-GT gene will be lethal or whether it will affect pig development or lead to phenotypic cha

Method used

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  • Tissue products derived from animals lacking any expression of functional alpha 1,3 galactosyltransferase
  • Tissue products derived from animals lacking any expression of functional alpha 1,3 galactosyltransferase
  • Tissue products derived from animals lacking any expression of functional alpha 1,3 galactosyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0284] Generating pig cells that are heterozygous for the α-1,3-GT gene

[0285] Isolation and Transfection of Primary Pig Fetal Fibroblasts

[0286] Fetal fibroblasts (PCFF4-1 to PCFF4-10) were isolated from 10 fetuses of the same pregnancy on the 33rd day of pregnancy. After removing the head and internal organs, the fetus was washed with Hanks balanced salt solution (HBSS; Gibco-BRL, Rockville, MD), placed in 20ml HBSS, and cut into pieces with small surgical scissors. The tissue was pelleted and resuspended in a 50ml test tube containing 40ml DMEM and 100 U / ml collagenase per fetus (Gibco-BRL). The tube was incubated in a shaking water bath at 37°C for 40 minutes. The digested tissue is allowed to settle for 3-4 minutes, and the cell-rich supernatant is transferred to a new 50 ml test tube and pelleted. The cells were then resuspended in 40 ml DMEM containing 10% fetal calf serum (FCS), 1X non-essential amino acids, 1 mM sodium pyruvate and 2ng / ml bFGF, and inoculated into a 1...

Embodiment 2

[0298] Preparation of pig cells heterozygous for α-1,3-GT gene

[0299] Heterozygous α-1,3-GT knockout fetal fibroblasts (657A-I11 1-6) cells were isolated from the fetus on the 32nd day of pregnancy as described above (see also Dai et al. Nature Biotechnology 20: 451 (2002)). After removing the head and internal organs, some fetuses were washed with Hanks balanced salt solution (HBSS; Gibco-BRL, Rockville, MD), placed in 20ml HBSS, and cut into pieces with small surgical scissors. The tissue is pelleted and resuspended in a 50ml test tube containing 40ml DMEM and 100U / ml collagenase (Gibco-BRL) per fetus. The test tube was incubated in a shaking water bath at 37°C for 40 minutes. The digested tissue is allowed to settle for 3-4 minutes, and the cell-rich supernatant is transferred to a new 50 ml test tube and pelleted. Then resuspend the cells in 40ml containing 10% fetal calf serum (FCS), 1X non-essential amino acids, 1mM sodium pyruvate (Gibco-BRL) and 2ng / ml basic fibroblast g...

Embodiment 3

[0301] Selection of pig cells homozygous for α-1,3-GT gene with Clostridium difficile toxin A

[0302] Toxin A cytotoxicity curve

[0303] Porcine cells (PCFF4-6) were exposed to a 10-fold serial dilution of toxin A (0.00001μg / ml-10μg / ml) for 1 hour or overnight. Culture the cells in a 24-well plate and incubate with toxin at 37°C for 1 hour or overnight. The results of this exposure are detailed in Table 2. Obviously, exposure to 1 μg / ml toxin A for 1 hour resulted in a cytotoxic effect on >90% of cells. Therefore, a toxin A concentration of 1 μg / ml or slightly higher than 1 μg / ml is selected to select genetically altered cells.

[0304] Table 2. Toxin A toxicity after 1 hour and overnight exposure

[0305] [Toxin A], μg / ml

Incubate for 1 hour

Incubate overnight

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Abstract

The present invention provides tissues derived from animals, which lack any expression of functional alpha 1,3 galactosyltransferase (alpha-1,3.GT). Such tissues can be used in the field of xenotransplantation, such as orthopedic reconstruction and repair, skin repair and internal tissue repair or as medical devices.

Description

[0001] This application claims the priority of US Provisional Patent Application No. 60 / 553,895 filed on March 17, 2004 and US Provisional Patent Application No. 60 / 559,816 filed on April 6, 2004. Invention field [0002] The present invention provides tissues derived from animals lacking any functional α1,3 galactosyltransferase (α1,3GT) expression. The tissue can be used in the field of xenotransplantation, such as plastic surgery reconstruction and repair, skin repair and internal tissue repair, or as a medical device. Background of the invention [0003] Ruminants such as pigs, sheep and cattle are considered to be the source of xenotransplanted organs and tissues. Pig xenografts have received the most attention because the supply of pigs is sufficient, reproduction procedures have been established, and their size and physiology are compatible with humans. Other ruminant sources such as cattle or sheep have also been suggested as sources of hard and soft tissue xenografts. How...

Claims

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Application Information

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IPC IPC(8): A01K67/00A61F2/02A61F2/08A61F2/10A61F2/28
Inventor D·阿亚里斯P·罗里克
Owner REVIVICOR INC
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