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Unintended Effects of Genetic Manipulation
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Difficulties were encountered when trying to express human collagen in barley.

Manipulated Organism: Barley (Hordeum vulgare).

Inserted Transgenes: Barley plants were transformed with eight different genetic constructs. Two constructs contained a version of the rCIA1 gene for human collagen I alpha-1. The remaining six constructs contained only part of the rCIA1 gene that codes for a fragment of the larger collagen protein. Three different promoters were tested: a maize ubiquitin promoter (Ubi-1), which is constitutively active throughout the plant; a seed (endosperm)-specific promoter from the rice glutelin gene; and an alpha-amylase promoter from barley, which is active during germination.

Background: "The pharmaceutical and food industries use collagen and its derivative gelatin (a denatured form of collagen) extensively in various applications. . . . The traditional way of gelatin production is by extraction from collagen-rich tissues of animals. . . . Using recombinant technology, it may be possible to design and express gelatin molecules with predetermined characteristics to match the requirements in a variety of pharmaceutical applications" (p. 657-8).

Goal of This Study: Explore the "feasibility of using transgenic barley seeds as an expression system for recombinant collagen-related protein products" (p. 658).

Intended Effect: Several transgenic barley lines accumulated high levels of the human collagen fragment in their seeds.

Unintended Effects:
  • Microprojectile bombardment of barley cells was attempted with six genetic constructs, but transgenic plants could be created only with three of these for reasons that were unknown.

  • The full-length collagen protein was found at low but detectable levels in the seeds of five transgenic (Ubi-1) plants regenerated from tissue culture, but the next generation had fertility problems (low yield) and produced seeds with no detectable human collagen.

  • The collagen fragment protein was purified from one of the transgenic lines and analyzed for certain modifications. In humans, roughly half the proline amino acids in collagen are modified to hydroxyproline, which is required for the formation of collagen's unique triple helix structure. The transgenic collagen fragment in barley had only 10% of its proline residues hydroxylated. When the purified collagen fragment was analyzed, three different versions of it were detected, possibly due to inconsistent patterns of proline hydroxylation.

  • In addition to propagating the transgenic barley plants by seed, a procedure known as doubled haploid breeding was used to create plants with two copies of the collagen gene. However, doubled haploid plants could not be regenerated for one of the more promising transgenic lines, for reasons that were unknown.

Source: Eskelin, K., A. Ritala, T. Suntio, S. Blumer et al. (2009). "Production of a Recombinant Full-Length Collagen Type I Alpha-1 and of a 45-kDa Collagen Type I Alpha-1 Fragment in Barley Seeds," Plant Biotechnology Journal vol. 7, pp. 657-72.

Author Affiliations: University of Helsinki, Finland; VTT Research Centre, Finland; FibroGen Inc., California.

Funding: Technology Development Agency and Ministry of Agriculture, Finland.

Product Status: Not on the market as of 2009.

Copyright 2009 The Nature Institute.

This document: http://natureinstitute.org/nontarget/reports/barley_003.php

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