Genetically Modified Organisms and Genetic Engineering in Research and Therapy

P. Piguet, P. Poindron

• Éditeur: S. Karger
• Année: 2012
• ISBN:
  9783805590662
• Sujet: Medicina
• Sous-sujet: Psiquiatría y salud mental

Genetically modified organisms (GMO) raise societal, political and ethical concerns. They inspire strong resistance or, conversely, enthusiastic assent. The aim of this publication is to give an overview of genetic engineering, starting with the history of the discovery of restriction enzymes continuing with technical aspects of transgenesis to its applications in research and ethical considerations. Be it the use of single engineered cells or GMO, these applications cover a broad array, ranging from disease-oriented research (but not only), to the promising perspectives of gene therapy. Historical and technical aspects give insights into the problems inherent to the creation of GMO, and illustrate the links and limits between genetic engineering, GMOs and gene therapy. A summary article in English and French structures the links between the different chapters and concepts.Scientists interested in genetic engineering of single cells or animal models, as well as in gene therapy, will find an up-to-date review on the use and perspectives of transgenesis. However, this publication is also recommended to the public interested in the definition of GMO, which encompasses a much broader array than the genetically modified crops covered by media.

Piguet P, Poindron P (eds): Genetically Modified Organisms and Genetic Engineering in Research and Therapy. BioValley Monogr. Basel, Karger, 2012, vol 3, pp 1–32

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Genetically Modified Organisms: Concepts and Methods

Pascale Piguet^a · Philippe Poindron^b

^aPharmazentrum, University of Basel, Basel, Switzerland; ^bUniversité de Strasbourg, Strasbourg, France

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Abstract

Genetically modified organisms (GMO) raise societal, political and ethical concerns. They inspire strong resistance or, conversely, enthusiastic assent. What problems are faced in the field of genetic modification, and what scientific questions does it contribute to answer? The aim of this volume is to give an overview of genetic engineering from the technical aspects of transgenesis to their applications in research. Be it through the use of individual transfected cells or GMO, these applications cover a broad array ranging from disease-oriented research (but not only) to important perspectives for therapy. Gene therapy is fully described in full-length articles devoted to the use of this technique in cancer and in muscular disease. In this section, we shall define the following concepts: organisms, modification of germen and modification of soma, vectors and vector of expression, and transgene. We will then briefly describe the methods currently used to create GMO. A short description of the main transgenic organisms will be given in the first chapter. We do not intend to be exhaustive, but demonstrative and to give our readers objective elements for building their own opinion. Genetically modified plants are not covered in this chapter.

Copyright © 2012 S. Karger AG, Basel

Defining and Illustrating Basic Concepts

Organism

In his introductory lecture to the International Congress for Virology held in Strasbourg, in August 1981, André Lwoff brilliantly defined what an organism is. For this purpose, he compared the pertinent properties of several biological entities (table 1). According to Lwoff [1], the main and specific properties of an organism are the capacity to evolve in an independent manner, contrary to cells which are, as organisms, living entities, but cannot evolve outside them. However, it should be mentioned that cultured cells, especially cell lines, or cell strains can modify their properties all along the passages. However, they are not organisms since these alterations depend on experimental but not natural conditions.

Table 1. Respective properties of several biological entities

	Growth	Division
Protists, Fungi, Planta, and Animalia	+	+
Cells	+	+
Mitochondria, chloroplasts	+	+
Cytoplasmic membrane	+	+
Chromosomes, plasmids	0	0
Molecules, virus, and cellular organelles	0	0
Adapted from Lwoff [1].

It results from this definition that organisms, properly said, are biological entities belonging to all kingdoms of living creatures: animals, plants, fungi, protists.

Modification of Germen and Modification of Soma

An organism is able to multiply and reproduce itself. It results that, properly said, a GMO is an organism the germen of which has been altered either by insertion of one or several foreign genes originating from the same species or genus, from other more or less further-related organisms (transgenic organisms), or by deletion or inactivation of selected genes (knock-out (KO) organisms). We will focus this paper mainly on the first class of GMO, i.e. organisms modified by the insertion of new genes into their germinal genome.

It is also possible to modify the soma of an organism. The modification may theoretically affect all somatic cells, a given tissue, or only a specific cell type. This strategy is at the basis of gene therapy but has also been used for research purposes, i.e. to screen for specific genes - such as cancer genes - within a given tissue [2-4]. Since both types of modification (germen and soma) result in genetic modifications, we will briefly discuss the methods used in these two cases since they differ widely.

It is conceptually important not to confuse organisms with vectors, and especially viruses such as Lentivirus or Adenovirus that are frequently used for introducing gene sequences into target cells, as in therapy or transgenesis. Viruses are not organisms.

Gene Vectors or Gene Carriers for Gene Therapy

The name gene vector or gene carrier is often given to any entity that allows the introduction of genes into target tissues or cells. Numerous families of viruses have been used as vectors of genes. Alternatively, synthetic molecules have been designed to allow the entry of genes into cells. Such vectors should also be nontoxic, poorly or nonimmunogenic; they may be natural or synthetic polymers or consist of nanoparticles designed for entering all cells or a specific class of cells of an organism. This class of vector is preferentially named gene carrier. It is associated with gene sequences and other elements that improve both the penetration and guidance of selected genes toward the nucleus. The conception of such artificial vectors resembles that of artificial viruses, without the adverse properties often associated to natural viruses that are immunogenicity and triggering of an immune response rapidly rendering them ineffective on reiterated administration.

Use of nude plasmids has also been proven efficient. In this situation, only the gene vector is used.

Vector of Expression

The name vector of expression rather applies to gene sequences especially designed to allow correct expression of a given gene in a given tissue, in all tissues, or in a zygote. In the case of gene therapy, this implies that regulating sequences of bases located upstream of the coding sequences be chosen correctly. The situation is different when the aim of genetic modification is to create a GMO.

Tools for Genetic Modification

The Case for Genetically Modified Organisms

Transgenic animals are essential research tools, whether they are used to address basic biological questions or to develop preclinical models of human diseases. In particular, transgenic mice have played a fundamental role in investigating tissue-specific gene expression, oncogenesis, and developmental mutations [5].

In order to successfully achieve transgenesis of animal eukaryotic cells (the case for prokaryotic organisms and plants will not be envisaged here), it is necessary to make the selected transgene (a) enter the recipient cell and (b) be integrated into its genome. These two steps have undergone numerous improvements during the past decade. Genetic methods of transgenesis can be divided into (a) transgene overexpression and (b) gene knockout techniques. Although their aims are not identical, both involve the two steps cited above.

The first animals to carry experimentally inserted genes were mice produced by injecting retroviral Simian virus 40 (SV40) DNA into the cavity of blastocysts [6]. Even though a large percentage of animals derived from the embryos carried SV40-specific DNA sequences in some of their tissues (mosaic integration), they neither incorporated the SV40 DNA into their germ cells nor expressed the viral genes. Germ line incorporation was later obtained by exposing mouse embryos to an infectious strain of Moloney leukemia virus. This first strain of transgenic mice (transmitting the virus as Mendelian genes) developed leukemia after birth, suggesting that viral genes were retained and expressed in adult animals [7]. Moreover, this demonstrated that embryos could harbor foreign DNA and still develop to term.

Most cell transfections involved a selection strategy to identify the cells into which the foreign DNA was successfully incorporated [8]. In a next step, the possibility of directly injecting DNA into mammalian cells was explored. In 1980, Gordon et al. [9] introduced foreign DNA into mouse embryos by injecting it into pronuclei, and Gordon and Ruddle [10] subsequently demonstrated the transmission of the introduced foreign DNA to offspring.

How to Make a Transgene Enter a Recipient Cell?

Theoretically, three kinds of cells can be chosen for achieving transgenesis: in vitro fertilized egg, embryonic stem cell and spermatozoa.

Original Method. The original method developed to generate transgenic organisms therefore involved the direct microinjection of foreign DNA. With nuclear injection of DNA, investigators rapidly reported germline transmission of transgenes, expression of transgenes [11], and a phenotype associated with transgene expression [12]. Pronuclear microinjection consists of injection of a small volume of fluid containing the gene of interest into a pronucleus of a zygote. Then, the zygotes are transferred to a foster mother. The generation of transgenic animals through the injection of naked plasmid DNA into the male pronucleus of fertilized oocytes has been standard practice for several decades. Nevertheless, the success of this technique has been largely limited to mice [5]. Even though pronuclei are clearly visible in many species, it is necessary in some other species to centrifuge embryos to displace the optically opaque cytoplasm in order to see the pronuclei [13]. After problems concerning the embryo viability were solved, several research groups have been able to produce transgenic rabbits, sheep and pigs by pronuclear microinjection [14, 15]. In swine, although the detection of tran...