Cytokines & Cells Online Pathfinder Encyclopaedia
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Cytokines & Cells Online Pathfinder Encyclopaedia |
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The term "transgenic " describes organisms that contain in their chromosomes stably integrated copies of genes or gene constructs derived from other species or not normally found in the host animals. These so-called transgenes may or may not be expressed. Transgenic animals have been generated for many species and can be generated by introducing cloned DNA of the foreign genes into fertilized oocytes by micro-injection into pronuclei. These oocytes are subsequently transferred into the uterus of a pseudo-pregnant recipient animal and develop to term. Another strategy involves the use of embryonic stem cells (see: ES cells). These cells have the advantage that they can be manipulated easily in vitro before they are used to generate transgenic s.
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| Strategies to generate transgenic animals. In principle two strategies are available. The classical technique involves microinjection of gene constructs into fertilized oozytes obtained from a donor animal. These are then implanted into a pseudopregnant foster animal and carried out to term. Other strategies rely on in vitro manipulation of embryonic stem cells (ES cells) which can be grown as populations of pure totipotent cells in vitro in the presence of suitable feeder cell layers or LIF (leukemia inhibitory factor). Manipulated ES cells are either aggregated with morula stage cells or injected into blastocysts. Aggregated cells or manipulated blastocysts are again implanted into pseudopregnant animals where they can develop to term. Offspring obtained is tested by Southern blotting or Polymerase chain reaction for the presence of the transgene. Founder animals, which are hemizygous for the transgene, can then be crossed to obtain pure homozygous lines. Manipulation of ES cells is particularly useful for gene targeting experiments designed to create null mutations of an endogenous gene since all manipulations and also the successful disruption of a gene can be confirmed in vitro before the manipulated cells are used to generate live transgenic animals. |
Animals developing from manipulated cells contain the foreign gene in all somatic cells and also in germ-line cells if the foreign gene was integrated into the genome of the recipient cell before the first cell division. Integration at a later stage yields genetic mosaics consisting of normal cells with a normal genome and cells with a transgenome. Transgenes integrated into the genome of germ line cells of the transgenic animals are transmitted to their offspring according to the Mendel's Law.
Primary animals developing after gene transfer are always hemizygous because the transgene is not integrated into both copies of a homologous pair of chromosomes. Pure homozygous lines are obtained by crossing of two suitable hemizygous (founder) animals.
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| Construction of transgenic animals in different species. See text for details. EI signifies embryo injection. |
The importance of gene transfer techniques for biomedical and molecular biological basic research cannot be overemphasized. Transgenic animals have been used to determine the cis-acting DNA elements responsible for the tissue-specific expression of genes, and current technology now permits the targeted expression of a protein to a particular organ or cell type within an organ. It is a common feature that the transgene is expressed properly both spatially and temporally. So far, a large number of transgenic mice have been generated expressing, for example, Oncogene genes, viral genes, immunoglobulins, cytokines, and cell surface and MHC antigens. A wide variety of Oncogene genes from viral and cellular sources have been inserted into the germline of mice with subsequent development of neoplasia. Many of the published reports describe similarities between morphologic features of the transgenic mice tumors and those occurring naturally in humans. Transgenic mice bearing an oncogene targeted for the expression in a specific tissue can reveal how that oncogene influences differentiation and help to delineate the pathways to malignancy. Transgenic animals have been powerful models also for the investigation of various features of the immune system, particularly for studies of the differentiation and tolerance of lymphocytes.
In many instances transgenic animal models indirectly also provide an enormous increase in our knowledge of human diseases, providing opportunities to dissect the various molecular pathways involved in such processes. Transgenic animals are also studied to design gene constructs that function in a reproducible, predictable manner and that can be inserted at sites predetermined by the researchers as an important prerequisite for the ultimate goal: gene therapy (approximately 5 % of established transgenic lines carry insertional mutations, caused by the integration of the transgene per se, leading to inherited disorders and developmental abnormalities). In addition, the characterization of the insertion sites of exogenous sequences in transgenic mice can identify loci that are potentially useful for the genetic analysis of the mammalian genome.
The analyses of transgenic animal models can be carried out with a degree of sophistication unattainable by any other technique. The analysis of transgenic animals allows, for example to elucidate the control mechanisms underlying embryonic development and the development and differentiation of embryonic cells, cancer cells (see: Oncogene), T-cells, and B-cells. Transgenic animals also facilitate the analysis of the complex network of interactions between different genes including tissue-specific gene expression and additive polygenic actions of genes. Gene transfer techniques also allow studies of the effects and consequences of the expression of developmentally regulated genes the uncontrolled expression of which may be lethal. Such genes can be investigated if the expression is restricted to a specific tissue which can be achieved by choosing suitable regulatory elements (see also: gene expression, Response elements).
Transgenic animal models have now been established for many different cytokines (see subentry: Transgenic /Knock-out/Antisense studies for individual cytokines). These animal models either over-expressing or constitutively expressing a particular cytokine gene are of particular interest in research on cytokines because the analysis of these animals reveals at least some of the complex molecular biological effects of single cytokines in a living organism that cannot be elucidated by studying cell cultures. The identification and characterization of a growing number of key effectors demonstrates that seemingly diverse immune mediated diseases involve similar pathogenetic mechanisms. It is becoming possible to design and generate effective transgenic models for different diseases. The transgenic models can be used, for example, to study the cell-specific, tissue-specific, and developmentally regulated expression of individual cytokines and their pathophysiological consequences. Information from such experiments is applicable frequently directly to the understanding of pathogenetic mechanisms and will help to design new therapeutic approaches. It should be noted, however, there are also many examples of transgenic mouse models in which the particular consequences of transgene expression or non-expression are very different from the phenotypes associated with the human genetic diseases upon which these models were bases (for instance, Lesh-Nyhan syndrome, Duchenne muscular dystrophy, phenylketonuria).
Essentially the techniques used currently for the generation of transgenic animals expand the normal genome by the addition of a gene. Gene targeting technologies, i.e., the homologous recombination between specific chromosomal DNA sequences and exogenously introduced DNA sequences) have been developed and applied to pluripotent, mouse embryonic stem cells. Gene targeting provides the means to create mice of specifically altered genotype in which endogenous genes in their natural position in a cell genome are disrupted by the insertion of exogenous DNA. These transgenic animals carry Null mutations (loss-of-function mutations) of a particular gene and are referred to frequently as a Knock-out. Many of the phenotypes expressed by these animals are as expected from the in vitro assay data, but some are unexpected in that some animals with disrupted cytokine genes show an almost normal behavior. For techniques allowing studies of the effects of disrupted cytokine gene expression see: antisense RNA, genetic ablation.
See REFERENCES for entry transgenic
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