Frontiers in the Nutrition Sciences Proceedings of a Symposium (1989) / Chapter Skim
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The revolution in molecular biology has brought new opportunities and a fresh challenge for metabolic research. To date, research in molecular biology has focused on the characterization of specific genes and on the processes that alter their expression.
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It is ironic that it is now easier to isolate, clone, and sequence a gene than it is to characterize it by established physical methods. ' An excellent example of the great power of the combined techniques discussed above is the work of Robert Fletterick and colleagues on the characterization of glycogen phosphorylase (Sprang et al., 1987; S
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An understanding of the nature of the complex interaction between these regulatory molecules has been limited by the absence of a detailed structure-function analysis of glycogen phosphorylase. In a recent publication, Sprang et al.
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~ FIGURE 1 Schematic representation of changes in the dimeric glycogen phosphorylase a molecule. Abbreviations: S
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THE INTRODUCTION OF GENES INTO CELLS AND ANIMALS Construction of Chimeric Genes Many structural genes that code for proteins of metabolic interest have been isolated and characterized (Goodridge and Hanson, 1986) , including genes that code for key regulatory enzymes in metabolic pathways, hormones such as growth hormone, and insulin and a variety of receptors, to name only a few genes of interest.
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As the techniques~for stably introducing genes into cells and animals improve, it should be possible to reproducibly target genes for selected tissues in animals by using these chimeric genes. This is a new tool with great potential for metabolic studies, since it will permit, for the first time, a modification of an enzymatic step in a complex metabolic pathway without the use of inhibitors or other compounds that have a broad It also provides the potential to correct metabolic defects in humans, if the technology can be perfected to ensure a predicted site of integration into the human genome, as well as a normal level of expression of the newly introduced gene.
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. _,_ ~ The various hormone response elements in the promoter have been identified by gene transfection experiments in which chimeric genes containing segments of the promoter-regulatory region of the PEPCK gene were ligated to the structural gene for a selectable marker, such as the amino-3'-glycosyl phosphotransferase gene, which makes cells resistant to the cytotoxic compound, G418, or the Herpes virus thymidine kinase (TK)
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a, O a' ~ ~ o so a, - ~ to ~ o - ~Jo it ~o ~ ~ ~ / / ~v o ~ ~ no / ~no =^ ~ ~ <.
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/, + + -109 +1 +1800/-416 -61 ___~, ,* + + -109 +1 +1800~-61 -416 -S48 +73i-650 +1 -S48/-650 ~1 FIGURE 3 Functional analysis of the PEPCK promoter-regulatory region for cAMP and glucocorticoid regulatory elements.
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This element, which extends from positions -72 to -205, is highly conserved in all species studied and confers the correct developmental and regulatory properties on a linked structural gene. It has even been possible to induce neoplasia of the exocrine pancreas in fetal transgenic mice that contain a chimeric gene composed of 4.5 kilobases (kb)
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Despite its limitations, this procedure is widely used to measure the function and regulation of specific promoter elements in any cell line that can be maintained in culture. The usefulness of DNA transfection for metabolic studies is limited by its low efficiency of gene transfer and by the need to isolate and establish 13
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In certain cases, this obviates the need to subsequently select from a large population of cells individual cells that functionally express the transfected gene.- A wide variety of species-specific retroviral vectors are available that can accommodate DNA segments ranging from 1 to 15 kb. Finally, retroviral vectors can be used to infect both cells and animals, which makes retroviruses useful vectors for the introduction of genes into differentiating tissues in animals during development (Johnson et al., 1985; Soriano and Jaenisch, 1986; Van der Putten et al., 1985~.
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An example is the retroviral vector ply, shown in Figure SA, which is derived from the Maloney murine leukemia retrovirus (Korman et al., 1987~. To be functional, retroviral vectors of this type rely on the availability of bans functions from helper cells that have been infected with retroviruses or from a specially constructed cell that contains these functions stably integrated into its genome.
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A new chimeric gene composed of the some region of the PEPCK gene ligated to the bGH structural gene (without its own promoter) was inserted at the unique ClaI site in an orientation opposite the direction of transcription from that of the 5' LTR.
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estimated that of 60 eggs that survive microinjection, 6 of the implanted zygotes develop to term, and of these, 1 to 2 are tranagenic. METABOLIC STUDIES USING GENETICALLY MODIFIED CELLS AND ANIMALS The most common use of genetically modified cells and animals is for the analysis of gene expression control.
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The 550-bp segment of the promoter-regulatory region of the PEPCK gene used to construct these chimeric genes contains both the cAMP and glucocorticoid regulatory elements (Short et al., 1986; Wynshaw-Boris et al., 1984, 1986~. When Bt2cAMP or glucocorticoids were added to hepatoma cells (rat FTO-2B cells and mouse Hepa 1-6C cells)
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This is a high level of expression of an infected gene and indicates that the 550 bp segment of the PEPCK promoter-regulatory region used in this chimeric gene may be useful in future studies in which a strong, regulatable promoter is required for producing high levels of a specific gene product for metabolic studies. Use of Retroviral Vectors to Introduce Genes into Animals Retroviral vectors have been used to introduce genes into mammalian tissues, both during development and after 19
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The potential for these techniques for metabolic studies is illustrated by a series of experiments in which the murine retroviral vector, pLJPCKneo (see Figure 5B) , was injected as an infectious retrovirus into the peritoneal cavity of fetal rats between 16 and 20 days of gestation while the animals were still in utero (Hatzoglou et al., unpublished observations)
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The ability to direct the expression of a chimeric gene into the tissues of an animal relatively late in development, however, holds promise as a technique of potential usefulness for broader metabolic studies aimed at introducing modified genes of interest into animals to alter various metabolic and nutritional parameters.
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infection and its induction by Bt2cAMP. The infectious retrovirus pLJPCKneo was introduced into FTO-2B hepatoma cells and into the peritoneal cavities of 19 fetal rats in utero.
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. The promoter-regulatory region of the PEPCK gene contained 450 bp of 5'-flanking sequence, which included the cAMP and glucocartic regulatory elements.
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_ 1 1 Bt2AMP (90 min) starved High High CHO Protein No CHO 3 2 1 FIGURE 8 Regulation of the bGH concentration in the blood of transgenic mice by diet and Bt2cAMP administration.
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It should also be possible to use a segment of the PEPCK promoter, when it is linked to a structural gene of interest that contains its own core promoter but that lacks homologous tissue-specific elements, to direct the expression of this chimeric gene to the liver and kidney. POSSIBLE APPROACHES TO GENETICALLY BASED ALTERATIONS IN METABOLIC PROCESSES There are many metabolic models that can be used to test the potential of the techniques described above.
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Thus, birds recycle lactate from the muscle and red blood cells to the liver for gluconeogenesis It should be possible to test the metabolic role of the two forms of PEPCK directly in the hepatic cells isolated from chickens, in which the gene for the cytosolic form of the enzyme has been introduced via an infectious retrovirus by the techniques described above. The gene for the cystosolic form of PEPCK from chickens has been isolated, and a full-length cDNA is available (Hod et al., 1984 a,b)
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, will be an invaluable tool in these types of studies, since it permits infection with the murine retroviral vectors already developed and has been shown to effectively introduce functional and regulatable genes into other hepatoma cells. Since the cells are able to synthesize glucose from the gluconeogenic precursors contained in the culture medium, their growth in the absence of added glucose should be directly dependent on the efficiency of gluconeogenesis.
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A core technology centered around the ability to introduce specific genes into cells and animals in a directed and regulatable fashion is now developing. This technology will be generic in its usefulness in-biology and medicine and will extend from the possible correction of genetic defects to the genetic patterning of cells and animals for metabolic studies.
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Hollaendis, eds. Genetic Engineering: Principles and Methods, Vol.
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Hod, J., S Morris, and R.W.
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207:787-802. Van der Putten, H., F.M.
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