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Genetic Manipulation in Nutrition, Metabolism, and Obesity Research

Posted on: Thursday, 30 September 2004, 06:00 CDT

We summarize the current standard methods for overexpressing, inactivating, or manipulating genes, with special focus on nutritional and obesity research. These molecular biology procedures can be carried out with the maintenance of the genetic information to subsequent generations (transgenic technology) or devised to exclusively transfer the genetic material to a given target animal, which cannot be transmitted to the future progeny (gene therapy). On the other hand, the RNA interference (RNAi) approach allows for the creation of new experimental models by transient ablation of gene expression by degrading specific mRNA, which can be applied to assess different biological functions and mechanisms. The combination of these technologies contributes to the study of the function and regulation of different metabolism- and obesity- related genes as well as the identification of new pharmacologic targets for nutritional and therapeutic approaches.

Key words: transgenic, RNAi, gene therapy, rodents

2004 International Life Sciences Institute

doi: 10.1301/nr.2004.aug.321-330

Introduction

Obesity and other nutrition-related chronic diseases are becoming increasingly more prevalent, and much effort is being devoted to understanding their pathogenesis and treatment.1 One approach to investigate the mechanisms involved in metabolic disturbances is to overexpress, inactivate, or manipulate specific genes playing a role in the regulation of body weight and energy metabolism.2,3 Although gene transfer or blockade can be performed in tissue culture models, the interaction of the manipulated genes with the components of an intact organism provides a much more complete and physiologically relevant picture of the gene function than could be achieved any other way. Many of these techniques are fully established as powerful and routine tools4 and are increasingly being applied in order to understand endocrine problems and metabolic pathways, providing elegant models to study nutritional, physiologic, and disease situations. In addition, these approaches may be used to reveal new biologic functions and to identify new pharmacologic targets for the treatment of obesity or other genetically related diseases.

Among all of the possible animal species to be used for nutritional research and gene manipulation, the mouse and rat are the models of choice. The physiology, embryology, and genetics of these species are well studied and understood,4-6 and the relatively short life cycle and inbred strains of rodents provide the opportunity to study disease traits in a defined genetic background.4 In addition, a large genetic reservoir of potential models with metabolic implications has been generated through the identification of spontaneous, radiation-induced, or chemical- induced mutant loci in rodents.6 The mouse and rat models are made even more attractive because of the extensive and varied genetic tools available (Table 1). Another important reason for specifically using the mouse as an animal model for gene targeting lies in the possibility of isolating embryonic stem (ES) cells (cells derived from the inner cell mass of the blastocyst, which present multipotent differentiation potential), in which any gene can be modified to generate novel mutant mice.4

Several excellent reports regarding obesity and transgenic models of rodents have been recently published.5,7-9 Therefore, we aim to emphasize the current standard methods for manipulating genes- transgenic animals, gene transfer technology, and RNA interference (RNAi), which are focused on nutritional and obesity research-and to highlight newer strategies and goals in the field of gene transfer. Thus, more complex approaches are emerging, extending the potential of these technologies through the conditional (temporal and spatial) control of the genetic manipulation or by the ablation of the expression of specific genes by using RNAi, which can be applied to nutritional research.

Table 1. Some Web Pages about Resources for Rodent Genomics

Transgenic Technology

Depending on the method used to introduce the gene, as well as the time and site in which the process occurs, genetic manipulation can be preserved by the successive generations creating the germline transmission.10 In this method, usually termed transgenic technology (Figure 1), every cell of the animal carries the genetic manipulation. The two more commonly used strategies are the pronuclear microinjection of a fertilized oocyte and the transfection of ES cells.4,10

Pronuclear Microinjection

This technique is based on the injection of a DNA fragment into the pronuclei of a fertilized egg with the use of a microsyringe under a microscope. The technique is reliable, although only approximately 5% to 40% of mice developed from manipulated eggs contain the transgene that results in a stable chromosomal integration, which is crucial for the success of the technique.10 Although pronuclear microinjection has been performed in both mice and rats,11 it is more widely used in the first species because it is easiest to find the framework and background to develop a new model in mice.

Figure 1. Most frequently used techniques for genetic manipulation in rodents and obesity research.

The first visible phenotypic change using this technique was described in 1982 for animals expressing the rat growth hormone sequence under the control of a ubiquitous promoter, the mouse metallothionein-I, producing a model for gigantism.12 More directly related to nutrition and obesity were the pioneering experimental studies with GLUT413 and TGF[alpha].14 In the first one, the main advance in creating transgenic animals was the tissue-specific overexpression of a transgene (Figure 2A and 2B). Putting a tissue- specific promoter downstream to the cDNA of our interest can lead the expression of the transgene in a spatial manner. The most commonly used specific tissue-promoters involved in energy expenditure and control of feeding are myosin light chain (MLC-1), [alpha]-skeletal actin, and muscle creatine kinase (MCK) for skeletal muscle,15 adipocyte fatty acid-binding (aP2) for adipose tissue,16 albumin and liver enriched activator protein (LAP) for liver,17 and nestin, neuron-specific enolase (NSE), and glyal fibrillary acidic protein (GFAP)18 for brain. Other recent examples of this technique are the overexpression of the plasminogen activator inhibitor-1 gene, which attenuated a high-fat diet- induced obesity,19 and the overexpression of the uncoupling proteins- 2 (UCP2) and -3 (UCP3), which reduced fat mass and increased LDL cholesterol.20

Figure 2. Different models of controlling the over-expression of an exogenous gene in transgenic mice. (A) No control. (B) Spatial control of the gene of our interest. (C) Temporal control of the gene of our interest. (D) Spatial and temporal control of the gene of our interest.

Despite the efficiency and consistency of pronuclear microinjection in creating transgenic animals, the studies using this approach were hindered by three major problems: the inability to control the randomly appearing sites of integration into the genome; the inability to control the integrated copy numbers of transgenes; and embryonic lethality due to the toxic effects of certain gene expression.10 Random integration and multiple copies lead to unregulated expression of transgenes and cause side effects, while expression of some genes in an early or inconvenient moment can produce lethal consequences. To circumvent the latter, several conditional techniques, such as temporal control, have been developed. The temporal conditional system allows the researcher to reversibly control the time expression of a transgene at any point during development or postnatal life and, combined with spatial control, only in the desired cell type. Many systems are available for this type of conditional control, including tamoxifen, glucocorticoids, and tetracycline.21 The last, known as the tet system, is by far the most popular system and is routinely used in in vivo and in vitro models. This technique is based on the property of a bacterial operator tetO (TRE promoter) to be deactivated by the tetR-VP16 protein (tTA) in the presence of tetracycline.21 The successful application of this methodology requires the breeding of two different lines of mice (Figure 2). One line expresses the tTA- protein and the other expresses the transgene of our interest under the control of the TRE promoter. After breeding both lines, the whole body of the next generation carries the genetic information under the control of tetracycline. In order to mix temporal and spatial conditional control, tTA protein could be expressed under the control of a tissue-specific promoter (Figure 2D). Although this method is reliable and fully established, new possibilities are being investigated in order to overcome different problems such as leaking or low levels of tTA expression.21

Transfection of ES Cells

To avoid some of the technical problems affecting the pronuclear microinjection approach, genetic information can also be integrated in the genome of ES cells by means of homologous recombination (a process by which a fragment of exogenous genomi\c DNA introduced into a mammalian cell can be located and recombined with the endogenous homologous sequence).6,10 These cells are derived from the inner cell mass of the mouse blastocyst and thus have the potential to contribute to all tissues of the developing embryo.22 After the extraction of the cells, this method requires culture conditions that maintain the cells in an undifferentiated state. Since these cells are cultured, DNA can be introduced by transfection or viral transduction (see below for different methods), as in any other established cell line, and the transformed cells can be selected using standard markers.6 The recombinant cells are then introduced into the blastocoele of a host embryo at the blastocyst stage, where they mix with the inner cell mass. Unfortunately, ES cell technology is only available for the mouse model, despite the tremendous amount of effort that has been invested in other species. To bypass this technological barrier in rats, nuclear transfer can be used as an alternative approach. The genome of genetically modified cells is physically transferred to a rat enucleated oocyte, which develops into a genetically modified animal.23

The most common strategy using ES cell technology is to disrupt the function of a gene by introducing a DNA flanked by homologous regions that recognize the target gene.6 After the recombination and generation of the mouse, a transgenic model is created with a null allele of the selected gene, a strategy that is termed gene knockout (KO). Early obesity-related models were the transforming growth factor-[beta] 1 KO24 and a mouse lacking functional lipoprotein lipase (LPL).25 Today, the use of KO mice in obesity research is still effective, and recent developments include the fatty acid synthase (FAS),26 adiponectin,27 ghrelin,28 and mall (FABP5) genes.29 Recent reviews7-9 give a complete list of KO models used for metabolism research. When the global removal of a gene of interest using conventional KO methods results in embryonic lethality, investigators can choose to produce a conditional tissue- specific (also time-specific if combined with the let system) KO line of mice in which deletions can still be studied in vivo. Examples of this technology applied to body weight regulation are the insulin receptor depletion in adipose tissue30 and GLUT4,31 and the muscle-specific PPAR[gamma]-deficient mouse.32

Gene Therapy

When the scientific aim in nutritional research is not to transfer genetic information to subsequent generations, the most commonly used method is gene transfer to somatic cells,33 which can be achieved by using in vivo or ex vivo approaches (Figure 1). While in vivo gene transfer delivers the genetic manipulation directly into the animal, ex vivo gene delivery refers to the transfer of the gene manipulation into cells/organs removed from a donor, expanded in vitro, and then subsequently reintroduced into the animal.34 In both cases, the cells lose the capacity to pass the gene manipulation on to subsequent generations, but the time, cost, and complexity of the experiments are reduced.35 Moreover, gene transfer can be transiently or permanently established, and is commonly used in gene therapy research for humans.34 The most commonly used methods for gene transfer in rodents and obesity research are viral transduction systems,36 mainly with adenovirus, and the direct injection of naked DNA.37

Viral Transduction

Due to the efficiency with which viruses can deliver their nucleic acid into cells, and the high levels of replication and gene expression, viruses have been repeatedly used as vectors not only for gene expression in cultured cells, but also for gene transfer to living animals.35 Several types of viral vectors have been developed in gene transfer. The main type of virus used for gene transfer in obesity is the adenovirus. Several examples of this gene transfer strategy in creating endocrine-modified models in rodents have been reported in the experiments of Muzzin et al.38 with leptin, the study by Nagamatsu et al.39 expressing preproinsulin in adipose tissue, or the injection of the liver glucokinase into skeletal muscle.40 More recently, the peripheral delivery of adiponectin41 has been published. This type of strategy presents high levels of transient gene expression, and adenoviruses can infect dividing and nondividing cells. While up to 7 to 8 kb DNA can be added in the adenoviral vector, it is not suitable for long-term expression of the transgene due to the lack of integration into the host genome, and because adenoviral vector particles are highly immunogenic. Despite its being a widely used technique, it could produce inflammatory and toxic reactions in the host, and this immunogenicity is responsible for the depletion of adenovirally transduced cells.35 For this reason, other non-pathogenic and non- toxic approaches were developed. For example, adeno-associated viruses (AAV) can infect a wide range of cells, including those that do not divide. However, a limited capacity for foreign genes (approximately 4 kb) is provided. They present viral genome integration into the cell genome, although there is a lack of specific integration, which may result in cell mutagenesis. Again, leptin has been a target gene for genetic transference using this type of virus.42

Other similar approaches with larger capacities are the herpes simplex virus and retrovirus.10 The first one has a maximum size capacity of 30 kb and a broad host range, although in natural human infections the virus is neurotropic and does not integrate into the cell genome. The applications of this type of virus in overexpressing lacZ in skeletal muscle43 and 5-HT1B receptors in neurons44 were reported. The latter, a retrovirus, only infects dividing cells, but the insertion capacity is 10 kb and presents stable gene expression due to viral genome integration into cell chromosomes (again, by random insertion, which may result in mutagenesis). The classic target tissue for gene transfer using retrovirus is the liver,45 although examples regarding obesity apparently have not yet been described.

Finally, the lentivirus (with 10 kb of capacity), a type of retroviral-based vector, solves some of the problems of retrovirus infecting nondividing cells, and maintains stable gene expression due to viral genome integration into cell chromosomes.35 Several experiments have been carried out using this type of virus in different target tissues, such as the heart,46 muscle,47 and neurons.48 Nonviral Vectors

Theoretically, nonviral vectors have no limit concerning the size of DNA to be incorporated into the cells, and they are suitable for oligonucleotide delivery, which is also applicable for RNAi transfer.49,50 In addition, these vectors are relatively non-toxic and non-infectious. However, the targeting is not specific and the effect is transient (several days), although new achievements have extended this period to weeks or months.35 Moreover, transfection in vivo is generally far less efficient than the adenoviruses and can induce an immunogenic response. Indeed, the use of nonviral vectors is extensive and generally applied, and multiple examples have been described. Typical nonviral techniques of gene transfer are the mechanical administration of naked DNA, electroporation, cationic liposomes, and DNA-protein complex.35 Our group, for example, has performed muscle gene transfer of UCP1,51 UCP2,52 and leptin53 by in vivo injection of naked DNA into the rodent. This approach had some implications in the regulation of body weight and energy metabolism. Transfection by electroporation is also simple, inexpensive, and safe, and by using this technique it is possible to enhance the transfection in vivo of a direct DNA injection in brain,54 muscle,55 or liver.56 Finally, recent studies have reported significant success in gene transfer by transfecting in vivo57 and ex vivo58 exogenous DNA with cationic liposomes or other composites, nonviral vector systems in liver,59 and other obesity target tissues of rodents, such as adipose tissue.60

RNA Interference (RNAi)

A new technology arising in the field of genetic and molecular manipulation is the antisense approach, which is being applied to inhibit the expression of a target gene in a sequence-specific manner.61 A possible breakthrough in these technologies is RNAi, which is used to investigate and decipher gene function by degrading a specific mRNA target, thus knocking down the level of an encoded protein.6 Key to the technique are double-stranded RNAs 21-25 nucleotides long, called short interfering RNAs (siRNAs), that interact in the cytoplasm with a multiprotein complex called RISC (RNA-induced silencing complex). Finally, the siRNA-induced activation of RISC binds this complex to the homologous mRNA by base- pairing interactions for cleavage and degradation of the cognate RNA (Figure 3).

Gene silencing mediated by RNAi can be achieved in two different ways. In the first one, in vitro-synthesized siRNAs are introduced into cells using microinjection, transfection, or electroporation to transiently suppress gene expression.61 A disadvantage of this approach is that synthetic siRNA is not stable and must be protected during shipping. In the second approach, siRNAs are expressed in vivo from DNA vectors and cassettes to create stably expressed siRNAs within the cells or transgenic animals.61 However, they require more hands-on time from the researcher. Three different RNA polymerase III promoters are currently used to drive the expression of a small hairpin siRNA in mammalian cells: human and mouse U6 and human H1. This strategy appears to be the best method for long-term studies and in cases in which antibiotic selection of siRNA- containing cells is desired to enrich the culture with cells that have taken up the plasmid.62

RNAi is a powerful tool for probing gene function. In regard to obesity\, RNAi was first assayed as a gene expression silencing endogenous gene function in the nematode Caenorhabditis elegans,63 in which it has been used to disrupt the expression of each of the 16,757 worm genes. In this animal, 305 gene inactivations caused reduced body fat, and 112 gene inactivations caused increased fat storage. RNAi has since been used to investigate different aspects of lipid metabolism homeostasis, such as the regulation of insulin signaling,64 the role for fatty acid synthase in cancer cells,65 the function of adiponectin receptors,66 and the physiologic action of the agouti-related peptide (AGRP).49 In cell cultures, after the use of RNAi for silencing genes in insulin-sensitive adipocytes, the role of these genes in insulin-signaling cascades has been demonstrated.64 In vivo hypothalamic administration of RNAi against AGRP has proven that this peptide reduces metabolic rate independently of food intake.49

There are several methods to introduce siRNA and nucleic acids into cultured cells: electroporation67; calcium phosphate precipitation68; oligofectamine69; lipofectamine70; adenovirus71; lentivirus72; oncoretrovirus73; and different synthetic polymers.74 However, the in vivo administration presents some problems and so far the results are not encouraging. Delivery remains a major obstacle; to deliver siRNA and si-encoding DNAs to the site of action on vertebrate cells, a gene therapy approach could be interesting, using local administration of naked DNA,49 cationic- liposomes,75 lentivirus,76 adenovirus,77 or hydrodynamic transfection.50 Even embryo (in ovo) electroporation has been tested.78 Nevertheless, the development of RNAi viral vectors (adeno- or lentivirus) by the inclusion of inducible or tissue-specific promoters could permit the therapeutic use of these specific RNAi tools in vivo.

Figure 3. The mechanism of RNA interference.

Finally, it is also possible to create transgenic animals by vector-mediated siRNA delivery in stem cells,79 oocytes,80 or blastocysts.81 Moreover, new methods to induce cell- and tissue- specific expression of RNAi are being developed.79,81

New Perspectives

In the field of gene manipulation, new technologies and improvements are providing new possibilities for the scientist and for metabolism and obesity researchers. These new achievements, together with the knowledge of a growing number of genes and molecules involved in the body weight control system (Table 2), are allowing for the in vivo study of the regulation and function of different endocrine- and obesity-related genes and the identification of new pharmacologic targets for therapeutic approaches. Once a basic level of understanding of the endocrine system is reached, new models of investigation must be carried out by performing the different genetic manipulation techniques described in this review: gene therapy to replace the ablated gene of a KO model82; inhibiting the expression of a gene using RNAi in transgenic mice76; controlling the suppression of genes using RNAi by conditional inducible systems83; or inhibiting/activating several genes expressed in different tissues and at different times to understand more complex pictures concerning nutrition-, metabolism- , and obesity-related issues.

Table 2. Some Examples of Rodent Genetic Manipulation in Nutrition, Metabolism, and Obesity Research

Acknowledgements

The authors thank LE/97 from the University of Navarra and Navarra Government funds for financial support, and Jane Hoashi for valuable help in the preparation of the manuscript.

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Javier Campin, Ph.D., Fermn I. Milagro, Ph.D., and J. Alfredo Martnez, Ph.D.

Drs. Campin, Milagro, and Martnez are with the Department of Physiology and Nutrition, University of Navarra, Pamplona, Spain.

Copyright International Life Sciences Institute and Nutrition Foundation Aug 2004

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