Genetic Manipulation in Nutrition, Metabolism, and Obesity Research

August 2004: 321–330Special Articles

Genetic Manipulation in Nutrition, Metabolism, and ObesityResearchJavier Campion, Ph.D., Fermın I. Milagro, Ph.D., and J. Alfredo Martınez, Ph.D.

We summarize the current standard methods foroverexpressing, inactivating, or manipulatinggenes, with special focus on nutritional and obe-sity research. These molecular biology proce-dures can be carried out with the maintenance ofthe genetic information to subsequent genera-tions (transgenic technology) or devised to exclu-sively transfer the genetic material to a giventarget animal, which cannot be transmitted to thefuture progeny (gene therapy). On the other hand,the RNA interference (RNAi) approach allows forthe creation of new experimental models by tran-sient ablation of gene expression by degradingspecific mRNA, which can be applied to assessdifferent biological functions and mechanisms.The combination of these technologies contrib-utes to the study of the function and regulation ofdifferent metabolism- and obesity-related genesas well as the identification of new pharmacologictargets for nutritional and therapeutic approaches.Key words: transgenic, RNAi, gene therapy, ro-dents© 2004 International Life Sciences Institute

doi: 10.1301/nr.2004.aug.321–330

Introduction

Obesity and other nutrition-related chronic diseases arebecoming increasingly more prevalent, and much effortis being devoted to understanding their pathogenesis andtreatment.1 One approach to investigate the mechanismsinvolved in metabolic disturbances is to overexpress,inactivate, or manipulate specific genes playing a role inthe regulation of body weight and energy metabolism.2,3

Although gene transfer or blockade can be performed intissue culture models, the interaction of the manipulatedgenes with the components of an intact organism pro-vides a much more complete and physiologically rele-vant picture of the gene function than could be achieved

any other way. Many of these techniques are fullyestablished as powerful and routine tools4 and are in-creasingly being applied in order to understand endo-crine problems and metabolic pathways, providing ele-gant models to study nutritional, physiologic, and diseasesituations. In addition, these approaches may be used toreveal new biologic functions and to identify new phar-macologic targets for the treatment of obesity or othergenetically related diseases.

Among all of the possible animal species to be usedfor nutritional research and gene manipulation, themouse and rat are the models of choice. The physiology,embryology, and genetics of these species are well stud-ied and understood,4–6 and the relatively short life cycleand inbred strains of rodents provide the opportunity tostudy disease traits in a defined genetic background.4 Inaddition, a large genetic reservoir of potential modelswith metabolic implications has been generated throughthe identification of spontaneous, radiation-induced, orchemical-induced mutant loci in rodents.6 The mouseand rat models are made even more attractive because ofthe extensive and varied genetic tools available (Table1). Another important reason for specifically using themouse as an animal model for gene targeting lies in thepossibility of isolating embryonic stem (ES) cells (cellsderived from the inner cell mass of the blastocyst, whichpresent multipotent differentiation potential), in whichany gene can be modified to generate novel mutantmice.4

Several excellent reports regarding obesity andtransgenic models of rodents have been recently pub-lished.5,7–9 Therefore, we aim to emphasize the currentstandard methods for manipulating genes—transgenicanimals, gene transfer technology, and RNA interference(RNAi), which are focused on nutritional and obesityresearch—and to highlight newer strategies and goals inthe field of gene transfer. Thus, more complex ap-proaches are emerging, extending the potential of thesetechnologies through the conditional (temporal and spa-tial) control of the genetic manipulation or by the abla-tion of the expression of specific genes by using RNAi,which can be applied to nutritional research.

Drs. Campion, Milagro, and Martınez are with theDepartment of Physiology and Nutrition, University ofNavarra, Pamplona, Spain.

321Nutrition Reviews�, Vol. 62, No. 8

Transgenic Technology

Depending on the method used to introduce the gene, aswell as the time and site in which the process occurs,genetic manipulation can be preserved by the successivegenerations creating the germline transmission.10 In thismethod, usually termed transgenic technology (Figure1), every cell of the animal carries the genetic manipu-lation. The two more commonly used strategies are thepronuclear microinjection of a fertilized oocyte and thetransfection of ES cells.4,10

Pronuclear MicroinjectionThis technique is based on the injection of a DNAfragment into the pronuclei of a fertilized egg with theuse of a microsyringe under a microscope. The techniqueis reliable, although only approximately 5% to 40% ofmice developed from manipulated eggs contain the trans-gene 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 firstspecies because it is easiest to find the framework andbackground to develop a new model in mice.

The first visible phenotypic change using this tech-nique was described in 1982 for animals expressing therat growth hormone sequence under the control of aubiquitous promoter, the mouse metallothionein-I, pro-ducing a model for gigantism.12 More directly related tonutrition and obesity were the pioneering experimentalstudies with GLUT413 and TGF�.14 In the first one, themain advance in creating transgenic animals was thetissue-specific overexpression of a transgene (Figure 2Aand 2B). Putting a tissue-specific promoter downstreamto the cDNA of our interest can lead the expression of thetransgene in a spatial manner. The most commonly usedspecific tissue-promoters involved in energy expenditureand control of feeding are myosin light chain (MLC-1),�-skeletal actin, and muscle creatine kinase (MCK) forskeletal muscle,15 adipocyte fatty acid-binding (aP2) foradipose tissue,16 albumin and liver enriched activator

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

Table 1. Some Web Pages about Resources for Rodent Genomics

http://www.ncbi.nlm.nih.gov/genome/guide/mouse/ Mouse Genome Resourceshttp://tbase.jax.org/ Transgenic/Targeted Mutation Databasehttp://jaxmice.jax.org/library/models/index.html Jackson Laboratory Libraryhttp://www.med.umich.edu/tamc/index.html Transgenic Animal Model Corehttp://www.ensembl.org/Mus_musculus/ Mouse Genome Sequencing Consortiumhttp://www.mshri.on.ca/nagy/ Nagy Labhttp://www.anex.med.tokushima-u.ac.jp/rat/index-e.html Rat Data Basehttp://rgd.mcw.edu/ Rat Genome Databasehttp://www.bioscience.org/knockout/knochome.htm Database of Gene Knockoutshttp://ratmap.org/index.html Ratmaphttp://www.atcc.org/ ATCC Global Bioresource Centerhttp://www.zmg.uni-mainz.de/tetmouse/tet.htm Laboratory of Molecular Mouse Genetics

322 Nutrition Reviews�, Vol. 62, No. 8

protein (LAP) for liver,17 and nestin, neuron-specificenolase (NSE), and glyal fibrillary acidic protein(GFAP)18 for brain. Other recent examples of this tech-nique are the overexpression of the plasminogen activa-tor inhibitor-1 gene, which attenuated a high-fat diet-induced obesity,19 and the overexpression of theuncoupling proteins-2 (UCP2) and -3 (UCP3), whichreduced fat mass and increased LDL cholesterol.20

Despite the efficiency and consistency of pronuclearmicroinjection in creating transgenic animals, the studiesusing this approach were hindered by three major prob-lems: the inability to control the randomly appearingsites of integration into the genome; the inability tocontrol the integrated copy numbers of transgenes; andembryonic lethality due to the toxic effects of certaingene expression.10 Random integration and multiple cop-ies lead to unregulated expression of transgenes andcause side effects, while expression of some genes in anearly or inconvenient moment can produce lethal conse-quences. To circumvent the latter, several conditionaltechniques, such as temporal control, have been devel-oped. The temporal conditional system allows the re-searcher to reversibly control the time expression of atransgene at any point during development or postnatallife and, combined with spatial control, only in thedesired cell type. Many systems are available for thistype of conditional control, including tamoxifen, glu-cocorticoids, and tetracycline.21 The last, known as thetet system, is by far the most popular system and isroutinely used in in vivo and in vitro models. This

technique is based on the property of a bacterial operatortetO (TRE promoter) to be deactivated by the tetR-VP16protein (tTA) in the presence of tetracycline.21 Thesuccessful application of this methodology requires thebreeding of two different lines of mice (Figure 2). Oneline expresses the tTA-protein and the other expressesthe transgene of our interest under the control of the TREpromoter. After breeding both lines, the whole body ofthe next generation carries the genetic information underthe control of tetracycline. In order to mix temporal andspatial conditional control, tTA protein could be ex-pressed under the control of a tissue-specific promoter(Figure 2D). Although this method is reliable and fullyestablished, new possibilities are being investigated inorder to overcome different problems such as leaking orlow levels of tTA expression.21

Transfection of ES CellsTo avoid some of the technical problems affecting thepronuclear microinjection approach, genetic informationcan also be integrated in the genome of ES cells bymeans of homologous recombination (a process bywhich a fragment of exogenous genomic DNA intro-duced into a mammalian cell can be located and recom-bined with the endogenous homologous sequence).6,10

These cells are derived from the inner cell mass of themouse blastocyst and thus have the potential to contrib-ute to all tissues of the developing embryo.22 After theextraction of the cells, this method requires culture con-ditions that maintain the cells in an undifferentiated state.

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


Since these cells are cultured, DNA can be introduced bytransfection or viral transduction (see below for differentmethods), as in any other established cell line, and thetransformed cells can be selected using standard mark-ers.6 The recombinant cells are then introduced into theblastocoele 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 mousemodel, despite the tremendous amount of effort that hasbeen invested in other species. To bypass this technolog-ical barrier in rats, nuclear transfer can be used as analternative approach. The genome of genetically modi-fied cells is physically transferred to a rat enucleatedoocyte, which develops into a genetically modified ani-mal.23

The most common strategy using ES cell technologyis to disrupt the function of a gene by introducing a DNAflanked by homologous regions that recognize the targetgene.6 After the recombination and generation of themouse, a transgenic model is created with a null allele ofthe selected gene, a strategy that is termed gene knockout(KO). Early obesity-related models were the transform-ing growth factor-� 1 KO24 and a mouse lacking func-tional lipoprotein lipase (LPL).25 Today, the use of KOmice in obesity research is still effective, and recentdevelopments include the fatty acid synthase (FAS),26

adiponectin,27 ghrelin,28 and mal1 (FABP5) genes.29

Recent reviews7–9 give a complete list of KO modelsused for metabolism research. When the global removalof a gene of interest using conventional KO methodsresults in embryonic lethality, investigators can choose toproduce a conditional tissue-specific (also time-specificif combined with the tet system) KO line of mice inwhich deletions can still be studied in vivo. Examples ofthis technology applied to body weight regulation are theinsulin receptor depletion in adipose tissue30 andGLUT4,31 and the muscle-specific PPAR�-deficientmouse.32

Gene Therapy

When the scientific aim in nutritional research is not totransfer genetic information to subsequent generations,the most commonly used method is gene transfer tosomatic cells,33 which can be achieved by using in vivoor ex vivo approaches (Figure 1). While in vivo genetransfer delivers the genetic manipulation directly intothe animal, ex vivo gene delivery refers to the transfer ofthe gene manipulation into cells/organs removed from adonor, expanded in vitro, and then subsequently re-introduced into the animal.34 In both cases, the cells losethe capacity to pass the gene manipulation on to subse-quent generations, but the time, cost, and complexity ofthe experiments are reduced.35 Moreover, gene transfercan be transiently or permanently established, and is

commonly used in gene therapy research for humans.34

The most commonly used methods for gene transfer inrodents and obesity research are viral transduction sys-tems,36 mainly with adenovirus, and the direct injectionof naked DNA.37

Viral TransductionDue to the efficiency with which viruses can deliver theirnucleic acid into cells, and the high levels of replicationand gene expression, viruses have been repeatedly usedas vectors not only for gene expression in cultured cells,but also for gene transfer to living animals.35 Severaltypes of viral vectors have been developed in genetransfer. The main type of virus used for gene transfer inobesity is the adenovirus. Several examples of this genetransfer strategy in creating endocrine-modified modelsin rodents have been reported in the experiments ofMuzzin et al.38 with leptin, the study by Nagamatsu etal.39 expressing preproinsulin in adipose tissue, or theinjection of the liver glucokinase into skeletal muscle.40

More recently, the peripheral delivery of adiponectin41

has been published. This type of strategy presents highlevels of transient gene expression, and adenoviruses caninfect dividing and nondividing cells. While up to 7 to 8kb DNA can be added in the adenoviral vector, it is notsuitable for long-term expression of the transgene due tothe lack of integration into the host genome, and becauseadenoviral vector particles are highly immunogenic. De-spite its being a widely used technique, it could produceinflammatory and toxic reactions in the host, and thisimmunogenicity is responsible for the depletion of ad-enovirally transduced cells.35 For this reason, other non-pathogenic and non-toxic approaches were developed.For example, adeno-associated viruses (AAV) can infecta wide range of cells, including those that do not divide.However, a limited capacity for foreign genes (approxi-mately 4 kb) is provided. They present viral genomeintegration into the cell genome, although there is a lackof specific integration, which may result in cell mutagen-esis. Again, leptin has been a target gene for genetictransference using this type of virus.42

Other similar approaches with larger capacities arethe herpes simplex virus and retrovirus.10 The first onehas a maximum size capacity of 30 kb and a broad hostrange, although in natural human infections the virus isneurotropic and does not integrate into the cell genome.The applications of this type of virus in overexpressinglacZ in skeletal muscle43 and 5-HT1B receptors in neu-rons44 were reported. The latter, a retrovirus, only infectsdividing cells, but the insertion capacity is 10 kb andpresents stable gene expression due to viral genomeintegration into cell chromosomes (again, by randominsertion, which may result in mutagenesis). The classictarget tissue for gene transfer using retrovirus is the


liver,45 although examples regarding obesity apparentlyhave not yet been described.

Finally, the lentivirus (with 10 kb of capacity), atype of retroviral-based vector, solves some of the prob-lems of retrovirus infecting nondividing cells, and main-tains stable gene expression due to viral genome integra-tion into cell chromosomes.35 Several experiments havebeen carried out using this type of virus in different targettissues, such as the heart,46 muscle,47 and neurons.48

Nonviral VectorsTheoretically, nonviral vectors have no limit concerningthe size of DNA to be incorporated into the cells, andthey are suitable for oligonucleotide delivery, which isalso applicable for RNAi transfer.49,50 In addition, thesevectors are relatively non-toxic and non-infectious. How-ever, the targeting is not specific and the effect is tran-sient (several days), although new achievements haveextended this period to weeks or months.35 Moreover,transfection in vivo is generally far less efficient than theadenoviruses and can induce an immunogenic response.Indeed, the use of nonviral vectors is extensive andgenerally applied, and multiple examples have beendescribed. Typical nonviral techniques of gene transferare the mechanical administration of naked DNA, elec-troporation, cationic liposomes, and DNA-protein com-plex.35 Our group, for example, has performed musclegene transfer of UCP1,51 UCP2,52 and leptin53 by in vivoinjection of naked DNA into the rodent. This approachhad some implications in the regulation of body weightand energy metabolism. Transfection by electroporationis also simple, inexpensive, and safe, and by using thistechnique it is possible to enhance the transfection invivo of a direct DNA injection in brain,54 muscle,55 orliver.56 Finally, recent studies have reported significantsuccess in gene transfer by transfecting in vivo57 and exvivo58 exogenous DNA with cationic liposomes or othercomposites, nonviral vector systems in liver,59 and otherobesity target tissues of rodents, such as adipose tissue.60

RNA Interference (RNAi)

A new technology arising in the field of genetic andmolecular manipulation is the antisense approach, whichis being applied to inhibit the expression of a target genein a sequence-specific manner.61 A possible break-through in these technologies is RNAi, which is used toinvestigate and decipher gene function by degrading aspecific mRNA target, thus knocking down the level ofan encoded protein.62 Key to the technique are double-stranded RNAs 21–25 nucleotides long, called shortinterfering RNAs (siRNAs), that interact in the cyto-plasm with a multiprotein complex called RISC (RNA-induced silencing complex). Finally, the siRNA-inducedactivation of RISC binds this complex to the homologous

mRNA by base-pairing interactions for cleavage anddegradation of the cognate RNA (Figure 3).

Gene silencing mediated by RNAi can be achievedin two different ways. In the first one, in vitro-synthe-sized siRNAs are introduced into cells using microinjec-tion, transfection, or electroporation to transiently sup-press gene expression.61 A disadvantage of this approachis that synthetic siRNA is not stable and must be pro-tected during shipping. In the second approach, siRNAsare expressed in vivo from DNA vectors and cassettes tocreate stably expressed siRNAs within the cells or trans-genic animals.61 However, they require more hands-ontime from the researcher. Three different RNA polymer-ase III promoters are currently used to drive the expres-sion of a small hairpin siRNA in mammalian cells:human and mouse U6 and human H1. This strategyappears to be the best method for long-term studiesand in cases in which antibiotic selection of siRNA-containing cells is desired to enrich the culture withcells 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 geneexpression silencing endogenous gene function in thenematode Caenorhabditis elegans,63 in which it has beenused to disrupt the expression of each of the 16,757worm genes. In this animal, 305 gene inactivationscaused reduced body fat, and 112 gene inactivationscaused increased fat storage. RNAi has since been usedto investigate different aspects of lipid metabolism ho-meostasis, such as the regulation of insulin signaling,64

the role for fatty acid synthase in cancer cells,65 thefunction of adiponectin receptors,66 and the physiologicaction of the agouti-related peptide (AGRP).49 In cellcultures, after the use of RNAi for silencing genes ininsulin-sensitive adipocytes, the role of these genes ininsulin-signaling cascades has been demonstrated.64 Invivo hypothalamic administration of RNAi againstAGRP has proven that this peptide reduces metabolicrate independently of food intake.49

There are several methods to introduce siRNA andnucleic acids into cultured cells: electroporation67; cal-cium phosphate precipitation68; oligofectamine69; lipo-fectamine70; adenovirus71; lentivirus72; oncoretrovirus73;and different synthetic polymers.74 However, the in vivoadministration presents some problems and so far theresults are not encouraging. Delivery remains a majorobstacle; to deliver siRNA and si-encoding DNAs to thesite of action on vertebrate cells, a gene therapy approachcould be interesting, using local administration of nakedDNA,49 cationic-liposomes,75 lentivirus,76 adenovirus,77

or hydrodynamic transfection.50 Even embryo (in ovo)electroporation has been tested.78 Nevertheless, the de-velopment of RNAi viral vectors (adeno- or lentivirus)by the inclusion of inducible or tissue-specific promoters


could permit the therapeutic use of these specific RNAitools in vivo.

Finally, it is also possible to create transgenic ani-mals by vector-mediated siRNA delivery in stem cells,79

oocytes,80 or blastocysts.81 Moreover, new methods toinduce cell- and tissue-specific expression of RNAi arebeing developed.79,81

New Perspectives

In the field of gene manipulation, new technologies andimprovements are providing new possibilities for thescientist and for metabolism and obesity researchers.These new achievements, together with the knowledge ofa growing number of genes and molecules involved inthe body weight control system (Table 2), are allowing

for the in vivo study of the regulation and function ofdifferent endocrine- and obesity-related genes and theidentification of new pharmacologic targets for thera-peutic approaches. Once a basic level of understandingof the endocrine system is reached, new models ofinvestigation must be carried out by performing thedifferent genetic manipulation techniques described inthis review: gene therapy to replace the ablated geneof a KO model82; inhibiting the expression of a geneusing RNAi in transgenic mice76; controlling the sup-pression of genes using RNAi by conditional induciblesystems83; or inhibiting/activating several genes ex-pressed in different tissues and at different times tounderstand more complex pictures concerning nutri-tion-, metabolism-, and obesity-related issues.

Figure 3. The mechanism of RNA interference.


Table 2. Some Examples of Rodent Genetic Manipulation in Nutrition, Metabolism, and Obesity ResearchGene Name Type of Manipulation

Lipid Metabolism Lipoprotein Lipase (LPL) KOHormone-sensitive Lipase (HSL) KOCD36 tgFatty Acid Binding Protein 4 (FABP4) KOAcyl CoA: Diglycerol Acyltransferase (DGAT) KOAcetyl Coenzyme A Carboxylase 2 (ACC2) KOGlycerol Phosphate Dehydrogenase (GPD) tgAcyl Coenzyme A Oxidase (ACOX) KOFatty Acid Synthase (FAS) KO, siFatty Acid Binding Protein 5 (FABP5) KOPhosphoenolpyruvate carboxykinase (PEPCK) tg

Insulin Signaling Insulin Receptor (IR) KOInsulin Receptor Substrate 2 (IRS2) KOTubby KOProtein Tyrosine Phosphatase 1B (PTP1B) KOGLUT4 KO, tgAkt si

Adipokines Adiponectin KOLeptin tgTNF-� KOTGF-� tg

Appetite Regulation Neuropeptide Y (NPY) KOAgouti-related Protein (AGRP) tg, siOrexins 1 and 2 KOPro-opiomelanocortin (POMC) KOMelanin-concentrating Hormone (MCH) KOGhrelin KOCorticotropin-releasing Hormone (CRH) KO

Thermogenesis Uncoupling Protein 1 (UCP1) KO, tgUncoupling Protein 2 (UCP2) KO, tgUncoupling Protein 3 (UCP3) KO, tg

Transcription Factors PPAR� KOPPAR� KOSREBP1C KO, tgC/EBP� KOC/EBP� KO

Receptors Melanocortin 4 Receptor (MC4R) KOMelanocortin 3 Receptor (MC3R) KOBombesin Receptor KOCorticotropin-releasing Hormone 1 Receptor (CRH1R) KOCholecystokinin A Receptor KOMelanin-concentrating Hormone 1 Receptor (MCH1R) KONeuropeptide 1 Receptor (NPY-1R) KONeuropeptide 2 Receptor (NPY-2R) KONeuropeptide 5 Receptor (NPY-5R) KOVLDL Receptor KO�2-Adrenergic Receptor tg�1-Adrenergic Receptor tg�3-Adrenergic Receptor KOAdiponectin Receptor 1 and 2 si

Others Perilipin KOCaveolin 1 KOMetallothionein 1 and 2 KO

KO � knockout, tg � transgenic overexpression, si � siRNA. From references 7–9.


Acknowledgements

The authors thank LE/97 from the University of Navarraand Navarra Government funds for financial support, andJane Hoashi for valuable help in the preparation of themanuscript.

1. Martinez JA. Body-weight regulation: causes ofobesity. Proc Nutr Soc. 2000;59:337–345.

2. Moreno-Aliaga MJ, Marti A, Garcia-Foncillas J, Al-fredo Martinez J. DNA hybridization arrays: a pow-erful technology for nutritional and obesity research.Br J Nutr. 2001;86:119–122.

3. Marti A, De Miguel C, Jebb SA, et al. Methodolog-ical approaches to assess body-weight regulationand aetiology of obesity. Proc Nutr Soc. 2000;59:405–411.

4. van der Weyden L, Adams DJ, Bradley A. Tools fortargeted manipulation of the mouse genome.Physiol Genomics. 2002;11:133–164.

5. Valet P, Tavernier G, Castan-Laurell I, Saulnier-Blache JS, Langin D. Understanding adipose tissuedevelopment from transgenic animal models. JLipid Res. 2002;43:835–860.

6. Robinson SW, Dinulescu DM, Cone RD. Geneticmodels of obesity and energy balance in the mouse.Annu Rev Genet. 2000;34:687–745.

7. Palou A, Pico C, Bonet ML. The molecular basis ofbody weight control. In: Elmadfa I, Anklam E, KonigJS, eds. Modern Aspects of Nutrition: PresentKnowledge and Future Perspectives. InternationalCongress of Nutrition, Vienna, August 2001. Basel:S. Karger AG; 2003;56:164–168.

8. Fruhbeck G, Gomez-Ambrosi J. Control of bodyweight: a physiologic and transgenic perspective.Diabetologia. 2003;46:143–172.

9. Snyder EE, Walts B, Perusse L, et al. The humanobesity gene map: the 2003 update. Obes Res.2004;12:369–439.

10. Primrose SB, Twyman R, Old RW. Principles ofGene Manipulation. 6th ed. Oxford, England: Black-well Publishing; 2001.

11. Swift GH, Hammer RE, MacDonald RJ, Brinster RL.Tissue-specific expression of the rat pancreaticelastase I gene in transgenic mice. Cell. 1984;38:639–646.

12. Palmiter RD, Brinster RL, Hammer RE, et al. Dra-matic growth of mice that develop from eggs mi-croinjected with metallothionein-growth hormonefusion genes. Nature. 1982;300:611–615.

13. Shepherd PR, Gnudi L, Tozzo E, Yang H, Leach F,Kahn BB. Adipose cell hyperplasia and enhancedglucose disposal in transgenic mice overexpressingGLUT4 selectively in adipose tissue. J Biol Chem.1993;268:22243–22246.

14. Luetteke NC, Lee DC, Palmiter RD, Brinster RL,Sandgren EP. Regulation of fat and muscle devel-opment by transforming growth factor alpha intransgenic mice and in cultured cells. Cell GrowthDiffer. 1993;4:203–213.

15. Bothe GW, Haspel JA, Smith CL, Wiener HH, Bur-den SJ. Selective expression of Cre recombinase inskeletal muscle fibers. Genesis. 2000;26:165–166.

16. Imai T, Jiang M, Chambon P, Metzger D. Impairedadipogenesis and lipolysis in the mouse upon se-

lective ablation of the retinoid X receptor alphamediated by a tamoxifen-inducible chimeric Crerecombinase (Cre-ERT2) in adipocytes. Proc NatlAcad Sci U S A. 2001;98:224–228.

17. Schonig K, Schwenk F, Rajewsky K, Bujard H. Strin-gent doxycycline dependent control of CRE recom-binase in vivo. Nucleic Acids Res. 2002;30:e134.

18. Barton MD, Dunlop JW, Psaltis G, Kulik J, De-Gennaro L, Kwak SP. Modified GFAP promoterauto-regulates tet-activator expression for in-creased transactivation and reduced tTA-associ-ated toxicity. Brain Res Mol Brain Res. 2002;101:71–81.

19. Lijnen HR, Maquoi E, Morange P, et al. Nutritionallyinduced obesity is attenuated in transgenic miceoverexpressing plasminogen activator inhibitor-1.Arterioscler Thromb Vasc Biol. 2003;23:78–84.

20. Horvath TL, Diano S, Miyamoto S, et al. Uncouplingproteins-2 and 3 influence obesity and inflammationin transgenic mice. Int J Obes Relat Metab Disord.2003;27:433–442.

21. Ryding AD, Sharp MG, Mullins JJ. Conditionaltransgenic technologies. J Endocrinol. 2001;171:1–14.

22. Babinet C, Cohen-Tannoudji M. Genome engineer-ing via homologous recombination in mouse embry-onic stem (ES) cells: an amazingly versatile tool forthe study of mammalian biology. An Acad BrasCienc. 2001;73:365–383.

23. Hayes E, Galea S, Verkuylen A, et al. Nuclear trans-fer of adult and genetically modified fetal cells of therat. Physiol Genomics. 2001;5:193–204.

24. Kulkarni AB, Huh CG, Becker D, et al. Transforminggrowth factor beta 1 null mutation in mice causesexcessive inflammatory response and early death.Proc Natl Acad Sci U S A. 1993;90:770–774.

25. Weinstock PH, Bisgaier CL, Aalto-Setala K, et al.Severe hypertriglyceridemia, reduced high densitylipoprotein, and neonatal death in lipoprotein lipaseknockout mice: mild hypertriglyceridemia with im-paired very low density lipoprotein clearance in het-erozygotes. J Clin Invest. 1995;96:2555–2568.

26. Chirala SS, Chang H, Matzuk M, et al. Fatty acidsynthesis is essential in embryonic development:fatty acid synthase null mutants and most of theheterozygotes die in utero. Proc Natl Acad SciU S A. 2003;100:6358–6363.

27. Ma K, Cabrero A, Saha PK, et al. Increased beta-oxidation but no insulin resistance or glucose intol-erance in mice lacking adiponectin. J Biol Chem.2002;277:34658–34661.

28. Sun Y, Ahmed S, Smith RG. Deletion of ghrelinimpairs neither growth nor appetite. Mol Cell Biol.2003;23:7973–7981.

29. Maeda K, Uysal KT, Makowski L, et al. Role of thefatty acid binding protein mal1 in obesity and insulinresistance. Diabetes. 2003;52:300–307.

30. Bluher M, Michael MD, Peroni OD, et al. Adiposetissue selective insulin receptor knockout protectsagainst obesity and obesity-related glucose intoler-ance. Dev Cell. 2002;3:25–38.

31. Abel ED, Peroni O, Kim JK, et al. Adipose-selectivetargeting of the GLUT4 gene impairs insulin actionin muscle and liver. Nature. 2001;409:729–733.

32. Norris AW, Chen L, Fisher SJ, et al. Muscle-specific


PPARgamma-deficient mice develop increased ad-iposity and insulin resistance but respond to thiazol-idinediones. J Clin Invest. 2003;112:608–618.

33. Kelley VR, Sukhatme VP. Gene transfer in the kid-ney. Am J Physiol. 1999;276:F1–F9.

34. Fauci AS, Braunwald E, Isselbacher KJ, et al. Har-rison’s Principles of Internal Medicine. 14th ed. NewYork: McGraw Hill; 1998.

35. Romano G, Michell P, Pacilio C, Giordano A. Latestdevelopments in gene transfer technology: achieve-ments, perspectives, and controversies over thera-peutic applications. Stem Cells. 2000;18:19–39.

36. Larcher F, Del Rio M, Serrano F, et al. A cutaneousgene therapy approach to human leptin deficien-cies: correction of the murine ob/ob phenotype us-ing leptin-targeted keratinocyte grafts. FASEB J.2001;15:1529–1538.

37. Utvik JK, Nja A, Gundersen K. DNA injection intosingle cells of intact mice. Hum Gene Ther. 1999;10:291–300.

38. Muzzin P, Cusin I, Charnay Y, Rohner-JeanrenaudF. Single intracerebroventricular bolus injection of arecombinant adenovirus expressing leptin results inreduction of food intake and body weight in bothlean and obese Zucker fa/fa rats. Regul Pept. 2000;92:57–64.

39. Nagamatsu S, Nakamichi Y, Ohara-Imaizumi M, etal. Adenovirus-mediated preproinsulin gene transferinto adipose tissues ameliorates hyperglycemia inobese diabetic KKA(y) mice. FEBS Lett. 2001;509:106–110.

40. Jimenez-Chillaron JC, Newgard CB, Gomez-FoixAM. Increased glucose disposal induced by adeno-virus-mediated transfer of glucokinase to skeletalmuscle in vivo. FASEB J. 1999;13:2153–2160.

41. Shklyaev S, Aslanidi G, Tennant M, et al. Sustainedperipheral expression of transgene adiponectin off-sets the development of diet-induced obesity inrats. Proc Natl Acad Sci U S A. 2003;100:14217–14222.

42. Dube MG, Beretta E, Dhillon H, Ueno Nm, Kalra PS,Kalra SP. Central leptin gene therapy blocks high-fat diet-induced weight gain, hyperleptinemia, andhyperinsulinemia: increase in serum ghrelin levels.Diabetes. 2002;51:1729–1736.

43. Huard J, Goins WF, Glorioso JC. Herpes simplexvirus type 1 vector mediated gene transfer to mus-cle. Gene Ther. 1995;2:385–392.

44. Neumaier JF, Vincow ES, Arvanitogiannis A, WiseRA, Carlezon WA Jr. Elevated expression of 5-HT1Breceptors in nucleus accumbens efferents sensi-tizes animals to cocaine. J Neurosci. 2002;22:10856–10863.

45. Aubert D, Pichard V, Durand S, Moullier P, Ferry N.Cytotoxic immune response after retroviral-medi-ated hepatic gene transfer in rat does not precludeexpression from adeno-associated virus 1 trans-duced muscles. Hum Gene Ther. 2003;14:473–481.

46. Fleury S, Simeoni E, Zuppinger C, et al. Multiplyattenuated, self-inactivating lentiviral vectors effi-ciently deliver and express genes for extended pe-riods of time in adult rat cardiomyocytes in vivo.Circulation. 2003;107:2375–2382.

47. O’Rourke JP, Hiraragi H, Urban K, Patel M, OlsenJC, Bunnell BA. Analysis of gene transfer and ex-

pression in skeletal muscle using enhanced EIAVlentivirus vectors. Mol Ther. 2003;7:632–639.

48. Blomer U, Naldini L, Kafri T, Trono D, Verma IM,Gage FH. Highly efficient and sustained gene trans-fer in adult neurons with a lentivirus vector. J Virol.1997;71:6641–6649.

49. Makimura H, Mizuno TM, Mastaitis JW, Agami R,Mobbs CV. Reducing hypothalamic AGRP by RNAinterference increases metabolic rate and de-creases body weight without influencing food in-take. BMC Neurosci. 2002;3:18.

50. McCaffrey AP, Meuse L, Pham TT, Conklin DS,Hannon GJ, Kay MA. RNA interference in adultmice. Nature. 2002;418:38–39.

51. Larrarte E, Margareto J, Novo FJ, Marti A, AlfredoMartinez J. UCP1 muscle gene transfer and mito-chondrial proton leak mediated thermogenesis.Arch Biochem Biophys. 2002;404:166–171.

52. Marti A, Larrarte E, Novo FJ, Garcia M, Martinez JA.UCP2 muscle gene transfer modifies mitochondrialmembrane potential. Int J Obes Relat Metab Disord.2001;25:68–74.

53. Marti A, Novo FJ, Martinez-Anso E, Zarategui M,Aguado M, Martinez JA. Leptin gene transfer intomuscle increases lipolysis and oxygen consumptionin white fat tissue in ob/ob mice. Biochem BiophysRes Commun. 1998;246:859–862.

54. Kondoh T, Motooka Y, Bhattacharjee AK, KokunaiT, Saito N, Tamaki N. In vivo gene transfer into theperiventricular region by electroporation. NeurolMed Chir (Tokyo). 2000;40:618–622.

55. Croze F, Prud’homme GJ. Gene therapy of strepto-zotocin-induced diabetes by intramuscular deliveryof modified preproinsulin genes. J Gene Med. 2003;5:425–437.

56. Liu F, Huang L. Electric gene transfer to the liverfollowing systemic administration of plasmid DNA.Gene Ther. 2002;9:1116–1119.

57. Matsuura M, Yamazaki Y, Sugiyama M, et al. Poly-cation liposome-mediated gene transfer in vivo.Biochim Biophys Acta. 2003;1612:136–143.

58. Lakey JR, Young AT, Pardue D, et al. Nonviraltransfection of intact pancreatic islets. Cell Trans-plant. 2001;10:697–708.

59. Kawakami S, Yamashita F, Nishida K, Nakamura J,Hashida M. Glycosylated cationic liposomes forcell-selective gene delivery. Crit Rev Ther Drug Car-rier Syst. 2002;19:171–190.

60. Kato N, Nakanishi K, Nemoto K, et al. Efficient genetransfer from innervated muscle into rat peripheraland central nervous systems using a non-viralhaemagglutinating virus of Japan (HVJ)-liposomemethod. J Neurochem. 2003;85:810–815.

61. Dykxhoorn DM, Novina CD, Sharp PA. Killing themessenger: short RNAs that silence gene expres-sion. Nat Rev Mol Cell Biol. 2003;4:457–467.

62. Hannon GJ. RNA interference. Nature. 2002;418:244–251.

63. Kamath RS, Fraser AG, Dong Y, et al. Systematicfunctional analysis of the Caenorhabditis elegansgenome using RNAi. Nature. 2003;421:231–237.

64. Katome T, Obata T, Matsushima R, et al. Use ofRNA interference-mediated gene silencing and ad-enoviral overexpression to elucidate the roles of


AKT/protein kinase B isoforms in insulin actions.J Biol Chem. 2003;278:28312–28323.

65. De Schrijver E, Brusselmans K, Heyns W, Verho-even G, Swinnen JV. RNA interference-mediatedsilencing of the fatty acid synthase gene attenuatesgrowth and induces morphological changes andapoptosis of LNCaP prostate cancer cells. CancerRes. 2003;63:3799–3804.

66. Yamauchi T, Kamon J, Ito Y, et al. Cloning of adi-ponectin receptors that mediate antidiabetic meta-bolic effects. Nature. 2003;423:762–769.

67. Jiang ZY, Zhou QL, Coleman KA, Chouinard M,Boese Q, Czech MP. Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing. Proc Natl Acad Sci U S A.2003;100:7569–7574.

68. Donze O, Picard D. RNA interference in mammaliancells using siRNAs synthesized with T7 RNA poly-merase. Nucleic Acids Res. 2002;30:e46.

69. Elbashir SM, Harborth J, Lendeckel W, Yalcin A,Weber K, Tuschl T. Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammaliancells. Nature. 2001;411:494–498.

70. Gou D, Jin N, Liu L. Gene silencing in mammaliancells by PCR-based short hairpin RNA. FEBS Lett.2003;548:113–118.

71. Shen C, Buck AK, Liu X, Winkler M, Reske SN. Genesilencing by adenovirus-delivered siRNA. FEBSLett. 2003;539:111–114.

72. An DS, Xie Y, Mao SH, Morizono K, Kung SK, ChenIS. Efficient lentiviral vectors for short hairpin RNAdelivery into human cells. Hum Gene Ther. 2003;14:1207–1212.

73. Brummelkamp TR, Bernards R, Agami R. A systemfor stable expression of short interfering RNAs inmammalian cells. Science. 2002;296:550–553.

74. Lewis DL, Hagstrom JE, Loomis AG, Woolff JA,

Herweijer H. Efficient delivery of siRNA for inhibitionof gene expression in postnatal mice. Nat Genet.2002;32:107–108.

75. Sorensen DR, Leirdal M, Sioud M. Gene silencingby systemic delivery of synthetic siRNAs in adultmice. J Mol Biol. 2003;327:761–766.

76. Rubinson DA, Dillon CP, Kwiatkowski AV, et al. Alentivirus-based system to functionally silencegenes in primary mammalian cells, stem cells andtransgenic mice by RNA interference. Nat Genet.2003;33:401–406.

77. Zhao LJ, Jian H, Zhu H. Specific gene inhibition byadenovirus-mediated expression of small interferingRNA. Gene. 2003;316:137–141.

78. Pekarik V, Bourikas D, Miglino N, Joset P, PreiswerkS, Stoeckli ET. Screening for gene function inchicken embryo using RNAi and electroporation.Nat Biotechnol. 2003;21:93–96.

79. Carmell MA, Zhang L, Conklin DS, Hannon GJ,Rosenquist TA. Germline transmission of RNAi inmice. Nat Struct Biol. 2003;10:91–92.

80. Stein P, Svoboda P, Schultz RM. Transgenic RNAiin mouse oocytes: a simple and fast approach tostudy gene function. Dev Biol. 2003;256:187–193.

81. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D.Germline transmission and tissue-specific expres-sion of transgenes delivered by lentiviral vectors.Science. 2002;295:868–872.

82. Jiang XC, Qin S, Qiao C, et al. Apolipoprotein Bsecretion and atherosclerosis are decreased in micewith phospholipid-transfer protein deficiency. NatMed. 2001;7:847–852.

83. Wiznerowicz M, Trono D. Conditional suppressionof cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J Virol. 2003;77:8957–8961.


Genetic Manipulation in Nutrition, Metabolism, and Obesity Research

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