Perspective

In this brief review, the applications of TG and gene KO technologies to investigate the pathogenesis of diabetic neuropathy have been discussed. The results of the genetic analyses on the polyol pathway are in complete agreement with findings from ARI studies, thus unambiguously confirming the role of the polyol pathway in the pathogenesis of this disease. These studies also demonstrated that the polyol pathway-induced oxida-tive stress rather than osmotic stress is the cause of the diabetes-induced toxicity, and that the mechanism involves the activation of PARP. The experiments with RAGE null mice demonstrated that interaction between plasma AGE and RAGE is the main source of toxicity induced by nonenzymatic glycation. The contribution by intracellular AGE and AGE attached to extracellular matrix to diabetic neuropathy remains unclear. The finding that the development of diabetic neuropathy is accelerated in NF-deficient mice is difficult to interpret because these mice already showed functional and structural abnormalities in the nerve before the induction of diabetes. Perhaps a more appropriate model would be mice lacking the kinase that phosphorylates NF under hyperglycemia.

Genetic analysis of the pathogenic mechanism of a disease has its advantages and disadvantages. The main advantage is that it avoids the uncertainty of drug specificity and availability. Further, with the advent of genome sequence information and DNA array and proteomic analysis of diseases, there will be exponential increase in the number of genes implicated in various diseases that need to be verified. Currently, it is faster to inactivate gene functions by gene KO, antisense RNA, or siRNA technology than by developing specific chemical inhibitors of the gene products. However, gene manipulation by conventional TG and gene KO technologies might affect tissue development, making them unsuitable for adult disease studies. The NF-deficient mice described earlier illustrate this point. More sophisticated TG and gene KO technologies developed in recent years circumvent some of these problems (67). Several inducible transgene expression systems have been developed. In these systems the expression of the transgenes is under the control of a promoter that is regulated by a ligand-activated tran-scriptional enhancer. Usually, the ligands are cell-permeable small molecules not found in mammalian cells such as tetracycline, ecdysone, or isopropyl-P-thiogalactosidase. Although expression systems based on hormone receptor-activated promoters have also been developed. Thus, tissue specificity (determined by the specificity of the promoter that express the ligand-activated transcriptional enhancer) and time of induction (determined by the administration of the ligand) of the transgene can be selected as desired. However, development of these inducible transgene expression systems is quite

Fig. 1. Inducible transgene expression. Mouse line 1 carries the LAE gene that is under the control of a tissue-specific promoter. The transcription termination signal is indicated by (pA). Mouse 2 carries the transgene of interest that is under the control of the minimal promoter (P) together with the enhancer responsive element to which the activated LAE binds. Mating of mouse 1 and 2 brings the two transgenes together. Administration of the ligand to the mice activates the LAE, which then binds to enhancer responsive element to initiate the transcription of the transgene in tissues where LAE is expressed.

Fig. 1. Inducible transgene expression. Mouse line 1 carries the LAE gene that is under the control of a tissue-specific promoter. The transcription termination signal is indicated by (pA). Mouse 2 carries the transgene of interest that is under the control of the minimal promoter (P) together with the enhancer responsive element to which the activated LAE binds. Mating of mouse 1 and 2 brings the two transgenes together. Administration of the ligand to the mice activates the LAE, which then binds to enhancer responsive element to initiate the transcription of the transgene in tissues where LAE is expressed.

cumbersome because two separate TG lines have to be developed (Fig. 1) one carrying the ligand-activated enhancer (LAE) (Fig. 1, Mouse 1), and the other the transgene under the control of the ligand/enhancer inducible promoter (Fig. 1, Mouse 2). Mating between these two TG lines brings the two transgenes into the same host. Conditional gene KO methods have also been developed (Fig. 2). Again this involves the development of two separate mouse lines. First, by conventional gene-targeting technique, the site-specific recombination sequence loxP is introduced into both sides of the gene to be removed (Fig. 2, Mouse 1). Second, by mating of the loxP mice with a TG mouse line that carries the recombinase (Cre) (Fig. 2, mouse 2) will activate recombination between two loxP sequences resulting in the removal of the gene in between. Ablation of the gene in a specific tissue can be obtained by engineering the expression of the Cre transgene in the target tissue using an appropriate promoter. Temporal specificity of gene ablation can be obtained by engineering the Cre transgene expression under the control of an inducible promoter.

There are other genetically engineered mice that are useful for analyzing the pathogenic mechanisms of diabetic neuropathy. An example is the mice carrying a reporter gene under the control of the NF-kB promoter (68). Abnormal NF-kB activity is thought to be an important part of the mechanisms leading to diabetic neuropathy (69-71). In this case the reporter gene P-globin, is not normally found in nerve tissues, and its transcript is readily detectable by reverse transcriptase-polymerase chain reaction. Generally, this type of reporter transgene is applicable to study the transcriptional regulation of transcription factors, structural proteins, or enzymes that do not have a convenient assay to determine their activity or abundance. Other reporter genes such as P-galactosidase (72), luciferase (73), or fluorescence proteins (74) provide even more convenient in vitro and in situ assessment of gene induction or suppression. However, some of these reporter genes do have toxic effect in some cells. Another useful tool is

Fig. 2. Conditional gene knockout. For mouse line 1, by conventional homologous gene replacement technique, two site-specific recombination sequences (lox P) are introduced into each side of the sequence to be deleted. Open boxes with numbers indicate the introns. Conventional transgenic technology generates mouse line 2, where the site-specific Cre gene is under the control of a tissue-specific promoter. The transcription termination signal for the transgene is indicated by (pA). Mating between mouse 1 and 2 brings the Cre and lox P together. Expression of Cre in the target tissue activates the removal of the intron 1 as indicated.

Fig. 2. Conditional gene knockout. For mouse line 1, by conventional homologous gene replacement technique, two site-specific recombination sequences (lox P) are introduced into each side of the sequence to be deleted. Open boxes with numbers indicate the introns. Conventional transgenic technology generates mouse line 2, where the site-specific Cre gene is under the control of a tissue-specific promoter. The transcription termination signal for the transgene is indicated by (pA). Mating between mouse 1 and 2 brings the Cre and lox P together. Expression of Cre in the target tissue activates the removal of the intron 1 as indicated.

the line of TG mice that express the yellow fluorescence protein (YFP) specifically in the neurons (75). The transgene in these mice is the YFP cDNA under the control of the thymas cell antigen (thy 1.2) promoter. All sensory and motor neurons in these thy1.2-YFP mice emit yellow fluorescence when viewed under the fluorescence microscope. When the hairs of these mice are shaved to expose their skin, the dermal nerve fibers are visible under fluorescence microscope without sectioning of tissue. When induced to become diabetic, the loss of fluorescence nerve fibers was evident (76). Thus, these mice provide a noninvasive method of monitoring small fiber degeneration and regeneration during the progression of diabetes, and they will be useful for testing the efficacy of drugs in the treatment and prevention of diabetic neuropathy (Fig. 3).

The advantage of having a variety of techniques to manipulate their genome made mice a favorite animal model to study various biomedical problems. Consequently, a large number of TG and KO mice have been developed, and many more will be developed in the future. The RAGE, PARP, and NF null mice were not originally developed to study diabetic neuropathy. Undoubtedly, many other mutant mice will also be useful for investigating the pathogenesis of this disease. Although, mice do not exhibit the full spectrum of the pathology of diabetic neuropathy as in human, using them to determine the mechanisms leading to common pathology, and find out the reasons for the differences, will surely help us better understand the pathogenesis of this debilitating disease in human.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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