Intermittent Hypoxic Conditioning Rescues Cognition and Mitochondrial Bioenergetic Profile in the Triple Transgenic Mouse Model of Alzheimer's Disease.

The lack of effective disease-modifying therapeutics to tackle Alzheimer's disease (AD) is unsettling considering the actual prevalence of this devastating neurodegenerative disorder worldwide. Intermittent hypoxic conditioning (IHC) is a powerful non-pharmacological procedure known to enhance brain resilience. In this context, the aim of the present study was to investigate the potential long-term protective impact of IHC against AD-related phenotype, putting a special focus on cognition and mitochondrial bioenergetics and dynamics. For this purpose, six-month-old male triple transgenic AD mice (3×Tg-AD) were submitted to an IHC protocol for two weeks and the behavioral assessment was performed at 8.5 months of age, while the sacrifice of mice occurred at nine months of age and their brains were removed for the remaining analyses. Interestingly, IHC was able to prevent anxiety-like behavior and memory and learning deficits and significantly reduced brain cortical levels of amyloid-ß (Aß) in 3×Tg-AD mice. Concerning brain energy metabolism, IHC caused a significant increase in brain cortical levels of glucose and a robust improvement of the mitochondrial bioenergetic profile in 3×Tg-AD mice, as mirrored by the significant increase in mitochondrial membrane potential (ΔΨm) and respiratory control ratio (RCR). Notably, the improvement of mitochondrial bioenergetics seems to result from an adaptative coordination of the distinct but intertwined aspects of the mitochondrial quality control axis. Particularly, our results indicate that IHC favors mitochondrial fusion and promotes mitochondrial biogenesis and transport and mitophagy in the brain cortex of 3×Tg-AD mice. Lastly, IHC also induced a marked reduction in synaptosomal-associated protein 25 kDa (SNAP-25) levels and a significant increase in both glutamate and GABA levels in the brain cortex of 3×Tg-AD mice, suggesting a remodeling of the synaptic microenvironment. Overall, these results demonstrate the effectiveness of the IHC paradigm in forestalling the AD-related phenotype in the 3×Tg-AD mouse model, offering new insights to AD therapy and forcing a rethink concerning the potential value of non-pharmacological interventions in clinical practice.

S1P3 receptor influences key physiological properties of fast-twitch extensor digitorum longus muscle.

To examine the role of sphingosine 1-phosphate (S1P) receptor 3 (S1P3) in modulating muscle properties, we utilized transgenic mice depleted of the receptor. Morphological analyses of extensor digitorum longus (EDL) muscle did not show evident differences between wild-type and S1P3-null mice. The body weight of 3-mo-old S1P3-null mice and the mean cross-sectional area of transgenic EDL muscle fibers were similar to those of wild-type. S1P3 deficiency enhanced the expression level of S1P1 and S1P2 receptors mRNA in S1P3-null EDL muscle. The contractile properties of S1P3-null EDL diverge from those of wild-type, largely more fatigable and less able to recover. The absence of S1P3 appears responsible for a lower availability of calcium during fatigue. S1P supplementation, expected to stimulate residual S1P receptors and signaling, reduced fatigue development of S1P3-null muscle. Moreover, in the absence of S1P3, denervated EDL atrophies less than wild-type. The analysis of atrophy-related proteins in S1P3-null EDL evidences high levels of the endogenous regulator of mitochondria biogenesis peroxisome proliferative-activated receptor-γ coactivator 1α (PGC-1α); preserving mitochondria could protect the muscle from disuse atrophy. In conclusion, the absence of S1P3 makes the muscle more sensitive to fatigue and slows down atrophy development after denervation, indicating that S1P3 is involved in the modulation of key physiological properties of the fast-twitch EDL muscle.

Orexin gene therapy restores the timing and maintenance of wakefulness in narcoleptic mice.

STUDY OBJECTIVES: Narcolepsy is caused by selective loss of the orexin/hypocretin-producing neurons of the hypothalamus. For patients with narcolepsy, chronic sleepiness is often the most disabling symptom, but current therapies rarely normalize alertness and do not address the underlying orexin deficiency. We hypothesized that the sleepiness of narcolepsy would substantially improve if orexin signaling were restored in specific brain regions at appropriate times of day. DESIGN: We used gene therapy to restore orexin signaling in a mouse model of narcolepsy. In these Atx mice, expression of a toxic protein (ataxin-3) selectively kills the orexin neurons. INTERVENTIONS: To induce ectopic expression of the orexin neuropeptides, we microinjected an adeno-associated viral vector coding for prepro-orexin plus a red fluorescence protein (AAV-orexin) into the mediobasal hypothalamus of Atx and wild-type mice. Control mice received an AAV coding only for red fluorescence protein. Two weeks later, we recorded sleep/wake behavior, locomotor activity, and body temperature and examined the patterns of orexin expression. MEASUREMENTS AND RESULTS: Atx mice rescued with AAV-orexin produced long bouts of wakefulness and had a normal diurnal pattern of arousal, with the longest bouts of wake and the highest amounts of locomotor activity in the first hours of the night. In addition, AAV-orexin improved the timing of rapid eye movement sleep and the consolidation of nonrapid eye movement sleep in Atx mice. CONCLUSIONS: These substantial improvements in sleepiness and other symptoms of narcolepsy demonstrate the effectiveness of orexin gene therapy in a mouse model of narcolepsy. Additional work is needed to optimize this approach, but in time, AAV-orexin could become a useful therapeutic option for patients with narcolepsy.

Precise pattern of recombination in serotonergic and hypothalamic neurons in a Pdx1-cre transgenic mouse line.

BACKGROUND: Multicellular organisms are characterized by a remarkable diversity of morphologically distinct and functionally specialized cell types. Transgenic techniques for the manipulation of gene expression in specific cellular populations are highly useful for elucidating the development and function of these cellular populations. Given notable similarities in developmental gene expression between pancreatic ß-cells and serotonergic neurons, we examined the pattern of Cre-mediated recombination in the nervous system of a widely used mouse line, Pdx1-cre (formal designation, Tg(Ipf1-cre)89.1Dam), in which the expression of Cre recombinase is driven by regulatory elements upstream of the pdx1 (pancreatic-duodenal homeobox 1) gene. METHODS: Single (hemizygous) transgenic mice of the pdx1-creCre/0 genotype were bred to single (hemizygous) transgenic reporter mice (Z/EG and rosa26R lines). Recombination pattern was examined in offspring using whole-mount and sectioned histological preparations at e9.5, e10.5, e11.5, e16.5 and adult developmental stages. RESULTS: In addition to the previously reported pancreatic recombination, recombination in the developing nervous system and inner ear formation was observed. In the central nervous system, we observed a highly specific pattern of recombination in neuronal progenitors in the ventral brainstem and diencephalon. In the rostral brainstem (r1-r2), recombination occurred in newborn serotonergic neurons. In the caudal brainstem, recombination occurred in non-serotonergic cells. In the adult, this resulted in reporter expression in the vast majority of forebrain-projecting serotonergic neurons (located in the dorsal and median raphe nuclei) but in none of the spinal cord-projecting serotonergic neurons of the caudal raphe nuclei. In the adult caudal brainstem, reporter expression was widespread in the inferior olive nucleus. In the adult hypothalamus, recombination was observed in the arcuate nucleus and dorsomedial hypothalamus. Recombination was not observed in any other region of the central nervous system. Neuronal expression of endogenous pdx1 was not observed. CONCLUSIONS: The Pdx1-cre mouse line, and the regulatory elements contained in the corresponding transgene, could be a valuable tool for targeted genetic manipulation of developing forebrain-projecting serotonergic neurons and several other unique neuronal sub-populations. These results suggest that investigators employing this mouse line for studies of pancreatic function should consider the possible contributions of central nervous system effects towards resulting phenotypes.

Multilineage differentiation of adipose-derived stromal cells from GFP transgenic mice.

Functional engineering of musculoskeletal tissues generally involves rapid expansion of progenitor cells in vitro while retaining their potential for further differentiation and then induction in specific culture conditions. The autologous adipose-derived stromal cells (ASCs) are considered to contain pluripotent mesenchymal stem cells. Imaging with expression of green fluorescent protein (GFP) facilitates the detailed research on ASCs physiological behavior during differentiation into a variety of cell lineages both in vitro and in vivo. In this study, we aimed to confirm the trans-germ plasticity of homogeneously marked ASCs from GFP transgenic mice. Simultaneously, the term and intensity of GFP expression in ASCs were also focused on during variant inductions, when cells were incubated with multiple growth factors and adjuvant. ASCs were harvested from inguinal fat pads of transgenic nude mice, passaged 3 times in monolayer cultures, and then transferred to osteogenic, adipogenic, neurogenic, and myogenic medium. The morphological characterization of inductive cells was observed using phase-contrast microscopy and histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation. The expression of marker genes or proteins was measured using RT-PCR and immunocytochemical analysis. Collagen type I, osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, peroxisome proliferator-activated receptor(PPAR)-gamma2 and lipoprotein lipase (LPL) were positive in adipogenic ones, glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) were positive in neurogenic ones, and alpha-smooth muscle actin (alpha-SMA) was positive in myogenic ones. Moreover, the results of fluorescence microscopic imaging suggested that there was no significant decline of GFP expression during ASCs differentiation and the level of GFP maintained stable till differentiated ASCs showed apoptotic phenotype. So the endogenous GFP and multilineage potential of transgenic ASCs had no influences on each other. Since the population of GFP ASCs can be easily identified, it is proposed that they may be promising candidate seed cells for further studies on ASCs tissue engineering, especially the study on engineered tissues formed in vivo.

Protection by dietary zinc in ALS mutant G93A SOD transgenic mice.

Mutations to the copper, zinc superoxide dismutase (SOD) gene are responsible for 2-3% of amyotrophic lateral sclerosis (ALS) cases. These mutations result in the protein having a reduced affinity for zinc. SOD becomes toxic to motor neurons when zinc is missing from its active site. Recently, high dosages of zinc (75 and 375 mg/kg/day) have been paradoxically reported to increase the death of G93A-mutant SOD transgenic mice [G.J. Groeneveld, J. de Leeuw van Weenen, F.L. van Muiswinkel, H. Veldman, J.H. Veldink, J.H. Wokke, P.R. Bar, L.H. van den Berg, Zinc amplifies mSOD1-mediated toxicity in a transgenic mouse model of amyotrophic lateral sclerosis, Neurosci. Lett. 352 (2003) 175-178]. In contrast, we have found that moderate supplementation of zinc (approximately 12 mg/kg/day) delayed death in G93A-mutant SOD mice by 11 days compared to mice on a zinc-deficient diet. Supplementing zinc with even 18 mg/kg/day resulted in a more rapid death of some mice, consistent with the results of Groenevelt et al. However, large amounts of zinc competitively inhibit copper absorption, which inhibits the copper-dependent ceruloplasmin, and can cause a lethal anemia. We found that supplementing the 18 mg/kg/day dosage of zinc with 0.3 mg/kg/day of copper prevented the early death from zinc treatment alone. These data support a role for moderate levels of dietary zinc potentially protecting against the toxicity of ALS-associated SOD and the protection does not result from depleting copper.

Techniques: reporter mice - a new way to look at drug action.

During the past decade remarkable progress in molecular genetics and the possibility of manipulating cells so that the expression of genes can directly 'report' on drug activity has produced major changes in drug development strategies. The recent description and pharmacological validation of reporter mice for in vivo analysis of hormone receptor activity opens new horizons for drug discovery. These novel animal models, in association with in vivo imaging technologies, provide a global view of the target tissues of drug action following acute and repeated drug treatment, thus enabling the prediction of potential side-effects in the early phase of preclinical studies. It is anticipated that further improvements of transgene architecture will lead to models that combine pharmacokinetic, pharmacodynamic and toxicological studies in a single step, which should provide a tremendous saving in time and, paradoxically, the number of animals to be sacrificed in the development of novel pharmacologically active molecules.

Overexpression of corticotropin-releasing hormone in transgenic mice and chronic stress-like autonomic and physiological alterations.

To gain a greater insight into the relationship between hyperactivity of the corticotropin-releasing hormone (CRH) system and autonomic and physiological changes associated with chronic stress, we developed a transgenic mouse model of central CRH overproduction. The extent of central and peripheral CRH overexpression, and the amount of bioactive CRH in the hypothalamus were determined in two lines of CRH-overexpressing (CRH-OE) mice. Furthermore, 24 h patterns of body temperature, heart rate, and activity were assessed using radiotelemetry, as well as cumulative water and food consumption and body weight gain over a 7-day period. CRH-OE mice showed increased amounts of CRH peptide and mRNA only in the central nervous system. Despite the presence of the same CRH transgene in their genome, only in one of the two established lines of CRH-OE mice (line 2122, but not 2123) was overexpression of CRH associated with increased levels of bioactive CRH in the hypothalamus, increased body temperature and heart rate (predominantly during the light (inactive) phase of the diurnal cycle), decreased heart rate variability during the dark (active) phase, and increased food and water consumption, when compared with littermate wildtype mice. Because line 2122 of the CRH transgenic mice showed chronic stress-like neuroendocrine and autonomic changes, these mice appear to represent a valid animal model for chronic stress and might be valuable in the research on the consequences of CRH excess in situations of chronic stress.

Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control.

We have developed transgenic mouse models to determine whether endogenous expression of phytase transgenes in the digestive tract of monogastric animals can increase the bioavailability of dietary phytate, a major but indigestible form of dietary phosphorus. We constructed phytase transgenes composed of the appA phytase gene from Escherichia coli regulated for expression in salivary glands by the rat R15 proline-rich protein promoter or by the mouse parotid secretory protein promoter. Transgenic phytase is highly expressed in the parotid salivary glands and secreted in saliva as an enzymatically active 55 kDa glycosylated protein. Expression of salivary phytase reduces fecal phosphorus by 11%. These results suggest that the introduction of salivary phytase transgenes into monogastric farm animals offers a promising biological approach to relieving the requirement for dietary phosphate supplements and to reducing phosphorus pollution from animal agriculture.

Somatostatin and behaviour: the need for genetically engineered models.

Somatostatin was originally characterised as a hypothalamic neurohormone responsible for the inhibition of pituitary Growth Hormone secretion. In mammals two genes encode for somatostatin-related peptides, somatostatin 14 and 28, and cortistatins, respectively. All peptides bind with similar affinities to the five cloned somatostatin receptors (sst), which belong to the GPCR family. Despite numerous studies, no clear behavioural function has yet been attributed to somatostatin-related peptides. This is due to the lack of good pharmacological tools (selective antagonists) and animal models. This review will focus on the recent development of such tools.

On the relationships of high-frequency hearing loss and cochlear pathology to the acoustic startle response (ASR) and prepulse inhibition of the ASR in the BXD recombinant inbred series.

The measurement of the acoustic startle response (ASR) and prepulse inhibition (PPI) of the ASR in many inbred strains of mice, including C57BL/6 and DBA/2, may be complicated by age-related high-frequency hearing loss (HFHL) and the associated cochlear pathology. Willott and Erway (1998) have recently reported on the age-related changes of the acoustic brain response in the BXD recombinant inbred (RI) series. Based on these data, the RI series was divided into three groups: juvenile-, intermediate-, and adult-onset HFHL. Each of these groups was tested using paradigms which varied the frequency or intensity of the auditory startle and prepulse stimuli. The results obtained in adolescent mice (6-8 weeks) demonstrate that ASR performance is independent of HFHL; there was no group-dependent decline in the ASR amplitudes for high-frequency stimuli. The expected effect of HFHL on PPI is to increase the salience of the still-audible tones. In response to a white-noise prepulse stimulus, the PPI in the juvenile-onset group (which shows marked HFHL at 6 weeks) was similar to that in the adult-onset group. However, when the prepulse stimulus was a pure tone, the juvenile group showed a decrease in salience across all frequencies tested (5-20 kHz). The data point out the need for carefully constructing auditory tasks in the BXD RI series, to avoid the confounding effects of HFHL.

AlphaMUPA mice: a transgenic model for increased life span.

AlphaMUPA is a line of transgenic mice that, compared with their wild type (WT) counterparts, spontaneously eat less (approximately 20%) and live longer (average approximately 20%), thus resembling dietary-restricted (DR) mice. Here, we show that body temperature was significantly reduced in alphaMUPA compared with WT throughout a wide range of ages. Plasma corticosterone was significantly higher in young alphaMUPA compared to young WT; however, it significantly declined in aged alphaMUPA, but not in aged WT. In addition, age-associated thymus involution occurred in alphaMUPA as it did in WT. Thus alphaMUPA mice appear to largely resemble, but also to somewhat differ from diet-restricted animals. We also report on four new transgenic lines that, like alphaMUPA, produced in the brain the mRNA that encodes the extracellular protease urokinase (uPA); however, transgenic uPA expression was most extensive and widespread in the alphaMUPA brain, where it also occurred in the hypothalamus. AlphaMUPA was also the only line that ate less, but also showed another characteristic, high frequency leg muscle tremor seen only at unstable body states. We hypothesize that transgenic uPA in the brain could have caused the alphaMUPA phenotypic alterations. Thus alphaMUPA offers a unique transgenic model of inherently reduced eating to investigate the homeostatic state of delayed aging at the systemic and single-cell levels.

Apolipoprotein E*3-Leiden transgenic mice as a test model for hypolipidaemic drugs.

Apolipoprotein (APO) E*3-Leiden mice with impaired chylomicron and VLDL (very low density lipoprotein) remnant metabolism display hyperlipidaemia and atherosclerosis. In the present study, these mice were used for testing the hypolipidaemic effect of two marketed agents, lovastatin (CAS 75330-75-5) and gemfibrozil (CAS 25812-30-0) as well as a novel compound, SB 204990 (the 5-ring lactone of +/-(3R*,5S*) 3-carboxy-11-(2,4-dichlorophenyl)-3,5-dihydroxyundecanoic acid, CAS 154566-12-8), a potent inhibitor of cholesterol and fatty acid synthesis at the level of ATP-citrate lyase. APOE*3-Leiden mice were fed a saturated fat and cholesterol-rich diet supplemented with either 0.05 or 0.1% w/w of lovastatin, 0.1 or 0.2% w/w of gemfibrozil or 0.1 or 0.2% w/w of SB 204990. Lovastatin showed a dose-related decrease in plasma cholesterol levels (up to -20%) due to a lowering of LDL and HDL (low density resp. high density lipoprotein)-cholesterol (-20 and -18%, respectively), while plasma triglyceride levels were unaffected. Gemfibrozil had no effect on plasma total cholesterol levels but gave significant dose-dependent decreases in plasma (VLDL) triglyceride levels (up to -53%). SB 204990 resulted in a dose-dependent reduction of plasma cholesterol (up to -29%) by lowering VLDL, LDL and HDL-cholesterol (-50, -20 and -20%, respectively). In addition, a strong dose dependent reduction of plasma (VLDL) triglycerides up to -43% was observed with this compound. Although the effects of gemfibrozil and SB 204990 were not simply explained by changes in a single determinant of VLDL metabolism--no effects of these drugs were seen on post-heparin plasma lipoprotein lipase activity, in vivo rate of VLDL synthesis or hepatic apoC-III mRNA levels--APOE*3-Leiden mice were found to give robust hypolipidaemic responses to these test compounds. The responsiveness to hypolipidaemic therapy combined with a clear relationship between aortic lesion size and plasma cholesterol exposure, as demonstrated previously, makes this mouse an attractive model for the testing of anti-atherosclerotic properties of hypolipidaemic drugs.

Control of dopamine release in the retina: a transgenic approach to neural networks.

Dopaminergic, interplexiform amacrines (DA cells) were labeled in transgenic mice with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. It was therefore possible to investigate their activity in vitro after dissociation of the retina with whole-cell current and voltage clamp, as well as their connections in the intact retina with the electron microscope. DA cells generate action potentials even in the absence of synaptic inputs. This activity is abolished by the amacrine cell transmitters GABA and glycine, which induce an inward current carried by chloride ions, and is stimulated by kainate, an agonist at the receptor for the bipolar cell transmitter glutamate, which opens nonselective cation channels. Since DA cells are postsynaptic to amacrine and bipolar cells, we suggest that the spontaneous discharge of DA cells is inhibited in the dark by GABAergic amacrines that receive their input from off-bipolars. Upon illumination, the GABA-inhibition is removed, DA cells generate action potentials, and their firing is modulated by the excitation received from on-bipolars.

Delayed tumor onset in transgenic mice fed an amino acid-based diet supplemented with red wine solids.

Increased consumption of vegetable foods (cereals, legumes, fruits) and some beverages (tea, cider, wine) is associated with reduced risk of cancer. Polyphenols in these foods and beverages are thought to be responsible, based on data from in vitro assays and from in vivo studies that used animals pretreated with carcinogen and given tea or polyphenol-spiked water to drink. We tested the hypothesis that dehydrated-dealcoholized red wine (wine solids), when consumed as part of a precisely defined complete diet, would delay tumor onset in transgenic mice that spontaneously develop externally visible tumors without carcinogen pretreatment. Sibling transgenic mice were weaned onto an amino acid-based diet alone or supplemented with red wine solids. Mice were examined daily; the age at which a first tumor appeared was recorded as the age of tumor onset. The concentration of the major polyphenol of red wine (catechin) in blood serum was also measured at the end of the study. The supplemented diet was fed continuously for three generations to ensure that it supported normal growth and reproduction. We discovered that the wine solid supplement delayed tumor onset, that intact catechin was absorbed, and that the supplemented diet supported normal growth and reproduction for three generations. Also, our simple experimental protocol offers an alternate and/or complementary way to identify foods, beverages, and their constituents that delay tumor onset and to investigate possible mechanisms involved.

Skeletal muscle growth of oMTla-oGH transgenic mice.

Forty-eight transgenic mice carrying an ovine metallothionein 1a-ovine growth hormone (oMTla-oGH) transgene and 48 littermate control mice were used to investigate the effect of GH transgene on the growth and biochemical characteristics of skeletal muscle. Transgene expression was initiated in the transgenic mice by the addition of zinc sulfate to the water at 21 d of age; control mice were also supplemented with zinc sulfate. These mice were maintained on zinc sulfate until 84 d of age. Groups of mice (16 controls, 16 transgenics) were killed at 21, 42 and 84 d of age and muscles from the hind leg were dissected, weighed and analyzed. At 84 d, male transgenics were 32% heavier than controls, while female transgenics were 47% heavier. Transgenic mice of both sexes had smaller (p < 0.01) muscles than controls at weaning (21 d). In spite of significantly heavier body weights of transgenic mice at 84 d of age, there were no significant differences in muscle weights. This was due to a significantly lower (p < 0.01) proportion of muscle, expressed as percentages of body weights, in transgenic mice compared with controls. Higher DNA and RNA concentrations at 42 d of age and elevated cathepsins C and H activities at 42 and 84 d of age indicate that muscle protein metabolism is more active in transgenic mice, which are growing at a greater rate than controls from weaning to 84 d of age. The fact that oMTla-oGH transgenic mice inherently have a lower proportion of muscle, compared to controls and that this proportion does not change in spite of transgene activation and 30 fold increase in plasma GH levels, suggests the hypothesis that muscle growth may be controlled by locally produced IGFs, which are essentially independent of circulating GH concentrations.

Premature ageing in transgenic mice expressing different growth hormone genes.

Transgenic mice expressing various growth hormone genes have markedly reduced life spans, with few animals surviving beyond 12 months in some of the lines. Except for an increased incidence of mammary tumours in female mice expressing human growth hormones, pathological findings in debilitated or moribund transgenic mice resemble the well-documented degenerative changes that occur at a much greater chronological age in normal rodents. This study demonstrates that 10-month-old male transgenic mice expressing bovine growth hormone (known as 25-copy PEPCK. bGH transgenic mice) also show age-related changes in hypothalamic and striatal neurotransmitter metabolism that are not seen in normal litter mate controls until at least 24 months of age. Female mice and male mice with a lower circulating concentration of bGH (known as 5-copy PEPCK. bGH transgenic mice) live longer and fail to show the same magnitude of change in neurotransmitter synthesis and release. Although more work needs to be done to determine the physiological significance of these changes and to determine their relationship to the general effects of ageing on the CNS, transgenic mice expressing various growth hormone genes may provide an interesting and valuable model with which to study the ageing process.

Transgenic animals in the evaluation of compound efficacy and toxicity: will they be as useful as they are novel?

1. Construction of transgenic mice is predicated upon inserting foreign DNA into native host DNA and having this expressed in the germline. This may be accomplished by nuclear injection, retroviral vectors or use of embryonic stem (ES) cells. 2. Expression of novel structural genes may be reasonably directed by the judicious use of an accompanying promoter/enhancer sequence. Insertion of foreign genes may be designed to result in phenotypic expression of a novel trait or ablation of a native gene or gene product. 3. Resulting transgenic mice offer significant utility as models of human diseases and a unique opportunity for investigating immune and metabolic pathways as well as for exploring mechanisms of development, mutagenesis and teratogenesis. 4. Use of transgenic animals in drug development has considerable potential although realization of this potential will take time. Constructing transgenics is only the first step in a complex series of events culminating in understanding the consequences of imposing novel genetic material on an intact, highly integrated living system. Practical use of transgenic animals will depend upon substantial effort being spent in investigating and validating the phenotypic consequences of gene transfer.