Transgenic mice carrying a tetracycline-inducible, truncated transforming growth factor beta receptor (TbetaRII)

The transforming growth factor-betas (TGFbetas) have multiple roles, making genetic analysis of their functions difficult. We therefore developed transgenic mouse lines to disrupt TGFbeta signaling using a mechanism that is inducible, reversible, and cell-type specific. The transgenic mouse lines carry an EGFP-pBi-DeltaTbetaRII construct (PTR). The DeltaTbetaRII element codes for a dominant-negative receptor that is known to disrupt TGFbeta signaling. The DeltaTbetaRII has a c-myc tag. The transgene was silent in the PTR mice, with expression of both EGFP and DeltaTbetaRII occurring when the PTR mice were crossed with mice that express the tetracycline transactivator (CMV-tTA). The expression of EGFP was repressed by the addition of doxycycline to the drinking water of the PTRxCMV-tTA mice. The PTR mice were then crossed with neuron-specific-tTA mice. Expression of the DeltaTbetaRII transgene in these mice led to an upregulation of native TGFbeta receptor expression, suggesting that neurons can modulate their responsiveness to TGFbetas.     

A lack of functional NK1 receptors explains most,but not all,abnormal behaviours of NK1R‐/‐ mice1

Mice lacking functional neurokinin‐1 receptors (NK1R‐/‐) display abnormal behaviours seen in Attention Deficit Hyperactivity Disorder (hyperactivity, impulsivity and inattentiveness). These abnormalities were evident when comparing the behaviour of separate (inbred: ‘Hom’) wildtype and NK1R‐/‐ mouse strains. Here, we investigated whether the inbreeding protocol could influence their phenotype by comparing the behaviour of these mice with that of wildtype (NK1R+/+) and NK1R‐/‐ progeny of heterozygous parents (‘Het’, derived from the same inbred strains). First, we recorded the spontaneous motor activity of the two colonies/genotypes, over 7 days. This continuous monitoring also enabled us to investigate whether the diurnal rhythm in motor activity differs in the two colonies/genotypes. NK1R‐/‐ mice from both colonies were hyperactive compared with their wildtypes and their diurnal rhythm was also disrupted. Next, we evaluated the performance of the four groups of mice in the 5‐Choice Serial Reaction‐Time Task (5‐CSRTT). During training, NK1R‐/‐ mice from both colonies expressed more impulsive and perseverative behaviour than their wildtypes. During testing, only NK1R‐/‐ mice from the Hom colony were more impulsive than their wildtypes, but NK1R‐/‐ mice from both colonies were more perseverative. There were no colony differences in inattentiveness. Moreover, a genotype difference in this measure depended on time of day. We conclude that the hyperactivity, perseveration and, possibly, inattentiveness of NK1R‐/‐ mice is a direct consequence of a lack of functional NK1R. However, the greater impulsivity of NK1R‐/‐ mice depended on an interaction between a functional deficit of NK1R and other (possibly environmental and/or epigenetic) factors.     

Severe impairment of cerebellum development in mice expressing a dominant-negative mutation inactivating thyroid hormone receptor alpha1 isoform

Thyroid hormone deficiency is known to deeply affect cerebellum post-natal development. We present here a detailed analysis of the phenotype of a recently generated mouse model, expressing a dominant-negative TRα1 mutation. Although hormonal level is not affected, the cerebellum of these mice displays profound alterations in neuronal and glial differentiation, which are reminiscent of congenital hypothyroidism, indicating a predominant function of this receptor isoform in normal cerebellum development. Some of the observed effects might result from the cell autonomous action of the mutation, while others are more likely to result from a reduction in neurotrophic factor production.     

Knock-down of RGS4 and beta tubulin in CHO cells expressing the human MT1 melatonin receptor prevents melatonin-induced receptor desensitization

Previously, it has been shown that chronic melatonin exposure in MT1-CHO cells results in receptor desensitization while at the same time producing drastic morphological changes. The addition of a depolymerizing agent during the melatonin pretreatment period prevents MT1 receptor desensitization and the changes in cellular morphology. The lack of morphological change in the presence of a depolymerizing agent is easily explained by the inability of the microtubules to polymerize, however, the prevention of receptor desensitization is a little more complex and may involve G-protein activation. The goal of this study was to determine whether melatonin-induced MT1 receptor desensitization is regulated by proteins known to regulate G-protein activation states, beta-tubulin and RGS4,using anti sense knockdown approaches. The expression of RGS4 mRNA in CHO cells was confirmed using RT PCR and successful knockdown of each was confirmed by western blot analysis or quantitative PCR. Pretreatment of MT1-CHO cells, transfected with the nonsense probes and exposed to melatonin, resulted in a desensitization of the receptor, an increase in forskolin-induced cAMP accumulation, an increase in 2-[125I]-iodomelatonin binding and no change in the affinity of melatonin for the MT1 receptor. However, knockdown of either beta-tubulin or RGS4 in MT1-CHO cells followed by pretreatment with melatonin attenuated the desensitization of melatonin receptors, decreased total 2-[125I]-iodomelatonin binding, and did not affect neither the forskolin response nor the affinity of melatonin for the MT1 receptor. Perhaps RGS4 and beta-tubulin modulate Galpha-GDP and Galpha-GTP states thus modulating MT1 melatonin receptor function.     

Malonate and 3-nitropropionic acid neurotoxicity are reduced in transgenic mice expressing a caspase-1 dominant-negative mutant

Increasing evidence implicates caspase-1-mediated cell death as a major mechanism of neuronal death in neurodegenerative diseases. In the present study we investigated the role of caspase-1 in neurotoxic experimental animal models of Huntington's disease (HD) by examining whether transgenic mice expressing a caspase-1 dominant-negative mutant are resistant to malonate and 3-nitropropionic acid (3-NP) neurotoxicity. Intrastriatal injection of malonate resulted in significantly smaller striatal lesions in mutant caspase-1 mice than those observed in littermate control mice. Caspase-1 was significantly activated following malonate intrastriatal administration in control mice but significantly attenuated in mutant caspase-1 mice. Systemic 3-NP treatment induced selective striatal lesions that were significantly smaller within mutant caspase-1 mice than in littermate control mice. These results provide further evidence of a functional role for caspase-1 in both malonate- and 3-NP-mediated neurotoxin models of HD.     

Fibroblast growth factor receptor signaling affects development and function of dopamine neurons - inhibition results in a schizophrenia-like syndrome in transgenic mice

Developing and mature midbrain dopamine (DA) neurons express fibroblast growth factor (FGF) receptor-1 (FGFR1). To determine the role of FGFR1 signaling in the development of DA neurons, we generated transgenic mice expressing a dominant negative mutant [FGFR1(TK-)] from the catecholaminergic, neuron-specific tyrosine hydroxylase (TH) gene promoter. In homozygous th(tk-)/th(tk-) mice, significant reductions in the size of TH-immunoreactive neurons were found in the substantia nigra compacta (SNc) and the ventral tegmental area (VTA) at postnatal days 0 and 360. Newborn th(tk-)/th(tk-) mice had a reduced density of DA neurons in both SNc and VTA, and the changes in SNc were maintained into adulthood. The reduced density of DA transporter in the striatum further demonstrated an impaired development of the nigro-striatal DA system. Paradoxically, the th(tk-)/th(tk-) mice had increased levels of DA, homovanilic acid and 3-methoxytyramine in the striatum, indicative of excessive DA transmission. These structural and biochemical changes in DA neurons are similar to those reported in human patients with schizophrenia and, furthermore, these th(tk-)/th(tk-) mice displayed an impaired prepulse inhibition that was reversed by a DA receptor antagonist. Thus, this study establishes a new developmental model for a schizophrenia-like disorder in which the inhibition of FGF signaling leads to alterations in DA neurons and DA-mediated behavior.     

Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor

IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')₂ (t_1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')₂, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t_1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t_1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.     

Studies on brain volume, Alzheimer-related proteins and cytokines in mice with chronic overexpression of IL-1 receptor antagonist

Inflammation is associated with both acute and chronic neurological disorders, including stroke and Alzheimer's disease (AD). Cytokines such as interleukin (IL)-1 have several activities in the brain both under physiological and pathophysiological conditions. The objective of this study was to evaluate consequences of the central blockade of IL-1 transmission in a previously developed transgenic mouse strain with brain-directed overexpression of human soluble IL-1 receptor antagonist (Tg hsIL-1ra). Effects on brain morphology and brain levels of the AD-related proteins beta-amyloid precursor protein (APP) and presenilin 1(PS1), as well as the levels of IL-1beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) were analysed in homozygotic and heterozygotic mice and wild type (WT) controls, of both genders and of young (30-40 days) and adult (13-14 months) age. A marked reduction in brain volume was observed in transgenic mice as determined by volumetry. Western blot analysis showed higher levels of APP, but lower levels of PS1, in adult animals than in young ones. In the cerebellum, heterozygotic (Tg hsIL-1ra(+/-)) mice had lower levels of APP and PS1 than WT mice. With one exception, there were no genotypic differences in the levels of IL-1beta, IL-6 and TNF-alpha. The cytokine levels were generally higher in adult than in young mice. In conclusion, the chronic blockade of IL-1 signalling in the brain was associated with an atrophic phenotype of the brain, and with modified levels of APP and PS1. Brain-directed overexpression of hsIL-1ra was not followed by major compensatory changes in the levels of pro-inflammatory cytokines.     

Anxiety and increased 5‐HT1A receptor response in NCAM null mutant mice

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety‐like behavior of homozygous (NCAM^−/−) and heterozygous (NCAM^+/−) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety‐like behavior was reduced in both NCAM^+/+ and NCAM^−/− mice by systemic administration of the benzodiazepine agonist diazepam and the 5‐HT_1A receptor agonists buspirone and 8‐OH‐DPAT. However, NCAM^−/− mice showed anxiolytic‐like effects at lower doses of buspirone and 8‐OH‐DPAT than NCAM^+/+ mice. Such increased response to 5‐HT_1A receptor stimulation suggests a functional change in the serotonergic system of NCAM^−/− mice, likely involved in the control of anxiety and aggression. However, 5‐HT_1A receptor binding and tissue content of serotonin and its metabolite 5‐hydroxyindolacetic acid were found unaltered in every brain area of NCAM^−/− mice investigated, indicating that expression of 5‐HT_1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM^−/− mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5‐HT_1A receptors and inwardly rectifying K⁺ channels as the respective effector systems. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 343–355, 1999     

Transgenic mice expressing a dual, CRE-inducible reporter for the analysis of axon guidance and synaptogenesis

Improved and modular tools are needed for the neuroanatomical dissection of CNS axonal tracts, and to study the cell-intrinsic and cell-extrinsic cues that govern their assembly and plasticity. Here we describe a general purpose transgenic tracer that can be used to visualize axonal tracts and synaptic terminals in any region of the embryonic neural tube or postnatal CNS, on any wild type or mutant genetic background. The construct permits CRE-inducible expression of a dicistronic axonal marker encoding two surface reporter proteins: a farnesylated GFP and the human Placental Alkaline Phosphatase (PLAP). Both proteins localize alongside the neuronal surface, permitting the concomitant detection of cell body, neurites, and presynaptic and postsynaptic sites in the same neuron. This provides a CRE-inducible dual system for imaging neural circuits in vivo, and to study their assembly and remodeling in cultured neurons, neural stem cells, and tissue explants derived from the reporter line. Unlike existing lines, this reporter does not encode a ubiquitously expressed, floxable LacZ gene, permitting the simultaneous analysis of beta galactosidase activity in mutant lines.     

Fusion protein consisting of the first immunoglobulin-like domain of porcine nectin-1 and Fc portion of human IgG1 provides a marked resistance against pseudorabies virus infection to transgenic mice

Nectin-1 is a Ca²⁺-independent Ig-like cell–cell adhesion molecule and an alphaherpesvirus receptor that binds to virion glycoprotein D by the first Ig-like domain. We have investigated the antiviral potentials of soluble forms of porcine nectin-1 to PRV infection by generating transgenic mice expressing different types of fusion protein. Previously, we reported that mice transgenic for a chimera that carried the entire ectodomain of porcine nectin-1 fused to the Fc portion of porcine IgG1 were more resistant than those transgenic for a chimera that carried the first Ig-like domain fused to the Fc portion. Recently, we generated transgenic mice expressing a fusion protein made of the first Ig-like domain fused to the Fc portion of human IgG1, and reported that they showed a microphthalmia. Here, two transgenic mouse lines expressing the fusion protein were challenged with PRV for comparing their resistances with those of transgenic mice expressing different types of fusion protein. Surprisingly, both transgenic mouse lines showed a high resistance to the viral infection, especially via the i.n. route. Significant resistance of the embryonic fibroblasts was also observed. Altogether, these findings indicated that the fusion protein consisting of the first Ig-like domain fused to the human Fc portion provided a marked resistance against PRV infection to the transgenic mice.     

Somatostatin receptor subtype 1-selective activation reduces cell growth and calcitonin secretion in a human medullary thyroid carcinoma cell line

Medullary thyroid carcinoma (MTC) is a rare and aggressive tumor and so far medical therapy has provided inconclusive results. In the human MTC cell line TT, expressing all somatostatin (SST) receptor subtypes, cell proliferation decreases with SST and SST receptor subtype 2 (sst(2)), but not sst(5), selective agonist treatment, whereas calcitonin (CT) expression and secretion are reduced by SST, but not by sst(2) and sst(5) agonists. The effectiveness of two new SST analogs, BIM-23926 and BIM-23745, selectively interacting with sst(1), was investigated in the TT cell line. DNA synthesis is significantly reduced by BIM-23926 (27-40% at 10(-10)-10(-6)M) and BIM-23745 (32-90% at 10(-8)-10(-6)M). Viable cell number is also significantly reduced by both BIM-23926 (40% at 10(-12)-10(-6)M) and BIM-23745 ( approximately 40% at 10(-10)-10(-6)M). Treatment with sst(1)-selective agonists significantly reduces CT secretion and gene expression, with a reduction of CREB phosphorylation. These findings suggest that potent sst(1)-selective agonists could have a therapeutic role in MTC.     

ADGRL1 haploinsufficiency causes a variable spectrum of neurodevelopmental disorders in humans and alters synaptic activity and behavior in a mouse model

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Neuroprotective effects of atypical D1 receptor agonist SKF83959 are mediated via D1 receptor-dependent inhibition of glycogen synthase kinase-3 beta and a receptor-independent anti-oxidative action

3-methyl-6-chloro-7,8-hydroxy-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959), a selective agonist for the putative phosphatidylinositol (PI)-linked dopamine receptor (DAR), has been shown to possess potent anti-Parkinson disease effects but produces less dyskinesia and motor fluctuation that are frequently observed in Parkinson disease drug therapies. The present study was designed to detect the neuroprotection of SKF83959 and its potential mechanism for the effect in cultured rat cortical cells. The presence of SKF83959 with a dose range of 0.1-30 micromol/L improved H2O2-reduced cell viability in a dose-dependent manner. The anti-apoptotic action of SKF83959 was partially abolished by pre-application of the D1 antagonist SCH23390 (30 micromol/L) and the PI 3-kinase (PI 3-K) inhibitor LY294002 but not by the MEK1/2 inhibitor PD98059 (30 micromol/L). Moreover, SKF83959 treatment significantly inhibited H2O2-activated glycogen synthase kinase-3beta (GSK-3beta) which was associated with the drug's neuroprotective effect, but this inhibition was attenuated by SCH23390 and a selective PI 3-K inhibitor. Moreover, the application of either SKF83959 or a pharmacological inhibitor of GSK-3beta attenuated the inhibition by H2O2 on the expression of inducible NO synthase and production of NO. This indicates that D1-like receptor, presumably PI-linked D1 receptor, -mediated alteration of PI 3-K/Akt/GSK-3beta pathway is involved in the neuroprotection by SKF83959. In addition, SKF83959 also effectively decreased the level of the lipid peroxidation and increased the activity of GSH-peroxidase altered by H2O2. These results suggest that SKF83959 exerts its neuroprotective effect through both receptor-dependent and independent mechanisms: Inhibition of GSK-3beta and consequently increasing the expression of inducible NO synthase via putative PI-linked DAR; and its anti-oxidative activity which is independent of DAR.     

Quantification of ErbB3 receptor density on human breast cancer cells, using a stable radio-labeled mutant of NRG1beta

ErbB3 transactivation can make tumor cells resistance to ErbB1/ErbB2 targeting drugs. This urges for a reliable method to determine cell surface ErbB3 levels, but in our hands iodinated NRG1β is unstable and tends to underestimate the number of ErbB3 receptors in a radio-receptor assay. Here we show by the use of a radio-labeled high affinity neuregulin mutant NRG/YYDLL that ErbB3 levels can be determined in a reliable manner by Scatchard analysis. Furthermore we show by differential competition with unlabeled NRG/YYDLL and betacellulin that the number of ErbB3 and ErbB4 receptors can be quantified separately on cultured human breast cancer cells.     

Hyperactivity,startle reactivity and cell‐proliferation deficits are resistant to chronic lithium treatment in adult Nr2e1frc/frc mice

The NR2E1 region on Chromosome 6q21‐22 has been repeatedly linked to bipolar disorder (BP) and NR2E1 has been associated with BP, and more specifically bipolar I disorder (BPI). In addition, patient sequencing has shown an enrichment of rare candidate‐regulatory variants. Interestingly, mice carrying either spontaneous (Nr2e1^frc) or targeted (Tlx^?) deletions of Nr2e1 (here collectively known as Nr2e1‐null) show similar neurological and behavioral anomalies, including hypoplasia of the cerebrum, reduced neural stem cell proliferation, extreme aggression and deficits in fear conditioning; these are the traits that have been observed in some patients with BP. Thus, NR2E1 is a positional and functional candidate for a role in BP. However, no Nr2e1‐null mice have been fully evaluated for behaviors used to model BP in rodents or pharmacological responses to drugs effective in treating BP symptoms. In this study we examine Nr2e1^frc/frc mice, homozygous for the spontaneous deletion, for abnormalities in activity, learning and information processing, and cell proliferation; these are the phenotypes that are either affected in patients with BP or commonly assessed in rodent models of BP. The effect of lithium, a drug used to treat BP, was also evaluated for its ability to attenuate Nr2e1^frc/frc behavioral and neural stem cell‐proliferation phenotypes. We show for the first time that Nr2e1‐null mice exhibit extreme hyperactivity in the open field as early as postnatal day 18 and in the home cage, deficits in open‐field habituation and passive avoidance, and surprisingly, an absence of acoustic startle. We observed a reduction in neural stem/progenitor cell proliferation in Nr2e1^frc/frc mice, similar to that seen in other Nr2e1‐null strains. These behavioral and cell‐proliferation phenotypes were resistant to chronic‐adult‐lithium treatment. Thus, Nr2e1^frc/frc mice exhibit behavioral traits used to model BP in rodents, but our results do not support Nr2e1^frc/frc mice as pharmacological models for BP.     

Exons and functional regions of the human vitamin D receptor gene around and within the main 1a promoter are well conserved among mammals

The human vitamin D receptor (hVDR) gene encompasses eight exons (2–9) in the so-called coding region and six more exons (1a–1f) in the so-called regulatory region, which contains several reported promoters. Evolutionary comparison performed on the VDR promoter sequences of a dozen of mammalian species shows a very high conservation of numerous regions around and in the 1a promoter, including exons 1e, 1a and 1d, and the Sp1 site region. This suggests that the so-called 1a promoter is well conserved among mammals. Homology among mammals also concerns three functional SNP site regions of the hVDR 1a promoter, the 1e-G-1739A SNP region (a Cdx-2 binding site), and both 1a-G-1521C and 1a-A-1012G sites, the 1a-1012A being located within a GATA site. Interestingly, the 1521G and 1012A nucleotides are being evolutionary conserved, suggesting that the 1521C/1012G haplotype, which is found in human chromosomes (43% of Caucasians), is a human specificity.     

The α1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca-ATPase

Membrane Ca²⁺-ATPase activity was stimulated in vitro separately by T₄ (10⁻¹⁰ M) and by epinephrine (10⁻⁶ M). In the presence of a fixed concentration of T₄, additions of 10⁻⁸ and 10⁻⁶ M epinephrine reduced the T₄ effect on the enzyme. β-Adrenergic blockade with propranolol (10⁻⁶ M) prevented stimulation by epinephrine of Ca²⁺-ATPase activity, but did not prevent the suppressive action of epinephrine on T₄-stimulable Ca²⁺-ATPase. In contrast α₁-adrenergic blockade with unlabelled prazosin restored the effect of T₄ on Ca²⁺-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulations by T₄ of red cell Ca²⁺-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (K_d, 1.2 × 10⁻⁸ M; B_max, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains α₁-radrenergic receptor sites that are capable of regulating Ca²⁺-ATPase activity.     

Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper-dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin

BACKGROUND: The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the coxsackievirus and adenovirus receptor (CAR) is very low in mature muscle. METHODS: We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette (HDAdCAR-dys). RESULTS: We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector to the host with pre-existing immunity to the same serotype of Ad. CONCLUSIONS: Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy, raising the prospects for gene therapy of other hereditary myopathies.