Constitutive activation of Nrf2 induces a stable reductive state in the mouse myocardium.

Redox homeostasis regulates key cellular signaling pathways in both physiology and pathology. The cell's antioxidant response provides a defense against oxidative stress and establishes a redox tone permissive for cell signaling. The molecular regulation of the well-known Keap1/Nrf2 system acts as sensor responding to changes in redox homeostasis and is poorly studied in the heart. Importantly, it is not yet known whether Nrf2 alone can serve as a master regulator of cellular redox homeostasis without compensation of the transcriptional regulation of antioxidant response element (ARE) genes through alternate mechanisms. Here, we addressed this question using cardiac-specific transgenic expression at two different levels of constitutively active nuclear erythroid related factor 2 (caNrf2) functioning independently of Keap1. The caNrf2 mice showed augmentation of glutathione (GSH), the key regulator of the cellular thiol redox state. The Trans-AM assay for Nrf2-binding to the antioxidant response element (ARE) showed a dose-dependent increase associated with upregulation of several major antioxidant genes and proteins. This was accompanied by a significant decrease in dihydroethidium staining and malondialdehyde (MDA) in the caNrf2-TG mice myocardium. Interestingly, caNrf2 gene-dosage dependent redox changes were noted resulting in generation of a multi-stage model of pro-reductive and reductive conditions in the myocardium of TG-low and TG-high mice, respectively. These data clearly show that Nrf2 levels alone are capable of serving as the master regulator of the ARE. These models provide an important platform to investigate the impact of the Nrf2 system independent of the need to regulate the activity of Keap1 and the consequent exposure to pro-oxidants or electrophiles, which have numerous off-target effects.

Transgene delivery via intracellular electroporetic nanoinjection.

Development of an effective cytoplasmic delivery technique has remained an elusive goal for decades despite the success of pronuclear microinjection. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In this work we report a cytoplasmic delivery method termed intracellular electroporetic nanoinjection (IEN). IEN is unique in that it manipulates transgenes using electrical forces. The microelectromechanical system (MEMS) uses electrostatic charge to physically pick up transgenes and place them in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronuclei using electrical pulses. Standard electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm for transgene delivery from the cytoplasm to the pronucleus. In this report we describe the principles which allow localized electroporation of the pronuclei including: the location of mouse pronuclei between 21 and 28 h post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on electric-field-driven movement of transgenes. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.

Nanoinjection: pronuclear DNA delivery using a charged lance.

We present a non-fluidic pronuclear injection method using a silicon microchip "nanoinjector" composed of a microelectromechanical system with a solid, electrically conductive lance. Unlike microinjection which uses fluid delivery of DNA, nanoinjection electrically accumulates DNA on the lance, the DNA-coated lance is inserted into the pronucleus, and DNA is electrically released. We compared nanoinjection and microinjection side-by-side over the course of 4 days, injecting 1,013 eggs between the two groups. Nanoinjected zygotes had significantly higher rates of integration per injected embryo, with 6.2% integration for nanoinjected embryos compared to 1.6% integration for microinjected embryos. This advantage is explained by nanoinjected zygotes' significantly higher viability in two stages of development: zygote progress to two-cell stage, and progress from two-cell stage embryos to birth. We observed that 77.6% of nanoinjected zygotes proceeded to two-cell stage compared to 54.7% of microinjected zygotes. Of the healthy two-cell stage embryos, 52.4% from the nanoinjection group and 23.9% from the microinjected group developed into pups. Structural advantages of the nanoinjector are likely to contribute to the high viability observed. For instance, because charge is used to retain and release DNA, extracellular fluid is not injected into the pronucleus and the cross-sectional area of the nanoinjection lance (0.06 µm(2)) is smaller than that of a microinjection pipette tip (0.78 µm(2)). According to results from the comparative nanoinjection versus microinjection study, we conclude that nanoinjection is a viable method of pronuclear DNA transfer which presents viability advantages over microinjection.

The use of transgenic mouse models in the study of male infertility.

Over the past few decades with the rapid advances in embryo and embryonic stem cell manipulation techniques, transgenic mouse models have emerged as a powerful tool for the study of gene function and complex diseases including male infertility. In this review we give a brief history of the development of tools for the production of transgenic mouse models. This spans the advances from early pronuclear injection to the use of targeted embryonic stem cells to produce gene targeted, conditional, and inducible knockout mouse models. Lastly we provide a few examples to illustrate the utility of mouse models in the study of male infertility.

Role for the alpha7beta1 integrin in vascular development and integrity.

The alpha7beta1 integrin is a laminin receptor that has been implicated in muscle disease and the development of neuromuscular and myotendinous junctions. Studies have shown the alpha7beta1 integrin is also expressed in nonskeletal muscle tissues. To identify the expression pattern of the alpha7 integrin in these tissues during embryonic development, alpha7 integrin chain knockout mice were generated by a LacZ knockin strategy. In these mice, expression from the alpha7 promoter is reported by beta-galactosidase. From embryonic day (ED) 11.5 to ED14.5, beta-galactosidase was detected in the developing central and peripheral nervous systems and vasculature. The loss of the alpha7 integrin gene resulted in partial embryonic lethality. Several alpha7 null embryos were identified with cerebrovascular hemorrhages and showed reduced vascular smooth muscle cells and cerebral vascularization. The alpha7 null mice that survived to birth exhibited vascular smooth muscle defects, including hyperplasia and hypertrophy. In addition, altered expression of alpha5 and alpha6B integrin chains was detected in the cerebral arteries of alpha7 null mice, which may contribute to the vascular phenotype. Our results demonstrate for the first time that the alpha7beta1 integrin is important for the recruitment or survival of cerebral vascular smooth muscle cells and that this integrin plays an important role in vascular development and integrity.

Targeted inactivation of cystic fibrosis transmembrane conductance regulator chloride channel gene prevents ischemic preconditioning in isolated mouse heart.

BACKGROUND: Recent evidence suggests that chloride channels may be involved in ischemic preconditioning (IPC). In this study, we tested whether the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels, which are expressed in the heart and activated by protein kinase A and protein kinase C, are important for IPC in isolated heart preparations from wild-type (WT) and CFTR knockout (CFTR-/-) mice. METHODS AND RESULTS: Hearts were isolated from age-matched WT or CFTR-/- (B6.129P2-Cftr(tm1Unc) and STOCKCftr(tm1Unc)-TgN 1Jaw) mice and perfused in the Langendorff or working-heart mode. All hearts were allowed to stabilize for 10 minutes before they were subjected to 30 or 45 minutes of global ischemia followed by 40 minutes of reperfusion (control group) or 3 cycles of 5 minutes of ischemia and reperfusion (IPC group) before 30 or 45 minutes of global ischemia and 40 minutes of reperfusion. Hemodynamic indices were recorded to evaluate cardiac functions. Release of creatine phosphate kinase (CPK) in the samples of coronary effluent and infarct size of the ventricles were used to estimate myocardial tissue injury. In WT adult hearts, IPC protected cardiac function during reperfusion and significantly decreased ischemia-induced CPK release and infarct size. A selective CFTR channel blocker, gemfibrozil, abrogated the protective effect of IPC. Furthermore, targeted inactivation of the CFTR gene in 2 different strains of CFTR-/- mice also prevented IPC's protection of cardiac function and myocardial injury against sustained ischemia. CONCLUSIONS: CFTR Cl- channels may serve as novel and crucial mediators in mouse heart IPC.

Brain-derived neurotrophic factor is required for the maintenance of cortical dendrites.

Brain-derived neurotrophic factor (BDNF) is thought to be involved in neuronal survival, migration, morphological and biochemical differentiation, and modulation of synaptic function in the CNS. In the rodent cortex, postnatal BDNF expression is initially low but subsequently increases to reach maximal levels around weaning. Thus, BDNF expression peaks at a time when both structural and functional maturation of cortical circuitry occurs. Although the function of BDNF has been probed using many approaches, its requirements during this phase of life have not previously been examined genetically. To test the in vivo requirements for BDNF during this important phase of development we generated early-onset forebrain-specific BDNF mutant mice. Although these mice undergo forebrain-restricted deletion of BDNF by Cre-mediated recombination during embryogenesis, they are healthy, and we did not detect the loss of specific cortical excitatory or inhibitory neurons. However, the neocortex of 5-week-old mice was thinner, attributable at least partly to neuronal shrinkage. Importantly, although visual cortical layer 2/3 neurons in the mutants initially developed normal dendrite structure, dendritic retraction became apparent by 3 weeks of age. Thus, our observations suggest that cortically expressed BDNF functions to support the maintenance of cortical neuron size and dendrite structure rather than the initial development of these features. This is consistent with a role for BDNF in stabilizing the "survival" of circuitry during the phase of activity-dependent reorganization of cortical connectivity.