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Tight coordination between transcription and translation is crucial to maintaining the integrity of gene expression in bacteria, yet how bacteria manage to coordinate these two processes remains unclear. Possible direct physical coupling between the RNA polymerase and ribosome has been thoroughly investigated in recent years. Here, we quantitatively characterize the transcriptional kinetics of Escherichia coli under different growth conditions. Transcriptional and translational elongation remain coordinated under various nutrient conditions, as previously reported. However, transcriptional elongation was not affected under antibiotics that slowed down translational elongation. This result was also found by introducing nonsense mutation that completely dissociated transcription from translation. Our data thus provide direct evidence that translation is not required to maintain the speed of transcriptional elongation. In cases where transcription and translation are dissociated, our study provides quantitative characterization of the resulting process of premature transcriptional termination (PTT). PTT-mediated polarity caused by translation-targeting antibiotics substantially affected the coordinated expression of genes in several long operons, contributing to the key physiological effects of these antibiotics. Our results also suggest a model in which the coordination between transcriptional and translational elongation under normal growth conditions is implemented by guanosine tetraphosphate.

Beneficial and deleterious mutations change an organism's fitness but the distribution of these mutational effects on fitness are unknown. Several experimental, theoretical, and computational studies have explored this question but are limited because of experimental restrictions, or disconnect with physiology. Here we attempt to characterize the distribution of fitness effects (DFE) due to mutations in a cellular regulatory motif. We use a simple mathematical model to describe the dynamics of gene expression in the lactose utilization network, and use a cost-benefit framework to link the model output to fitness. We simulate mutations by changing model parameters and computing altered fitness to obtain the DFE. We find beneficial mutations distributed exponentially, but distribution of deleterious mutations seems far more complex. In addition, we find neither the starting fitness, nor the exact location on the fitness landscape, affecting these distributions qualitatively. Lastly, we quantify epistasis in our model and find that the distribution of epistatic effects remains qualitatively conserved across different locations on the fitness landscape. Overall, we present a first attempt at exploring the specific statistical features of the fitness landscape associated with a system, by using the specific mathematical model associated with it.

We describe an electrochemical measurement technique that enables bioelectronic measurements of reporter proteins in living cells as an alternative to traditional optical fluorescence. Using electronically programmable microfluidics, the measurement is in turn used to control the concentration of an inducer input that regulates production of the protein from a genetic promoter. The resulting bioelectronic and microfluidic negative-feedback loop then serves to regulate the concentration of the protein in the cell. We show measurements wherein a user-programmable set-point precisely alters the protein concentration in the cell with feedback-loop parameters affecting the dynamics of the closed-loop response in a predictable fashion. Our work does not require expensive optical fluorescence measurement techniques that are prone to toxicity in chronic settings, sophisticated time-lapse microscopy, or bulky/expensive chemo-stat instrumentation for dynamic measurement and control of biomolecules in cells. Therefore, it may be useful in creating a: cheap, portable, chronic, dynamic, and precise all-electronic alternative for measurement and control of molecules in living cells.

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Both loss-of-function and gain-of-function mechanisms have been proposed to underlie this disease, but the pathogenic pathways are not fully understood. To better understand the involvement of different cell types in the pathogenesis of ALS, we systematically analyzed the distribution of promoter activity of the mouse ortholog of C9orf72 in the central nervous system. We demonstrate that C9orf72 promoter activity is widespread in both excitatory and inhibitory neurons as well as in oligodendrocytes and oligodendrocyte precursor cells. In contrast, few microglia and astrocytes exhibit detectable C9orf72 promoter activity. Although at a gross level, the distribution of C9orf72 promoter activity largely follows overall cellular density, we found that it is selectively enriched in subsets of neurons and glial cells that degenerate in ALS. Specifically, we show that C9orf72 promoter activity is enriched in corticospinal and spinal motor neurons as well as in oligodendrocytes in brain regions that are affected in ALS. These results suggest that cell autonomous changes in both neurons and glia may contribute to C9orf72-mediated disease, as has been shown for mutations in superoxide dismutase-1 (SOD1).

When produced biologically, especially by photosynthetic organisms, hydrogen gas (H 2 ) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H 2 -producing enzymes is the absence of methods for the high-throughput screening of H 2 production in biological systems. Here, we re-engineered the natural H 2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H 2 . A lacZ gene was placed under the control of the hupA H 2 -inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H 2 production levels was found for all automatically selected strains. One of the selected H 2 -overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H 2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.

Homologous recombination-mediated genome engineering has been broadly applied in prokaryotes with high efficiency and accuracy. However, this method is limited in realizing larger-scale genome editing with numerous genes or large DNA fragments because of the relatively complicated procedure for DNA editing template construction. Here, we describe a CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ) strategy for the rapid and efficient inactivation of bacterial gene (s) in a homologous recombination-independent manner and without the use of selective marker. Our study show that CA-NHEJ can be used to delete large chromosomal DNA fragments in a single step that does not require homologous DNA template. It is thus a novel and powerful tool for bacterial genomes reducing and possesses the potential for accelerating the genome evolution.

Escherichia coli lac repressor (LacI) is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the adjacent lac promoter. In this article, we demonstrate that upon binding to the O1 and O2 operators at their native positions LacI constrains three (-) supercoils within the 401-bp DNA loop of the lac promoter and forms a topological barrier. The stability of LacI-mediated DNA topological barriers is directly proportional to its DNA binding affinity. However, we find that DNA supercoiling modulates the basal expression from the lac operon in E. coli. Our results are consistent with the hypothesis that LacI functions as a topological barrier to constrain free, unconstrained (-) supercoils within the 401-bp DNA loop of the lac promoter. These constrained (-) supercoils enhance LacI's DNA-binding affinity and thereby the repression of the promoter. Thus, LacI binding is superhelically modulated to control the expression of lacZYA in the lac operon under varying growth conditions.

Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer-fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8-64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.

One of the major challenges in the treatment of primary muscle disorders, which often affect many muscle groups, is achieving efficient, widespread transgene expression in muscle. In utero gene transfer can potentially address this problem by accomplishing the gene delivery when the tissue mass is small and the immune system is immature. Earlier studies with systemic in utero adeno-associated viral (AAV) vector serotype 1 gene delivery to embryonic day 16 (E-16) pups resulted in high levels of transduction in diaphragm and intercostal muscles, but no detectable transgene expression in limb muscles. Recently, newer AAV serotypes, such as AAV8, have shown widespread and high transgene expression in skeletal muscles and diaphragm by systemic delivery in adult and neonatal mice. We tested AAV8 vector gene delivery by intraperitoneal administration in E-16 mice in utero. Using an AAV8 vector carrying a lacZ reporter gene, we observed high-level transduction of diaphragm and intercostal muscles and more moderate transduction of multiple limb muscles and heart. Our current studies show the potential of AAV8 to achieve widespread muscle transduction in utero and suggest its therapeutic potential for primary muscle disorders.

The development of the amygdala, a central structure of the limbic system, remains poorly understood. We found that two spatially distinct and early-specified telencephalic progenitor pools marked by the homeodomain transcription factor Dbx1 are major sources of neuronal cell diversity in the mature mouse amygdala. We found that Dbx1-positive cells of the ventral pallium generate the excitatory neurons of the basolateral complex and cortical amygdala nuclei. Moreover, Dbx1-derived cells comprise a previously unknown migratory stream that emanates from the preoptic area (POA), a ventral telencephalic domain adjacent to the diencephalic border. The Dbx1-positive, POA-derived population migrated specifically to the amygdala and, as defined by both immunochemical and electrophysiological criteria, generated a unique subclass of inhibitory neurons in the medial amygdala nucleus. Thus, this POA-derived population represents a previously unknown progenitor pool dedicated to the limbic system.

Here, we characterized behavioral abnormalities induced by prolonged social isolation in adult rodents. Social isolation induced both anxiety- and anhedonia-like symptoms and decreased cAMP response element-binding protein (CREB) activity in the nucleus accumbens shell (NAcSh). All of these abnormalities were reversed by chronic, but not acute, antidepressant treatment. However, although the anxiety phenotype and its reversal by antidepressant treatment were CREB-dependent, the anhedonia-like symptoms were not mediated by CREB in NAcSh. We found that decreased CREB activity in NAcSh correlated with increased expression of certain K(+) channels and reduced electrical excitability of NAcSh neurons, which was sufficient to induce anxiety-like behaviors and was reversed by chronic antidepressant treatment. Together, our results describe a model that distinguishes anxiety- and depression-like behavioral phenotypes, establish a selective role of decreased CREB activity in NAcSh in anxiety-like behavior, and provide a mechanism by which antidepressant treatment alleviates anxiety symptoms after social isolation.

Transfer of healthy autologous tissue as a microvascular free flap facilitates reconstruction during ablative cancer surgery. In addition to filling surgical defects, free flaps might concentrate viral vectors at the tumour bed and mediate local therapeutic effects. We evaluated the magnitude, topography and duration of luciferase gene expression after plasmid and adenoviral delivery in rat superficial inferior epigastric (SIE) flaps. For plasmid delivery, luciferase expression was significantly increased by all transduction routes (topical, intraflap injection, intravascular) (P<0.01) at day 1, but not at day 7. The spread of luciferase expression was significantly different between the 4 groups at 1 day (P=0.026) and was greatest for flaps transduced by intravascular injection. For adenoviral transduction, total radiance was significantly different between the transduced groups at 1, 14 and 28 days (P<0.05 for all comparisons). The highest levels of radiance were seen in the intravascular group. There was a statistically significant difference in the spread of light emission between the 3 groups at 1 (P=0.009) and 14 (P=0.013) days, but this was no longer evident at 28 days. Intravascular adenoviral delivery yields high-level, diffuse and durable gene expression in rat SIE flaps and is suitable for examination in therapeutic models.

In an attempt to determine whether muscle-derived stem cells are distinct from satellite cells, we investigated whether muscle-derived stem cells could be isolated from the skeletal muscle of Pax7-deficient mice, which have been shown to be devoid of or to contain only a minimal number of satellite cells. Utilizing a technique that separates cells based on their adhesion characteristics (the preplate technique), several distinct populations of muscle-derived cells were isolated. In these mice, the Pax7 gene was knocked out with the insertion of the LacZ gene. One population was both rapidly adhering, LacZ-positive, and displayed a high myogenic index, but was rapidly lost to terminal differentiation when continuously replated. A second population, which persisted over 50 passages, was LacZ-negative and displayed a low myogenic index. Although Pax3 may have acted as a compensatory mechanism for the myogenic commitment of the LacZ-positive cells, the LacZ-negative cells, despite expressing Pax3, required Pax7 transduction to restore their myogenic capacity. We believe that these two populations of myogenic progenitor cells, each endowed with different adhesion characteristics, may help explain the discrepancy in the literature concerning the presence of myogenic cells found in Pax7-deficient mice.

Gene electrotransfer is an efficient and reproducible nonviral gene transfer technique useful for the nonpermanent expression of therapeutic transgenes. The present study established optimal conditions for the electrotransfer of reporter genes into mesenchymal stem cells (MSCs) isolated from rat bone marrow by their selective adherence to tissue-culture plasticware. The electrotransfer of the lacZ reporter gene was optimized by adjusting the pulse electric field intensity, electric pulse type, electropulsation buffer conductivity and electroporation temperature. LacZ electrotransfection into MSCs was optimal at 1500 V cm(-1) with pre-incubation in Spinner's minimum essential medium buffer at 22 degrees C. Under these conditions beta-galactosidase expression was achieved in 29+/-3% of adherent cells 48 h post transfection. The kinetics of beta-galactosidase activity revealed maintenance of beta-galactosidase production for at least 10 days. Moreover, electroporation did not affect the MSC potential for multidifferentiation; electroporated MSCs differentiated into osteoblastic, adipogenic and chondrogenic lineages to the same extent as cells that were not exposed to electric pulses. Thus, this study demonstrates the feasibility of efficient transgene electrotransfer into MSCs while preserving cell viability and multipotency.

We evaluated whether the improved esophageal radiation tolerance following Manganese Superoxide Dismutase (MnSOD)-Plasmid Liposomes was explained by improved engraftment of bone marrow-derived progenitors. C57BL/6NHsd female mice pretreated with intraesophageal MnSOD-PL were irradiated to 29 Gy to the esophagus and intravenously transplanted with marrow from male B6. 129S7-Gt (ROSA) 26S OR/J ROSA (Lac-Z+, G418-resistant) mice. After 14 days, esophagi were removed and side population and non-side population cells evaluated for donor multilineage (endothelin/vimentin/F480) positive esophageal cells. Serial intravenous transplantability was tested in second generation 29 Gy esophagus-irradiated mice. Esophagi from recipients receiving swallowed MnSOD-PL 24 h prior to irradiation demonstrated significantly increased esophageal repopulation with donor bone marrow-derived Lac-Z+, G418+, Y-probe+ multilineage cells (37.8+/-1.8>50 cell Lac-Z+ foci per esophagus) compared to irradiated controls (19.8+/-1.8) P<0.0001. Serial transfer to second-generation irradiated C57BL/6NHsd mice of intravenously injected SP or NSP first generation recipient esophagus cells was also significantly enhanced by MnSOD-PL intraesophageal pretreatment (74.4+/-3.6 SP-derived Lac-Z+ foci per esophagus, 48.6+/-5.4 NSP-derived) compared to irradiation controls (23.4+/-1.8 SP, 6.0+/-3.0 NSP), P<0.0001. Thus, intraesophageal MnSOD-PL administration enhances engraftment of marrow-derived progenitors.

The bacterial lacZ gene encoding for beta-galactosidase (beta-gal) is a common reporter gene used in transgenic mice. Nonetheless, the absence of fluorigenic substrates usable in live animals greatly hampered the non-invasive follow-up of this reporter gene expression. We used far-red fluorescence for imaging beta-Gal expression in live cells in vitro or in vivo. The 9H-(1,3-dichloro-9,9-dimethylacridin- 2-one-7-yl) beta-D-galactopyranoside substrate was used to monitor beta-Gal expression as a reporter of tumor growth, or of the physiological levels of an endogenous gene or of gene transfer in lung. A quantitative evaluation of this method as well as a comparison of its sensitivity with Firefly Luciferase-based bioluminescence was also performed. In vivo measurements showed that 10(3) beta-Gal tumor cells located under the skin were detectable. In deeper organs like lung, as little as 5 ng of beta-Gal or Luciferase enzymes per mg of proteins were measured, confirming that both techniques reached similar sensibilities. Nonetheless, quantitative comparison of beta-Gal levels measured with far-red imaging or with a standardized enzymatic evaluation after killing revealed that the 2D-fluorescent reflectance imaging method is submitted to a color-dependent disparity of the organs and cannot supply quantitative measurements but that a simple correction can be applied.

Tissue damage and inflammation promote bone marrow stem cells (BMSCs) to differentiate into a variety of cell types in residing tissues. BMSCs can stably maintain their plasticity and are an ideal cell population for delivery of therapeutic genes to non-hematopoietic tissues. Using lacZ as a reporter gene, we demonstrated that the lung-specific human surfactant protein B (hSP-B) 1.5-kb promoter is able to deliver the lacZ gene into the lung of lysosomal acid lipase (LAL) gene-knockout (lal-/-) mice by beta-galactosidase staining, flow cytometry and double immunofluorescence staining. Around 10-18% alveolar type II epithelial cells (AT II cells) exhibited positive lacZ gene expression after 8 weeks of BMSC injection in recipient lal-/- mice. The wild-type mice exhibited no expression after the same treatment. BMSCs from hSP-B 1.5-kb lacZ transgenic mice entered and repopulated in lal-/- bone marrow. The study supports a concept that pulmonary inflammation caused by LAL deficiency can trigger BMSC residing in lal-/- bone marrow, migrating into the lung and converting into residential AT II cells. The hSP-B 1.5 kb promoter is an ideal tool to deliver therapeutic genes into AT II cells through BMSCs to cure pulmonary inflammation-triggered diseases.

Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors.

Recombinant adeno-associated (AAV) viruses have unique properties, which make them ideal vectors for gene transfer targeting the myocardium. Numerous serotypes of AAV have been identified with variable tropisms towards cardiac tissue. In the present study, we investigated the time course of expression of eight different AAV serotypes in rat myocardium and the nature of the immunity against these serotypes. We first assessed whether neutralizing antibodies (NAb) were present for any of the serotype in the rats. We injected 100 microl of each AAV 1-8 serotype (10(12) DNAse resistant particles/ml), encoding LacZ gene, into the apical wall of rat myocardium. At 1, 4, 12 and 24 weeks after gene delivery, the animals were killed and beta-galactosidase (beta-gal) activity was assessed by luminometry. Additionally, LacZ genomic copies and AAV capsids copies were measured through standard polymerase chain reaction analysis and cryo-sections from the area of viral injection were stained for X-gal detection at the same time points. No NAbs were detected against any of AAV serotypes. At all the time points studied, AAV1, 6 and 8 demonstrated the highest efficiency in transducing rat hearts in vivo. Parallel to the results with beta-gal activity, the highest levels LacZ and AAV DNA genomic copies were with AAV1, 6 and 8. The positive X-gal staining depicted by these serotypes confirmed these results. These results indicate that among the various AAV serotypes, AAV1, 6 and 8 have differential tropism for the heart unaffected by pre-existing NAb in the rat. Although AAV 1 and 6 vectors induced rapid and robust expression and reach a plateau at 4 weeks, AAV 8 continued increasing until the end of the study. AAV 2, 5 and 7 vectors were slower to induce expression of the reporter gene, but did reach levels of expression comparable to AAV1 and AAV6 vectors after 3 months.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SNi). As neurotrophic factors support the survival and enhance the function of dopaminergic neurons, gene therapy using neurotrophic factors has become the center of interest. Thus, we focused on hepatocyte growth factor (HGF) as a neurotrophic and angiogenic growth factor. At 7 days before injection of 6-hydroxydopamine into the SNi, stereotaxic transfection of human HGF or lacZ plasmid was performed into the unilateral striatum of rats. Expression of human HGF in the injected sites could be detected in rats transfected with HGF plasmid DNA, using immunohistochemical staining. Consistently, human immunoreactive HGF protein could be detected at least up to 12 days after transfection. Interestingly, PD rats transfected with lacZ demonstrated amphetamine-induced rotational asymmetry. However, transfection of HGF plasmid DNA resulted in significant inhibition of abnormal rotation up to 24 weeks in a dose-dependent manner. Over 90% of dopaminergic neurons were lost in PD rats transfected with lacZ, whereas over 70% survived in rats transfected with HGF, as assessed by immunohistochemical staining. Overall, the present study demonstrated that overexpression of HGF prevented neuronal death in a PD rat model, providing a potential novel therapy for PD.

Adeno-associated viral vectors (AAV) are attractive tool for gene therapy for coronary artery disease. However, gene expression in myocardium mediated by AAV serotype 2 (AAV2) does not peak until 4-6 weeks after gene transfer. This delayed gene expression may reduce its therapeutic potential for acute cardiac infarction. To determine whether earlier gene expression and better therapeutic effect could be achieved using a different serotype, CMV promoter driving the EPO gene (AAV-EPO) was packaged into AAV serotypes 1-5 capsids and injected into mouse myocardium. EPO expression was studied by measuring the hematocrits and EPO mRNA. After we found that AAV1 mediates the highest gene expression after 4 days of gene transduction, AAV-LacZ (CMV promoter driving LacZ gene expression) and MLCVEGF (hypoxia-inducible and cardiac-specific VEGF expression) were packaged into AAV1 and 2 capsids. LacZ expression was detected in AAV1-LacZ but not in AAV2-LacZ-injected hearts 1 day after vector injection. Compared to AAV2-MLCVEGF that mediated no significant VEGF expression, AAV1-MLCVEGF mediated 13.7-fold induction of VEGF expression in ischemic hearts 4 days after gene transduction and resulted in more neovasculatures, better cardiac function and less myocardial fibrosis. Thus, AAV1 mediates earlier and higher transgene expression in myocardium and better therapeutic effects.

While a number of virus-based delivery schemes have been developed for myocardial gene transfer, no technique has proven capable of modifying a majority of cardiac myocytes rapidly and homogeneously in the in vivo rat model, and most schemes result in significant infection of the liver and other organs. However, adenoviral delivery to the excised heart during retrograde perfusion can produce 67-92% efficient gene transfer. In this study, we adapt this isolation/perfusion scheme to the heart in vivo. We isolated the heart in vivo by simultaneously clamping all vessels to/from the heart. The heart was then continuously retrograde perfused through a catheter positioned in the aortic root. A second catheter in the right ventricle provided a path for efflux. After perfusing the heart for 7.5 min with calcium-free Tyrode solution followed by 90 s no-flow viral exposure (AdV.cmv.LacZ; 10(12) viral particles/ml), gene transfer efficiency was 60% compared to 5% by a conventional cross-clamp technique. Infection of peripheral organs was dramatically reduced. Given the prevalence of the rat in so many models of heart disease, this enhancement of infection represents an advancement in viral-based delivery of exogenous genes to heart for the study of gene therapy in vivo.

In this study, we explored the use of electroporation or media that promote lipoplex formation for nonviral gene transfer in the eye. There was no detectable staining for LacZ after subretinal, intravitreous, or periocular injection of a plasmid containing a CMV promoter expression cassette for LacZ, but when plasmid injection in each of the three sites was combined with electroporation, there was efficient transduction. Specific staining for LacZ was seen in retinal pigmented epithelial (RPE) cells after subretinal injection of a plasmid containing a vitelliform macular dystrophy 2 (VMD2) promoter expression cassette, demonstrating that this approach can be used to evaluate purported tissue-specific promoters in vivo. Electroporation with 10 V/mm resulted in strong LacZ staining, but was damaging to photoreceptors; substantial transduction with no evidence of retinal damage was seen using 3.4 V/mm. Staining for LacZ was also seen after subretinal or periocular, but not intravitreous, injection of plasmid DNA in medium containing 40% Lipofectamine2000 (Lf). Injection of 40% Lf into the subretinal space caused damage to photoreceptors, but subretinal injection of plasmid DNA in medium containing 10% NeuroPorter resulted in transduction of RPE cells with no adverse effects on retinal morphology or function as assessed by electroretinograms (ERGs). After either electroporation or lipofection, LacZ staining was detectable for at least 14 days, and could be reinduced by a second procedure. These data suggest that electroporation or lipofection can be used as experimental tools for ocular gene transfer to evaluate tissue-specific promoter fragments or to evaluate the effects of transgene expression in the retina. Also, with additional optimization, nonviral gene transfer may prove to be a valuable approach for the treatment of retinal and choroidal diseases.

In view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Oncolytic vesicular stomatitis virus (VSV) is a promising novel therapeutic agent for the treatment of cancer. The aim of this study was to evaluate the potential of recombinant VSV containing the M51R mutation in the matrix (M) protein gene administered intravenously as an effective and safe therapeutic agent for treating mice with experimental breast cancer metastases. Recombinant VSV(M51R)-LacZ was generated and characterized in vitro on human and murine breast cancer cells. Breast cancer metastases were established in immune-competent Balb/c mice by intravenous injection of syngeneic 4T1 cells. The vector was infused into the tumor-bearing animals via the tail vein, and productive infection of pulmonary breast cancer lesions was assessed by X-gal stainings of frozen lung sections. To evaluate potential systemic toxicity, histology of major organs and serum chemistries were analyzed. To assess effectiveness, buffer- or vector-treated tumor-bearing mice were followed for survival and the results were analyzed by the Kaplan-Meier method and the log-rank test. We found that VSV(M51R)-LacZ efficiently replicated and lysed human breast cancer cells but was partially attenuated in 4T1 cells in vitro. We also demonstrated that its maximum tolerated dose after intravenous infusion in normal Balb/c mice was elevated by at least 100-fold over that of the parental VSV vector containing the wild-type M gene. When VSV(M51R)-LacZ was repeatedly injected intravenously into mice bearing syngeneic 4T1 tumors, the virus was able to infect multiple breast cancer lesions in the lungs without apparent toxicities, which led to significant prolongation of animal survival (P=.003). In conclusion, systemic administration of M mutant VSV is both effective and safe in the treatment of experimental breast cancer metastases in immune-competent mice, suggesting that further development of this approach may have potential for clinical application in patients.

Aim: To construct a recombinant adeno-associated virus (rAAV) vector containing gene encoding phospholamban antisense RNA (asPLB), and analyse its effect on expression of PLB, expression and activity of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and the change of intracellular free Ca2+ concentration ([Ca2+]i) in rat cardiomyocytes., Methods: The target gene encoding PLB antisense RNA was inserted inversely into the adeno-associated virus plasmid pAAV-MCS digested by corresponding restricted endonuclease enzyme. The recombinant plasmid and pAAV-RC and pHelper were co-transfected into 293 cell. At the same time, a viral production positive control (rAAV-LacZ) and negative control were performed. The recombinant viruses were used to transfect the cultured rat cardiomyocytes. Site beta-Galactosidase staining were performed to observe the transfer efficiency. Reverse transcription-PCR and Western blot were used to determine the mRNA and protein expression of PLB and SERCA. The activity of SERCA and the [Ca2+]i were measured., Results: The rAAV vectors were constructed successfully and were transfected into rat cardiomyocytes effectively. The PLB mRNA and protein expression were reduced in rat cardiomyocytes transfected by rAAV-asPLB compared with controls. The activity of SERCA was increased. In rest state, the level of [Ca2+]i in the rAAV-asPLB transfected group decreased. The level of [Ca2+]i increased when induced by isoproterenol., Conclusion: AAV-asPLB vector was constructed successfully, which disrupted the expression of PLB, enhanced the activity of SERCA, reduced the resting [Ca2+]i, and improved the cardiac function.

Gene transfer complexes containing poly-L-lysine (poly-K) and DNA with ligands directed at the serpin enzyme complex receptor (sec-R) deliver reporter genes to receptor-bearing cells in vivo. Expression lasts for about 30 days, when complexes containing long-chain poly-K are used. Extending the duration of expression would be desirable if correction of genetic defects is the goal. To test whether the mechanism by which expression is extinguished was due to an immune response to the transgene, or the loss of the transgene, we conducted two experiments. In the first, we injected sec-R-targeted lacZ complexes intravenously (i.v.) into mice genetically engineered to express this gene briefly during development. These mice, who should recognize the protein as 'self', also extinguished lacZ expression after 30 days. In a second experiment, we injected immunodeficient animals with sec-R-targeted human factor IX complexes. A similar temporal pattern of expression was observed in Rag-1 -/- mice, in whom expression also extinguished by 40 days. Moreover, factor IX plasmid DNA was detected in the lung and spleen 50 days after injection of complexes, suggesting that not all cells which had taken up the transgene had been destroyed. Thus, the host's immune response to the transgene may not account for the loss of reporter gene expression from these molecular conjugates. We further tested whether repeat administration of sec-R-targeted complexes will be limited by host immune responses. Mice were pre-dosed twice with sec-R-targeted complexes containing lacZ over a 40-day period. We then injected the animals i.v. with sec-R-targeted human factor IX complexes and measured gene expression and antibody production. Although 14 of 36 animals displayed low-titer antibodies to the ligand in targeted complex, expression levels were unaffected compared with virgin dosing. When the complexes were administered three times intranasally (n=10), no antibodies against the complex were detected in blood. Plasma from mice dosed with saline, nontargeted complex or naked DNA did not react with the ligand, ligand-poly K conjugate or targeted complex. All animals exhibiting human factor IX expression developed antibodies to that transgene by 21 days. Thus, at least three repeat administrations of sec-R-directed molecular conjugates are possible, provided that immune responses to the transgene itself are not limiting.

Somatic gene repair of disease-causing chromosomal mutations is a novel approach for gene therapy. This method would ensure that the corrected gene is regulated by its endogenous promoter and expressed at physiological levels in the appropriate cell types. A reporter mouse, Gtrosa26(tm1Col), was generated by targeting a mutated LacZ gene to the Rosa26 locus in mouse embryonic stem (ES) cells. The LacZ gene contains a G to A point mutation, resulting in a Glu to Lys amino-acid substitution at position 461, which abrogates enzymatic activity. The gene is expressed in ES cells, primary embryonic fibroblasts, and in all tissues examined in the adult mouse, including the lung, liver, kidney, spleen, heart, brain and smooth muscle. This transgenic mouse will allow testing of gene repair strategies in vivo and identification of which cell types can be successfully targeted by chromosomal gene repair. Although low levels of gene repair were achieved in the ES cells used to generate the Gtrosa26(tm1Col) mouse, preliminary attempts at gene repair in vivo were unsuccessful, thus highlighting the difficulties that will have to be overcome to get this approach to work.

O6-methylguanine methyltransferase, Mgmt, constitutes the first line of defense against O6-alkylguanine, which can result in G : C to A : T transitions upon DNA replication. Mgmt has been found in organisms as diverse as archaebacteria and mammals. This evolutionary conservation suggests that all organisms may be exposed to either endogenous or environmental alkylating agents. We thus hypothesized that tissues of Mgmt-/- mice would exhibit elevated mutant frequencies. Employing the Big Blue trade mark transgenic system, we evaluated lacI mutants rescued from liver and small intestinal DNA of young Mgmt-/- mice. Interestingly, while there was a small difference between Mgmt-/- mice and controls with respect to lacI mutant frequency, no differences attributable to Mgmt deficiency were apparent in the mutational spectra. Although mutations stemming from O6-guanine alkylations would be predicted to be cumulative, we found no evidence of an Mgmt-dependent alteration in mutation spectrum in DNA samples from 12 month-old mice. To optimize our ability to detect mutations resulting from O6-alkylguanine-induced G : T mismatches, mice with combined deficiencies of Mgmt and the DNA mismatch repair molecule, Msh6, were analysed. In spite of this strategy, we observed no significant differences between Mgmt-/- Msh6-/- and Msh6-/- mouse lacI mutations, except for a trend towards a greater percentage (of total transitions) of G : C to A : T changes in Mgmt-/-Msh6-/- livers. Therefore, despite the striking evolutionary conservation of Mgmt, deficiency of this gene did not significantly impact the spontaneous lacI mutational spectrum in vivo.

The use of viral vectors for gene delivery to motor neurons in vivo has been hampered by the need to perform invasive surgery to inject directly the vector into the anterior horn of the spinal cord. Here, we have characterized the features of herpes simplex virus-1 (HSV)-derived vectors, in terms of gene mutations and promoter constructs, that are required to allow efficient transduction of motor neurons following a relatively noninvasive peripheral administration via sciatic nerve or footpad injection. Owing to the wide variety of animal models used to study neurodegenerative diseases of motor neurons, we analysed the effectiveness of these vectors in adult mice and adult and neonatal rats. We tested viruses with differing degrees of disablement based on the 1764 backbone (deleted for ICP34.5 and an insertional inactivation in VP16) rendered completely replication incompetent by the deletion of the essential immediate-early genes ICP27 and/or ICP4. In the adult mouse, prolonged gene expression in motor neurons was obtained after sciatic nerve inoculation with a vector defective in ICP4 and ICP27. In the adult rat, both the vector defective in ICP4 and the vector defective in ICP4 and ICP27 were capable of transducing motor neurons for extended periods of time during viral latency. This study demonstrates the feasibility of using HSV vectors for persistent transgene expression in motor neurons in a safe and nontoxic manner following peripheral administration. These vectors are potentially useful tools to investigate the functions of genes involved in motor neuronal survival and regeneration in models of motor neuron diseases in vivo.