Search

Electroporation is a technique used in vitro, ex vivo, and in vivo to permeabilize cell membranes. The effect on the tissue describes a continuum ranging from mild perturbations to massive tissue damage. Thus care should be taken when choosing pulses for a given application. Here the effects of electroporation paradigms ranging from severe to very gentle permeabilization were investigated on soleus, mainly composed of slow-twitch fibers, and extensor digitorum longus (EDL) and tibialis anterior (TA), almost exclusively composed of fast-twitch fibers. Five key physiological parameters were studied: force, muscle [Na.sup.+], [K.sup.+], and [Ca.sup.2+] content, and plasma lactate dehydrogenase activity. Four-week-old Wistar rats were anesthetized, and the lower part of the hind leg was electroporated. Blood samples were collected from the tail vein, and at the times indicated animals were killed and TA, EDL, and soleus muscles were collected for analysis of force and ion contents. Muscles were given eight high-voltage pulses of 100-[micro]s duration (8HV) at varying field intensity, one short high-voltage pulse combined with one long low-voltage pulse (HVLV), or eight medium-voltage pulses of 20-ms duration (8MV). Intensity of the electrical field strength was determinant for the degree of changes observed in the muscle. Field strengths below 300 V/cm did not give rise to measurable changes, whereas 8HV pulses at high field intensities (1,200 V/cm) caused severe and long-lasting damage to the muscle. Interestingly, the damage was more pronounced in EDL and TA compared with soleus, possibly because of the difference in fiber type composition. HVLV only caused temporary changes, with force and ion content being normalized by 4 h, suggesting that this pulse combination may be useful for the introduction of ions and molecules (e.g., DNA) into muscle cells. electropermeabilization; extensor digitorum longus; tibialis anterior; soleus; fiber type doi: 10.1152/ajpregu.00692.2009.

Generation of cultured human cells stably expressing one or more recombinant gene sequences is a widely used approach in biomedical research, biotechnology, and drug development. Conventional methods are not efficient and have severe limitations especially when engineering cells to coexpress multiple transgenes or multiprotein complexes. In this report, we harnessed the highly efficient, nonviral, and plasmid-based piggyBac transposon system to enable concurrent genomic integration of multiple independent transposons harboring distinct protein-coding DNA sequences. Flow cytometry of cell clones derived from a single multiplexed transfection demonstrated approximately 60% (three transposons) or approximately 30% (four transposons) stable coexpression of all delivered transgenes with selection for a single marker transposon. We validated multiplexed piggyBac transposon delivery by coexpressing large transgenes encoding a multisubunit neuronal voltage-gated sodium channel (SCN1A) containing a pore-forming subunit and two accessory subunits while using two additional genes for selection. Previously unobtainable robust sodium current was demonstrated through 38 passages, suitable for use on an automated high-throughput electrophysiology platform. Cotransfection of three large (up to 10.8 kb) piggyBac transposons generated a heterozygous SCNIA stable cell line expressing two separate alleles of the pore-forming subunit and two accessory subunits (total of four sodium channel subunits) with robust functional expression. We conclude that the piggyBac transposon system can be used to perform multiplexed stable gene transfer in cultured human cells, and this technology may be valuable for applications requiring concurrent expression of multiprotein complexes. piggyBac | SCN1A | SCN1B | SCN2B | sodium channel doi/ 10.1073/pnas.0910383107

Agrobacterium tumefaciens is a Gram-negative plant-pathogenic bacterium that causes crown gall disease by transferring and integrating its transferred DNA (T-DNA) into the host genorne. We characterized the chromosomally encoded alpha-crystallin-type small heat-shock protein ([alpha]-Hsp) HspL, which was induced by the virulence (vir) gene inducer acetosyringone (AS). The transcription of hspL but not three other [alpha]-Hsp genes (hspC, hspAT1, hspAT2) was upregulated by AS. Further expression analysis in various vir mutants suggested that AS-induced hspL transcription is not directly activated by the VirG response regulator but rather depends on the expression of VirG-activated virB genes encoding components of the type IV secretion system (T4SS). Among the 11 virB genes encoded by the virB operon, HspL protein levels were reduced in strains with deletions of virB6, virB8 or virB11. VirB protein accumulation but not virB transcription levels were reduced in an hspL deletion mutant early after AS induction, implying that HspL may affect the stability of individual VirB proteins or of the T4S complex directly or indirectly. Tumorigenesis efficiency and the VirB/D4-rnediated conjugal transfer of an IncQ plasmid RSF1010 derivative between A. tumefaciens strains were reduced in the absence of HspL. In conclusion, increased HspL abundance is triggered in response to certain VirB protein(s) and plays a role in optimal VirB protein accumulation, VirB/D4-mediated DNA transfer and tumoriagenesis. DOI 10.1099/mic.0.030676-0

This study uses sequences from four genes, which are involved in the formation of the type III secretion apparatus, to determine the role of horizontal gene transfer in the evolution of virulence genes for the enterobacterial plant pathogens. Sequences of Erwinia, Brenneria, Pectobacterium, Dickeya and Pantoea were compared (a) with one another, (b) with sequences of enterobacterial animal pathogens, and (c) with sequences of plant pathogenic [gamma] and [beta] proteobacteria, to evaluate probable paths of lateral exchange leading to the current distribution of virulence determinants among these micro-organisms. Phylogenies were reconstructed based on hrcC, hrcR, hrcJ and hrcV gene sequences using parsimony and maximum-likelihood algorithms. Virulence gene phylogenies were also compared with several housekeeping gene loci in order to evaluate patterns of lateral versus vertical acquisition. The resulting phylogenies suggest that multiple horizontal gene transfer events have occurred both within and among the enterobacterial plant pathogens and plant pathogenic [gamma] and [beta] proteobacteria, hrcJ sequences are the most similar, exhibiting anywhere from 2 to 50% variation at the nucleotide level, with the highest degree of variation present between plant and animal pathogen sequences, hrcV sequences are conserved among plant and animal pathogens at the N terminus. The C-terminal domain is conserved only among the enterobacterial plant pathogens, as are the hrcC and hrcR sequences. Additionally, hrcJ and hrcV sequence phylogenies suggest that at least some type III secretion system virulence genes from enterobacterial plant pathogens are related more closely to those of the genus Pseudomonas, a conclusion neither supported nor refuted by hrcC or hrcR. DOI 10.1099/mic.0.029892-0

Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme [O.sup.6]-methylguanine-DNA methyltransferase (MGMT *). We transplanted 5 rhesus macaques with [CD34.sup.+] cells transduced with lentiviral vectors encoding MGMT * and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after [O.sup.6]-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT * lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT * vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen., Introduction Retroviral vector--mediated gene transfer into HSCs has the potential to improve treatment for various genetic, malignant, and infectious diseases (1), (2). However, a critical limitation to stem cell gene [...]

Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid--derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response., Introduction In the first phase I/II trial of hepatic gene transfer of adeno-associated virus 2 (AAV2) encoding Factor IX (AAV2-F9) in human hemophilia B subjects, transgene expression was demonstrable but [...]

Noonan syndrome (NS), the most common single-gene cause of congenital heart disease, is an autosomal dominant disorder that also features proportionate short stature, facial abnormalities, and an increased risk of myeloproliferative disease. Germline-activating mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, cause about half of NS cases; other causative alleles include KRAS, SOS1, and RAF1 mutants. We showed previously that knock-in mice bearing the NS mutant [Ptpn11.sup.D61G] on a mixed 129S4/SvJae X C57BL6/J background exhibit all major NS features, including a variety of cardiac defects, with variable penetrance. However, the cellular and molecular mechanisms underlying NS cardiac defects and whether genetic background and/or the specific NS mutation contribute to the NS phenotype remained unclear. Here, using an inducible knock-in approach, we show that all cardiac defects in NS result from mutant Shp2 expression in the endocardium, not in the myocardium or neural crest. Furthermore, the penetrance of NS defects is affected by genetic background and the specific Ptpn11 allele. Finally, ex vivo assays and pharmacological approaches show that NS mutants cause cardiac valve defects by increasing Erk MAPK activation, probably downstream of ErbB family receptor tyrosine kinases, extending the interval during which cardiac endocardial cells undergo endocardial-mesenchymal transformation. Our data provide a mechanistic underpinning for the cardiac defects in this disorder. cardiac development | human disease | signal transduction | protein tyrosine phosphatase | cardiac valves

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules--a process known as indirect recognition. As [CD4.sup.+][CD25.sup.+] Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating [CD4.sup.+][CD25.sup.+] cells from C57BL/6 mice ([H-2.sup.b]) with allogeneic DCs from BALB/c mice ([H-2.sup.d]). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for [H-2K.sup.d] presented by [H-2A.sup.b] MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential., Introduction Naturally occurring [CD4.sup.+][CD25.sup.+] Tregs are known to play a key role in preventing autoimmunity. In recent years, several groups have reported that this population of cells can also be [...]

Brucella genomic islands (GIs) share similarities in their genomic organization to pathogenicity islands from other bacteria and are likely acquired by lateral gene transfer. Here, we report the identification of a GI that is important for the pathogenicity of Brucella melitensis. The deletion of GI-1, GI-5, or GI-6 did not affect bacterial growth in macrophages as well as their virulence in interferon regulatory factor 1-deficient (IRF-[1.sup.-/-]) mice, suggesting that these islands do not contribute to Brucella virulence. However, the deletion of GI-2 resulted in the attenuation of bacterial growth in macrophages and virulence in [IRF-1.sup.-/-] mice. The GI-2 mutant also displayed a rough lipopolysaccharide (LPS) phenotype indicated by acriflavin agglutination, suggesting that in vitro and in vivo attenuation is a result of LPS alteration. Further, systematic analysis of the entire GI-2 revealed two open reading frames (ORFs), BMEI0997 and I0998, that encode hypothetical sugar transferases and contribute to LPS alteration, as the deletion of either of these ORFs resulted in a rough phenotype similar to that of the GI-2 mutant. Complementation analyses indicated that in addition to I0997 and I0998, I0999 is required to restore the smooth LPS in the GI-2 mutant as well as its full in vitro and in vivo virulence. The I0999 sequence analysis suggested that it might function as a transporter to help facilitate the transport or linking of the O antigen to the LPS. Our study also indicated that the rough LPS resulting from the GI-2 deletion may affect pathogen-associated molecular pattern recognition by Toll-like receptors.

ATP/ADP translocases are a hallmark of obligate intracellular pathogens related to chlamydiae and rickettsiae. These proteins catalyze the highly specific exchange of bacterial ADP against host ATP and thus allow bacteria to exploit their hosts' energy pool, a process also referred to as energy parasitism. The genome sequence of the obligate intracellular pathogen Lawsonia intracellularis (Deltaproteobacteria), responsible for one of the most economically important diseases in the swine industry worldwide, revealed the presence of a putative ATP/ADP translocase most similar to known ATP/ADP translocases of chlamydiae and rickettsiae (around 47% amino acid sequence identity). The gene coding for the putative ATP/ADP translocase of L. intracellularis (L. intracellularis nucleotide transporter 1 [[NTT1.sub.Li]]) was cloned and expressed in the heterologous host Escherichia coli. The transport properties of [NTT1.sub.Li] were determined by measuring the uptake of radioactively labeled substrates by E. coli. [NTT1.sub.Li] transported ATP in a counterexchange mode with ADP in a highly specific manner; the substrate affinities determined were 236.3 ([+ or -] 36.5) [micro]M for ATP and 275.2 ([+ or -] 28.1) [micro]M for ADP, identifying this protein as a functional ATP/ADP translocase. [NTT1.sub.Li] is the first ATP/ADP translocase from a bacterium not related to Chlamydiae or Rickettsiales, showing that energy parasitism by ATP/ADP translocases is more widespread than previously recognized. The occurrence of an ATP/ADP translocase in L. intracellularis is explained by a relatively recent horizontal gene transfer event with rickettsiae as donors.

Realtime optical bioluminescence imaging is a powerful tool for studies of gene regulation in living animals. To elucidate exercise-induced signaling/transcriptional control of the peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha] (Pgc-1[alpha]) gene in skeletal muscle, we combined this technology with electric pulse-mediated gene transfer to cotransfect the Pgc-1[alpha] reporter gene with plasmid DNA encoding mutant/deletion forms of putative regulatory factors and, thereby, assess the responsiveness of the promoter to skeletal muscle contraction. We show that each of the myocyte enhancer factor 2 sites on the Pgc-1[alpha] promoter is required for contractile activity-induced Pgc-1[alpha] transcription. The responsiveness of the Pgc-1[alpha] promoter to contractile activity could be completely blocked by overexpression of the dominant-negative form of activating transcription factor 2 (ATF2), the signaling-resistant form of histone deacetylase (HDAC) 5 (HDAC5), or protein kinase D (PKD), but not by HDAC4. These findings provide in vivo evidence for functional interactions between PKD/HDAC5 and ATF2 regulatory factors and the Pgc-1[alpha] gene in adult skeletal muscle. signal transduction; transcriptional control; reporter gene; optical biolunminescence imaging; electric pulse-mediated gene transfer

The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin. 2,3-butanedione monoximine; gene transfer; adenovirus

Multiple peptide resistance (MprF) virulence factors control cellular permeability to cationic antibiotics by aminoacylating inner membrane lipids. It has been shown previously that one class of MprF can use Lys-t[RNA.sup.Lys] to modify phosphatidylglycerol (PG), but the mechanism of recognition and possible role of other MprFs are unknown. Here, we used an in vitro reconstituted lipid aminoacylation system to investigate the two phylogenetically distinct MprF paralogs (MprF1 and MprF2) found in the bacterial pathogen Clostridium perfringens. Although both forms of MprF aminoacylate PG, they do so with different amino acids; MprF1 is specific for Ala-t[RNA.sup.Ala], and MprF2 utilizes Lys-t[RNA.sup.Lys]. This provides a mechanism by which the cell can fine tune the charge of the inner membrane by using the neutral amino acid alanine, potentially providing resistance to a broader range of antibiotics than offered by lysine modification alone. Mutation of t[RNA.sup.Ala] and t[RNA.sup.Lys] had little effect on either MprF activity, indicating that the aminoacyl moiety is the primary determinant for aminoacyl-tRNA recognition. The lack of discrimination of the tRNA is consistent with the role of MprF as a virulence factor, because species-specific differences in tRNA sequence would not present a barrier to horizontal gene transfer. Taken together, our findings reveal how the MprF proteins provide a potent virulence mechanism by which pathogens can readily acquire resistance to chemically diverse antibiotics. alanine | lysine | tRNA

Abstract: Gene amelioration is the hypothesis that genes acquired via lateral gene transfer will, over time, acquire the molecular characteristics of the host genome. Species for which multiple strains have [...]

We previously reported that mutations in the [mu]-opioid receptor (MOR), S196L or S196A, rendered MOR responsive to the opioid antagonist naloxone without altering the agonist phenotype. Subsequently, a mouse strain carrying the S196A mutation exhibited in vivo naloxone antinociceptive activity without the development of tolerance, in this study we investigated the possibility of combining the in vivo site-directed delivery of MORS196A and systemic naloxone administration as a paradigm for pain management. Double-stranded adenoassociated virus type 2 (dsAAV2) was used to deliver MORS196A-EGFP by injecting the virus into the spinal cord (S2/S3) dorsal horn region of ICR mice. MORS196A-EGFP fluorescence colocalized with some calcitonin gene-related peptide and neuron-specific protein immunoreactivity in the superficial layers of the dorsal horn 1 week after injection and lasted for at least 6 months. In mice injected with the mutant receptor, morphine induced similar antinociceptive responses and tolerance development or precipitated withdrawal symptoms and reward effects, similar to those in the control mice (saline injected into the spinal cord). Conversely, in the dsAAV2-injected mice, naloxone produced antinociceptive effects at the spinal level but not at the supraspinal level, whereas naloxone had no measurable effect on the control mice. Furthermore, the chronic administration of naloxone to mice injected with dsAAV2-MORS196A-EGFP did not induce tolerance, dependence, or reward responses. Thus, our current approach to activate a mutant receptor, but not the endogenous receptor, with an opioid antagonist represents an alternative to the use of traditional opioid agonists for pain management. morphine | [mu]-opioid receptor | naloxone

Gene transfer to vein gratis has therapeutic potential to prevent late graft failure; however, certain issues, including efficacy and safety, have hindered the clinical application of this treatment modality. Here, we report the successful and efficient gene transfer of plasmid DNA via ex vivo electroporation into veins as well as into vein grafts. Two approaches were used: one involved transluminal in situ gene transfer using a T-shaped electrode (the 'Lu' method), and the other was an adventitial ex vivo approach using an electroporation cuvette followed by vein grafting (the 'Ad' method). The Lu method was earned out at 10 V, with optimal gene transfer efficiency in the in situ jugular veins of rabbits, and transgene expression was observed primarily in endothelial cells. However, when these veins were grafted into the arterial circulation, no luciferase activity was detected; this effect was probably due to the elimination of the gene-transferred cells as a result of endothelial denudation. In contrast, optimal and satisfactory gene transfer was obtained with the vein grafts subjected to the Ad method at 30 V, and transgene expression was seen primarily in adventitial fibroblasts. Gene transfer of endothelial nitric oxide synthase cDNA to the vein graft via the Ad method successfully limited the extent of intimal hyperplasia, even under hyperlipidemic conditions, at 4 wk after grafting. We thus propose that the Ad method via ex vivo electroporation may provide a novel, safe, and clinically available technique for nonviral gene transfer to sufficiently prevent late graft failure. endothelial cell nitric oxide synthase; adventitia; endothelial cells; hyperlipidemia; plasmid

Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the vitally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any fluorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology. actin; actin depolymerizing factor; adenovirus; green fluorescent protein; intravital microscopy

Liposomes modified with TAT peptide (TATp-liposomes) showed fast and efficient translocation into the cell cytoplasm with subsequent migration into the perinuclear zone. TATp-liposomes containing a small quantity ([less than or equal to] 10 mol %) of a cationic lipid formed firm noncovalent complexes with DNA. Here, we present results demonstrating both in vitro and in vivo transfection with TATp-liposome--DNA complexes. Mouse NIH/3T3 fibroblasts and rat H9C2 cardiomyocytes were transfected with such complexes in vitro. The transfection with the TATp-liposome-associated pEGFP-N1 plasmid encoding for the green fluorescent protein (GFP) was high, whereas the cytotoxicity was lower than that of commonly used cationic lipid-based gene-delivery systems. Intratumoral injection of TATp-liposome--DNA complexes into the Lewis lung carcinoma tumor of mice also resulted in an expression of GFP in tumor cells. This transfection system should be useful for various protocols of cell treatment in vitro or ex vivo as well as for localized in vivo gene therapy. gene delivery | green fluorescent protein | intracellular trafficking

Resonance-like biological effects of millimeter-wave radiation at frequencies of approximately 42 GHz on the growth rate of E. coli and on DNA have been reported in several scientific publications. In order to explore these nonthermal effects, we have measured the growth rate and the absorption spectrum of E. coli, irradiated by millimeter waves in the frequency range from 41 to 43 GHz. In addition, the effect of this radiation on DNA was studied by measuring plasmid transformation efficiency. Both the growth rate variations with varying frequency and the variations in the result of the plasmid transformation efficiency experiments were found to be statistically insignificant. Resonance-like absorption features observed in the absorption spectrum of E. coli were identified as modes generated in the millimeter-wave system, when the sample was inserted. The experimental results indicate that resonance effects are unlikely in this particular frequency range. Index Terms--Absorption, biological effects, DNA, E. coli, growth rate, low-intensity millimeter waves, transformation efficiency.

Modern treatment of cardiac arrhythmias is limited to pharmacotherapy, radiofrequency ablation, or implantable devices. Antiarrhythmic medications suppress arrhythmias, but their systemic effects are often poorly tolerated and their proarrhythmic tendencies increase mortality. Radiofrequency ablation can cure only a limited number of arrhythmias. Implantable devices can be curative for bradyarrhythmias and lifesaving for tachyarrhythmias, but require a lifetime commitment to repeated procedures, are a significant expense, and may lead to severe complications. One possibility is the use of gene therapy as an antiarrhythmic strategy. As an initial attempt to explore this option, we focused on genetic modification of the atrioventricular node. First, we developed an intracoronary perfusion model for gene delivery, building on our previous work in isolated cardiac myocytes and hearts perfused ex vivo. Using this method, we infected porcine hearts with Ad[beta]gal (recombinant adenovirus expressing Escherichia coli [beta]-galactosidase) or with AdG[sub.i] (adenovirus encoding the G[alpha][sub.i2] subunit). We hypothesized that excess G[alpha][sub.i2] would mimic the effects of [beta]-adreneric antagonists, in effect creating a localized [beta]-blockade. G[alpha][sub.i2] overexpression suppressed baseline atrioventricular conduction and slowed the heart rate during atrial fibrillation without producing complete heart block. In contrast, expression of the reporter gene [beta]-galactosidase had no electrophysiological effects. Our results demonstrate the feasibility of using myocardial gene transfer strategies to treat common arrhythmias., Author(s): J. Kevin Donahue (corresponding author) [1, 2]; Alan W. Heldman [2]; Heather Fraser [1]; Amy D. McDonald [1]; Julie M. Miller [2]; Jeffrey J. Rade [2]; Thomas Eschenhagen [3]; [...]

An Agrobacterium-mediated transformation of Agaricus bisporus facilitating cocultivation of the bacteria and the fruting body gill tissue is described.

Byline: L.H. Keller (1), R. Vander Heide (1), K.A. Pebbles (1), T.J. L'Ecuyer (1) Keywords: Key words: Gene transfer -- Pulmonary artery -- Angioplasty -- Endothelium Abstract: Gene transfer techniques are increasingly being used to study blood vessel biology and develop models for gene therapy. To date, there are no reports of pulmonary vascular gene transfer performed either without adjunctive agents or during angioplasty. We sought to demonstrate the feasibility of recombinant gene transfer to the pulmonary artery of juvenile pigs using naked plasmid DNA delivered via percutaneous angioplasty techniques. Plasmid DNA directing the expression of [beta]-galactosidase was used to transfect one pulmonary artery while the contralateral vessel served as an untreated control. One delivery technique used a standard angioplasty balloon coated with a DNA--heparin mixture. The second involved infusion of DNA between an angioplasty balloon and a surrounding, microporous balloon. Vessels were harvested 3 or 4 days following gene delivery. Protein expression was demonstrated by immunohistochemical staining in transfected but not control vessels in 9/9 pigs. Vascular wall expression was limited to endothelial cells. Pulmonary artery gene transfer using naked plasmid DNA delivered via percutaneous angioplasty techniques is feasible. Using naked plasmid DNA removes the potential for toxicity associated with adjunctive agents. The described techniques provide novel methods for studying pulmonary vascular biology in vivo and for developing future gene therapies. Author Affiliation: (1) Children's Hospital of Michigan, 3901 Beaubien, Detroit, MI 48201-2196, USA, US

Mating Tetrahymena thermophila were bombarded with ribosomal DNA-coated particles at various times in development. Both macronuclear and micronuclear transformants were recovered. Optimal developmental stages for transformation occurred during meiosis for the micronucleus and during anlagen formation for the macronucleus. Evidence is given for transient retention of the introduced plasmid. Genetic and molecular tests confirmed that sexually heritable transformation was associated with integration at the homologous site in the recipient micronuclear chromosome.

A new electrotransformation system helps introduce exogenous DNA into Streptococcus suis type 2 with transformation efficiencies of ten to the seventh power transformants per microgram of plasmid DNA. Vectors that are located on the broad-host-range plasmid pWVO1 duplicate in S. suis type 2. S. suis chromosomal DNA-containing plasmid pBR322 vectors integrate into the chromosome. The integration events are single and double cross-overs, as revealed by Southern hybridization, with a frequency of 15% for the double cross-over events.

The optimum conditions for transforming Kluyveromyces lactis by electroporation were established. Using the Bio-Rad Gene Pulser and Pulse Controller, 10(super 7) transformants per microgram of DNA could be obtained by pretreatment with DTT and carefully selecting pulse time, which depended entirely on sample resistance. Maximum efficiency of transformation could also be assured by loading as many cells and as much plasmid DNA as possible in the cuvette.

A high-efficiency transformation system for Tetrahymena thermophila utilizing electorporation techniques was developed. The procedure involved the the use of conjugating rather than somatic T. thermophila cells during plasmid DNA electroporation. The procedure was used to transform T. thermophila with plasmid DNA containing a paromomycin-resistant rRNA gene. The results showed that the procedure could yield as much as 900 transformants per microgram of DNA.

A comparative genomic study shows that, during evolution, nucleus-containing cells acquired DNA from bacteria primarily by endosymbiosis--the uptake and integration of one cell by another. See Article p.427 Once controversial, [...]

The gene transfer routes for integration of single copy transgenes into tissues by infection or electroporation are evaluated. Fluorescence in situ hybridization technique indicates that a consistent, expression level-independent association potential is imparted to nuclear matrix by the retroviral infectants, whereas electroporated proviral inserts mostly localize to the loop fraction.

July 12 In a step toward developing safer vaccines incapable of spreading to most of the body, researchers transferred genes associated with DNA repair and cell division from cold-loving, Arctic-dwelling [...]