In vivo gene introduction into keratinocytes using jet injection.
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Successful keratinocyte gene therapy requires the development of efficient methods of gene transfer to keratinocytes. Jet injection of a solution containing DNA can be used to transfer genes to several tissues in vivo. In this article, we tried to introduce DNA into rat and human keratinocytes using this method. First, we fired a beta-gal expression vector into rat skin at several distances using a jet injector and examined beta-gal activity in the epidermal keratinocytes. The highest activity in keratinocytes was found when the plasmid was fired at 10 cm from the skin surface; the activity lessened as the firing distance became shorter than 10 cm. Next, we transplanted human skin on to a nude rat, fired the vector into the human skin from a distance of 10 cm and examined the beta-gal activity. We also injected the same amount of plasmid with a needle to compare jet with needle injections. The results showed that jet injection of the naked DNA could introduce and express DNA in human keratinocytes in vivo and that jet injection exhibited much higher activity than needle injection. Jet injection of the naked DNA will provide a method for keratinocyte gene therapy in the future. (+info)
The active digestion of uniparental chloroplast DNA in a single zygote of Chlamydomonas reinhardtii is revealed by using the optical tweezer.
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The non-Mendelian inheritance of organelle genes is a phenomenon common to almost all eukaryotes, and in the isogamous alga Chlamydomonas reinhardtii, chloroplast (cp) genes are transmitted from the mating type positive (mt(+)) parent. In this study, the preferential disappearance of the fluorescent cp nucleoids of the mating type negative (mt(-)) parent was observed in living young zygotes. To study the change in cpDNA molecules during the preferential disappearance, the cpDNA of mt(+) or mt(-) origin was labeled separately with bacterial aadA gene sequences. Then, a single zygote with or without cp nucleoids was isolated under direct observation by using optical tweezers and investigated by nested PCR analysis of the aadA sequences. This demonstrated that cpDNA molecules are digested completely during the preferential disappearance of mt(-) cp nucleoids within 10 min, whereas mt(+) cpDNA and mitochondrial DNA are protected from the digestion. These results indicate that the non-Mendelian transmission pattern of organelle genes is determined immediately after zygote formation. (+info)
BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1.
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This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVDeltapac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVDeltapac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVDeltapac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVDeltapac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVDeltapac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD(50). Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens. (+info)
Evaluation of tick-borne encephalitis DNA vaccines in monkeys.
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Tick-borne encephalitis is usually caused by infection with one of two flaviviruses: Russian spring summer encephalitis virus (RSSEV) or Central European encephalitis virus (CEEV). We previously demonstrated that gene gun inoculation of mice with naked DNA vaccines expressing the prM and E genes of these viruses resulted in long-lived homologous and heterologous protective immunity (Schmaljohn et al., 1997). To further evaluate these vaccines, we inoculated rhesus macaques by gene gun with the RSSEV or CEEV vaccines or with both DNA vaccines and compared resulting antibody titers with those obtained by vaccination with a commercial, formalin-inactivated vaccine administered at the human dose. Vaccinations were given at days 0, 30, and 70. All of the vaccines elicited antibodies detected by ELISA and by plaque-reduction neutralization tests. The neutralizing antibody responses persisted for at least 15 weeks after the final vaccination. Because monkeys are not uniformly susceptible to tick-borne encephalitis, the protective properties of the vaccines were assessed by passive transfer of monkey sera to mice and subsequent challenge of the mice with RSSEV or CEEV. One hour after transfer, mice that received 50 microl of sera from monkeys vaccinated with both DNA vaccines had circulating neutralizing antibody levels <20-80. All of these mice were protected from challenge with RSSEV or CEEV. Mice that received 10 microl of sera from monkeys vaccinated with the individual DNA vaccines, both DNA vaccines, or a commercial vaccine were partially to completely protected from RSSEV or CEEV challenge. These data suggest that DNA vaccines may offer protective immunity to primates similar to that obtained with a commercial inactivated-virus vaccine. (+info)
Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1beta converting enzyme cDNA.
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We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1beta converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-gamma. First, we compared the levels of IFN-gamma secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-gamma production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-gamma levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8(+) T cell-mediated and partially IFN-gamma-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8(+) T cells and IFN-gamma-dependent pathways. (+info)
A DNA immunization model study with constructs expressing the tick-borne encephalitis virus envelope protein E in different physical forms.
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We have conducted a DNA immunization study to evaluate how the immune response is influenced by the physical structure and secretion of the expressed Ag. For this purpose, we used a series of plasmid constructs encoding different forms of the envelope glycoprotein E of the flavivirus tick-borne encephalitis virus. These included a secreted recombinant subviral particle, a secreted carboxyl-terminally truncated soluble homodimer, a nonsecreted full-length form, and an inefficiently secreted truncated form. Mice were immunized using both i.m. injection and Gene Gun-mediated application of plasmids. The functional immune response was evaluated by determining specific neutralizing and hemagglutination-inhibiting Ab activities and by challenging the mice with a lethal dose of the virus. As a measure for the induction of a Th1 and/or Th2 response, we determined specific IgG subclasses and examined IFN-gamma, Il-4, and Il-5 induction. The plasmid construct encoding a secreted subviral particle, which carries multiple copies of the protective Ag on its surface, was superior to the other constructs in terms of extent and functionality of the Ab response as well as protection against virus challenge. As expected, the type of Th response was largely dependent on the mode of application (i.m. vs Gene Gun), but our data show that it was also strongly influenced by the properties of the Ag. Most significantly, the plasmid encoding the particulate form was able to partially overcome the Th2 bias imposed by the Gene Gun, resulting in a balanced Th1/Th2 response. (+info)
Particle-mediated DNA immunization of cattle confers long-lasting immunity against bovine herpesvirus-1.
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Particle-mediated delivery was used as a method to vaccinate ruminants with a DNA vaccine. The optimal conditions for gene gun-based delivery of gold particles into the epidermal layer of the skin were determined. After delivery of the gold particles, an inflammatory response was observed. This response occurred regardless of the presence of plasmid and therefore was a result of the physical disturbance of the skin by the gold particles. To identify transfected cells, a plasmid expressing a green fluorescent protein was delivered into the skin. Fluorescent cells were located primarily in the outermost layers of the epidermis and outside the core of gold particles deposited by the gene gun. Cattle were immunized by gene gun with a plasmid expressing a truncated, secreted form of bovine herpesvirus-1 glycoprotein D. Serum antibody responses, antigen-specific proliferation, and interferon-gamma secretion by peripheral blood lymphocytes were demonstrated. These immune responses were found to be of long duration and sufficient magnitude to protect cattle against challenge with bovine herpesvirus-1, which demonstrates the efficacy of gene gun-based delivery of DNA vaccines to target species. (+info)
Regulation of abscisic acid-induced stomatal closure and anion channels by guard cell AAPK kinase.
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Abscisic acid (ABA) stimulates stomatal closure and thus supports water conservation by plants during drought. Mass spectrometry-generated peptide sequence information was used to clone a Vicia faba complementary DNA, AAPK, encoding a guard cell-specific ABA-activated serine-threonine protein kinase (AAPK). Expression in transformed guard cells of AAPK altered by one amino acid (lysine 43 to alanine 43) renders stomata insensitive to ABA-induced closure by eliminating ABA activation of plasma membrane anion channels. This information should allow cell-specific, targeted biotechnological manipulation of crop water status. (+info)