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DNA Constructs (dna + construct)

Distribution by Scientific Domains

Life Sciences	50%
Medical Sciences	30%

Selected Abstracts

Ultrathin Multilayered Films that Promote the Release of Two DNA Constructs with Separate and Distinct Release Profiles,

ADVANCED MATERIALS, Issue 21 2008
Xianghui Liu
,Charge-shifting' cationic polymers are used to fabricate ultrathin multilayered films that promote the release of two different DNA constructs with separate and essentially nonoverlapping release profiles. This approach could contribute to the development of thin films and functional coatings capable of regulating the localized release of different DNA constructs (or other agents) to cells or tissues in a broad range of fundamental and applied contexts. [source]

GANP suppresses DNA recombination, measured by direct-repeat ,-galactosidase gene construct, but does not suppress the type of recombination applying to immunoglobulin genes in mammalian cells

GENES TO CELLS, Issue 10 2007
Mikoto Yoshida
Immunoglobulin V-region somatic hypermutation and C-region class-switch recombination are initiated by activation-induced cytidine deaminase (AID) in B-cells. AID-induced DNA damage at the immunoglobulin S-region is known to be repaired by non-homologous end-joining, but repair mechanisms at the V-region remain to be elucidated. In Saccharomyces cerevisiae, DNA homologous recombination is regulated by the expression of Sac3, involved in actin assembly, cell cycle transition and mRNA metabolism. Here, we demonstrate that the Sac3-homologue GANP suppresses DNA recombination in a direct-repeat ,-galactosidase gene construct in mammalian cells. Homozygous ganp gene knockout is embryonic lethal in mice. Embryonic fibroblasts immortalized from hetero-deficient ganp+/, mice showed more DNA recombination than wild-type. In contrast, over-expression of GANP suppressed either spontaneous DNA recombination or that caused by the introduction of aid cDNA into NIH3T3 cells (susceptible to I-sceI restriction enzyme cleavage but not to RAG-mediated immunoglobulin gene recombination). GANP suppresses the DNA recombination not only on the extrachromosomal DNA construct but also on the integrated DNA. The Sac3-homology portion is necessary for the suppressive activity, but the truncated carboxyl terminal MCM3-binding/acetylating region adversely augmented DNA recombination, acting as a dominant negative form. Expression of full-length GANP is critical for suppression of DNA hyper-recombination in mammalian cells. [source]

Induction of a protective capsular polysaccharide antibody response to a multiepitope DNA vaccine encoding a peptide mimic of meningococcal serogroup C capsular polysaccharide

IMMUNOLOGY, Issue 2 2003
Deborah M. Prinz
Summary Systemic infection by encapsulated organisms, such as Neisseria meningitidis, is a major cause of morbidity and mortality worldwide, especially in individuals less than 2 years of age. Antibodies directed at the capsular polysaccharide are shown to be protective against disease by inducing complement-dependent bactericidal activity. The current polysaccharide vaccine has been shown to be poorly immunogenic in high-risk groups and this is probably related to its T-independent properties. An alternative approach to eliciting a T-dependent serum immunoglobulin G (IgG) antibody response to encapsulated pathogens is DNA vaccination. We assessed the immunogenicity of a multiepitope DNA vaccine encoding a T-cell helper epitope and a peptide mimic of N. meningitidis serogroup C. The DNA construct induced a significant anti-polysaccharide antibody response that was bactericidal. Mice immunized with the DNA construct were subsequently protected against challenge with a lethal dose of N. meningitidis serogroup C. [source]

Transgenic mice replicating hepatitis B virus but lacking expression of the major HBsAg,

JOURNAL OF MEDICAL VIROLOGY, Issue 4 2008
Leonie Halverscheid
Abstract Hepatitis B Virus (HBV) transgenic mice replicating the viral genome at high level but lacking expression of the small envelope protein (HBsAg) have been produced using a terminally redundant viral DNA construct (HBV 1.4). The generation of viable infectious progeny was dependent on sex and age of mice. Viral mRNA was abundant in liver and kidneys and at low levels in other organs of the mice. No viral particles or HBV envelope proteins could be detected in sera of mice. Despite expression of non-secreted LHBs and MHBs proteins in the liver, there was no accumulation of viral particles in the endoplasmic reticulum of hepatocytes and no necroinflammatory hepatitis was observed. Therefore, these mice represent an excellent model for studies of the role of HBsAg in viral assembly, antiviral immune responses, the further understanding of HBV immunopathogenesis, and the development of antiviral vaccines. J. Med. Virol. 80:583,590, 2008. © 2008 Wiley-Liss, Inc. [source]

Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo

THE JOURNAL OF GENE MEDICINE, Issue 6 2006
Laurence Vaysse
Abstract Background To develop more efficient non-viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR-NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV-1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle. Methods We have produced a new LacZ minicircle compatible with the TetR system. After transfection of the minicircle in combination with TetR-NLS or TetR-TAT using different transfection agents, we first measured ,-galactosidase activity in vitro. We then used a special delivery technique, in which DOTAP/cholesterol liposomes and DNA/protein complexes are sequentially injected intravenously, to evaluate the activity of this system in vivo. Results In vitro results showed a 30-fold increase in transfection efficiency of the nuclear-targeted minicircle compared to normal plasmid lipofection. Results on cell cycle arrested cells seem to indicate a different mechanism between the TetR-NLS and TetR-TAT. Finally, we demonstrate a more than 6-fold increase in ,-galactosidase expression in the mouse lung using the minicircle and the TetR-TAT protein. This increase is specific for the peptide sequence and is not observed with the control protein TetR. Conclusions Our results indicate that the combination of a minicircle DNA construct with a TetR nuclear-targeting system is able to potentiate gene expression of non-viral vectors. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Direct DNA delivery into zebrafish embryos employing tissue culture techniques

GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 1 2001
Raquel Sussman
Abstract Summary: The production of transfected fish embryos requires expertise in injecting the fertilized eggs and/or expensive equipment for electroporation or microprojectiles. This article demonstrates that by exposure to DNA constructs conjugated with transfecting reagents dechorionated Danio rerio embryos are capable of acquiring extracellular DNA and expressing reporter genes. Embryos incubated with pCMVluc complexed with GeneJammer or GenePORTER expressed luciferase 24,48 h after exposure. pCMVGFP DNA mixed with the same agents generated embryos that exhibited differential patterns of expression of green fluorescent protein (GFP). Embryonic development varied depending on the procedure employed and the reporter gene utilized. Expression of the luciferase gene did not interfere with the subsequent development of the embryos. In contrast, the embryos expressing a high level of GFP were affected, probably due to a very active promoter. These results demonstrate the ease of obtaining transfected fish embryos, which facilitate the mass production of new genotypes and extend the procedure to laboratories with limited resources. genesis 31:1,5, 2001. © 2001 Wiley-Liss, Inc. [source]

Hepatitis B virus precore protein augments genetic immunizations of the truncated hepatitis C virus core in BALB/c mice,

HEPATOLOGY, Issue 1 2008
Guoyang Liao
DNA immunization has been used to induce either humoral or cellular immune responses against many antigens, including hepatitis C virus (HCV). In addition, DNA immunizations can be enhanced or modulated at the nucleotide level. Genetic immunizations were examined in BALB/c mice through the use of plasmids and chimeric DNA constructs encoding HCV core proteins and hepatitis B virus (HBV) precore (preC) regions. Plasmids encoding the truncated HCV core induced potent humoral and cellular responses to HCV; pcDNA3.0A-C154 produced a stronger antibody response than pcDNA3.0A-C191 (P < 0.01) and pcDNA3.0A-C69 (P < 0.05). HBV preC enhanced the humoral and cellular immune responses of BALB/c mice to HCV; however, pcDNA3.0A-C69preC resulted in a weak cytotoxic T lymphocyte (CTL) response. In addition, the humoral and cellular immune responses to HCV of groups immunized with pcDNA3.0A-C154preC and pcDNA3.0A-C191preC plasmids were higher than those of groups immunized with pcDNA3.0A-C154 and pcDNA3.0A-C191. In vivo CTL responses verified that mice immunized with preC core fused DNAs showed significantly high specific lysis compared with mice immunized with HCV cores only (P < 0.01). In our study, pcDNA3.0A-C154preC led to the highest immune response among all DNA constructs. Conclusion: DNA that encodes truncated HCV core proteins may lead to increased immune responses in vivo, and these responses may be enhanced by HBV preC. (HEPATOLOGY 2007.) [source]

Ultrathin Multilayered Films that Promote the Release of Two DNA Constructs with Separate and Distinct Release Profiles,

Comparative efficacy of the Schistosoma mansoni nucleic acid vaccine, Sm23, following microseeding or gene gun delivery

PARASITE IMMUNOLOGY, Issue 4 2002
Akram A. Da'dara
Summary Sm23 is an integral membrane protein expressed widely in the human parasitic worm Schistosoma mansoni. Sm23 has already been shown to elicit protective immune responses following immunization with peptides or DNA constructs. In this study, we evaluated the immunogenicity and the protective efficacy of the Sm23 DNA vaccine using two different intradermal DNA delivery methods: microseeding and gene gun. Using both techniques, all mice immunized with the Sm23-pcDNA construct generated Sm23-specific immunoglobulin (Ig)G antibody, while mice immunized with the control plasmid, pcDNA, did not. Antibody isotypes analysis revealed that microseeding elicited mainly IgG2a and IgG2b antibodies, with relatively low levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio was 0·03, indicative of a Th1 type immune response. In contrast, gene gun immunization resulted in significantly higher levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio in this case was 11, indicative of a Th2 type immune response. No significant difference in the levels of IgG2b was observed. Coimmunization with plasmid DNA encoding either interleukin (IL)-12 or IL-4 by microseeding did not affect the levels of IgG1, while the levels of IgG2a and IgG2b were reduced. On the other hand, the levels of IgG3 were significantly increased by IL-4, but unchanged by IL-12. Importantly, in all experiments, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection. Microseeding immunizations resulted in higher levels of protection (31,34% protection) than gene gun immunization (18% protection). This suggests that the Th1 type immune response elicited by microseeding immunization was responsible for the higher protection levels. However, the protective effect of the vaccine was not affected by coadministering plasmids encoding either IL-12 or IL-4 using the microseeding technique. [source]

Revealing frequent alternative polyadenylation and widespread low-level transcription read-through of novel plant transcription terminators

PLANT BIOTECHNOLOGY JOURNAL, Issue 7 2010
Aiqiu Xing
Summary Plant genetic engineering can create transgenic crops with improved characteristics by introducing trait genes through transformation. Appropriate regulatory elements such as promoters and terminators have to be present in certain configurations for the transgenes to be properly expressed. Five terminators native to soybean genes-encoding a MYB family transcription factor (MYB2), a Kunitz trypsin inhibitor (KTI1), a plasma membrane intrinsic protein (PIP1), a translation elongation factor (EF1A2) and a metallothionein protein (MTH1) were cloned and tested for their ability to enable transgene expression, mRNA polyadenylation and transcription termination. The terminators are as good as a control terminator of the potato proteinase inhibitor II gene (PINII) in conferring proper transgene expression, leading to mRNAs with various polyadenylation sites and terminating mRNA transcripts. RNA transcription read-through was detected in all transgenic plants and was quantified by qRT-PCR to be <1% at positions ,1 kb downstream of the 5, ends of different terminators. The detection of read-through RNA transcripts of the corresponding endogenous genes up to approximately 1 kb beyond the polyadenylation sites suggests that limited RNA transcription read-through is a normal phenomenon of gene expression. The study also provided more choices of terminators for plant genetic engineering when constructing DNA constructs containing multiple gene expression cassettes. [source]