scholarly journals The transmembrane domain of diphtheria toxin improves molecular conjugate gene transfer

1997 ◽  
Vol 321 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Krishna J. FISHER ◽  
James M. WILSON
Keyword(s):  
Gene Transfer ◽  
Diphtheria Toxin ◽  
Transmembrane Domain ◽  
Transfection Efficiency ◽  
Bacterial Protein ◽  
Protein Fusion ◽  
Membrane Perturbation ◽  
Membrane Bound ◽  
Rna Transcript

Vectors based on the formation of a soluble DNA–polycation complex are being developed for the treatment of human diseases. These complexes are rapidly taken up by receptor-mediated endocytosis, but are inefficiently delivered to the nucleus owing to entrapment in membrane-bound vesicles. In this study we introduced the transmembrane domain of diphtheria toxin into a DNA–polycation conjugate complex in an effort to increase gene transfer by membrane perturbation. The transmembrane domain of diphtheria toxin was expressed in Escherichia coli as a maltose-binding protein fusion and chemically coupled to high-molecular-mass poly-l-lysine. Incorporation of this conjugate into a traditional complex formed with a luciferase-containing plasmid with an asialo-orosomucoid–polycation conjugate significantly increased transfection efficiency in vitro in a manner proportional to the amount of diphtheria toxin incorporated. The delivery of luciferase RNA transcript was similarly increased when complexed with similar polycation conjugates. This study uses the structural biology of a bacterial protein to improve polycation-based gene delivery.

404 DNA TAKE-UP BY SPERM AND IN VITRO EMBRYO DEVELOPMENT BY SPERM-MEDIATED GENE TRANSFER IN THE PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 317
Author(s):  
T. S. Kim ◽  
Y. Cao ◽  
H. T. Cheong ◽  
B. K. Yang ◽  
C. K. Park
Keyword(s):  
Gene Transfer ◽  
Transfection Efficiency ◽  
The Other ◽  
Control Group ◽  
Dna Uptake ◽  
Alkaline Lysis ◽  
Exogenous Dna ◽  
Other Hand ◽  
Dna Complex

Sperm mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind and internalize exogenous DNA and transfer it into the oocytes at fertilization. The purpose of this study was to assess introducing exogenous DNA into boar spermatozoa by DNA solution or DNA/liposome complex under different conditions (period of incubation, exogenous DNA, liposome, and concentration of spermatozoa). Genomic DNA of sperm loaded with DNA by treatment was isolated by alkaline lysis. Quantitation of exogenous DNA amplified by PCR was analyzed by agarose electrophoresis densitometry. The quality of treated spermatozoa under the best conditions or no treatment (control) was evaluated during incubation (0, 2, 4, and 6 h) for viability (SYBR-14/PI), motility (Makler counting chamber), morphology (rose bengal staining), and acrosomal status (Coomassie staining). Sperm loaded with DNA also were used for in vitro fertilization. Immature oocytes incubated in TCM-199 medium for 44 h were fertilized in mTBM medium for 6 h and cultured in PZM-3. Cleavage and development of embryos were assessed on Days 2 and 7 of culture, respectively. Transfection rates at the blastocyst stage were assessed by PCR analysis. Data were evaluated by Duncan's multiple-range test using the GLM procedure. In the preliminary experiment, DNA uptake of spermatozoa by DNA solution and liposome/DNA complex was completed within 90-120 min. Transfection efficiency of spermatozoa was significantly (P < 0.05) higher in the 105 spermatozoa group than in the other groups (104, 106, and 107 spermatozoa). The transfection efficiency was gradually increased by increasing the concentration of exogenous DNA. On the other hand, viability of transfected spermatozoa by all treatments (control, DNA solution, and DNA/liposome) at 0 h (72.3 � 0.2, 70.8 � 1.8, and 68.0 � 2.2%, respectively) of storage was significantly (P < 0.05) lower than for fresh spermatozoa (83.3 � 1.7%). Survival and motility of all treatments after 4 h of storage were significantly (P < 0.05) lower than at 0 and 2 h. Both abnormality and acrosome reaction of spermatozoa were gradually increased with prolonged storage periods. On the other hand, the cleavage rate of embryos by DNA/liposome complex (56.3 � 2.3%) was significantly (P < 0.05) lower compared to both DNA solution (64.0 � 1.1%) and control (67.8 � 2.3%). The developmental rates of blastocysts were significantly (P < 0.05) lower in the liposome/DNA complex and DNA solution groups (9.1 � 1.3 and 11.3 � 0.8%) than in the control group (22.2 � 0.6%). The transfection rates of blastocysts were higher in the liposome/DNA group (54.3 � 12.0%) than in the DNA solution group (38.7 � 6.6%). These results show that the SMGT method under the control conditions efficiently transfers exogenous DNA into the porcine oocytes. This work was supported by the Research on the Production of Bio-organs (No. 2005 03020302) Ministry of Agriculture and Forestry, Republic of Korea

scholarly journals In Vitro Gene Delivery Mediated by Asialofetuin-Appended Cationic Liposomes Associated with γ-Cyclodextrin into Hepatocytes

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Keiichi Motoyama ◽  
Yoshihiro Nakashima ◽  
Yukihiko Aramaki ◽  
Fumitoshi Hirayama ◽  
Kaneto Uekama ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Gene Delivery ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Cationic Liposomes ◽  
Nonviral Vector ◽  
Liposomal Membrane ◽  
Transfer Activity ◽  
Potential Use

The purpose of this study is to evaluate in vitro gene delivery mediated by asialofetuin-appended cationic liposomes (AF-liposomes) associating cyclodextrins (CyD/AF-liposomes) as a hepatocyte-selective nonviral vector. Of various CyDs, AF-liposomes associated with plasmid DNA (pDNA) and γ-cyclodextrin (γ-CyD) (pDNA/γ-CyD/AF-liposomes) showed the highest gene transfer activity in HepG2 cells without any significant cytotoxicity. In addition, γ-CyD enhanced the encapsulation ratio of pDNA with AF-liposomes, and also increased gene transfer activity as the entrapment ratio of pDNA into AF-liposomes was increased. γ-CyD stabilized the liposomal membrane of AF-liposomes and inhibited the release of calcein from AF-liposomes. The stabilizing effect of γ-CyD may be, at least in part, involved in the enhancing gene transfer activity of pDNA/γ-CyD/AF-liposomes. Therefore, these results suggest the potential use of γ-CyD for an enhancer of transfection efficiency of AF-liposomes.

Liposome-Mediated Gene Transfer into Established CNS Cell Lines, Primary Glial Cells, and in Vivo

1998 ◽  
Vol 7 (2) ◽  
pp. 175-185 ◽  
Author(s):  
Patrick Kofler ◽  
Bettina Wiesenhofer ◽  
Christine Rehrl ◽  
Gottfried Baier ◽  
Günter Stockhammer ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Glial Cells ◽  
Transfection Efficiency ◽  
Primary Cultures ◽  
C6 Glioma Cells ◽  
C6 Cells ◽  
Adult Rats ◽  
Neuronal Precursor Cells

Sufficient gene transfer into CNS-derived cells is the most crucial step to develop strategies for gene therapy. In this study liposome-mediated gene transfer using a β-galactosidase (β-GAL) reporter gene was performed in vitro (C6 glioma cells, NT2 neuronal precursor cells, 3T3 fibroblasts, primary glial cells) and in vivo. Using Trypan blue exclusion staining, optimal lipid concentration was observed in the range of 10-12 μg/mL. Under optimal conditions (80,000 cells/16 mm well, incubation overnight, lipid/DNA ratio = 1:18) a high transfection rate was achieved (<9% for C6 cells; <1% for NT2 cells). In primary cultures of glial cells a fair amount of positive stained cells (glial cell) was found, but the transfection efficiency was lower (<0.1%). A “boost-lipofection” markedly increased (twice) lipofection efficiency in C6 cells. Expression of β-GAL reached a maximum after 3-5 days. When the liposome–DNA complexes were injected/infused directly into the brains of adult rats, several weakly stained cells could be observed in the brain region adjacent to the injection site. It is concluded that liposome-mediated gene transfer is an efficient method for gene transfer into CNS cells in vitro, but the transfection efficiency into the rat brain in vivo is far too low and therefore not applicable.

Folate Receptor-Mediated Cancer Cell Specific Gene Delivery Using Folic Acid-Conjugated Oligochitosans

2006 ◽  
Vol 6 (9) ◽  
pp. 2860-2866 ◽  
Author(s):  
Dongwon Lee ◽  
Richard Lockey ◽  
Shyam Mohapatra
Keyword(s):  
Folic Acid ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Cancer Cells ◽  
Transfection Efficiency ◽  
Folate Receptor ◽  
Weight Ratio ◽  
Specific Gene ◽  
Transfer Potential

Chitosan-mediated gene delivery has gained an increasing interest due to its ability to treat cancers and genetic diseases. However, low transfection efficiency and lack of target specificity limit its application for gene and drug delivery. In the present work, folic acid was covalently conjugated to chitosan as a targeting ligand in an attempt to specifically deliver DNA to folate receptor-overexpressing cancer cells. Folic acid-conjugated chitosan (FACN) was successfully synthesized and characterized by 1H-NMR and is biocompatible. In vitro gene transfer potential of FACN was evaluated in human epithelial ovarian cancer OV2008 cells and human breast cancer MCF-7 cells. FACN at a weight ratio of 10 : 1 exhibited significantly (< 0.01) enhanced gene transfer potential in folate receptor-overexpressing cancer cells as compared to unmodified chitosan. Transfection of FACN/pDNA nanocomplexes is competitively inhibited by free folic acid, suggesting the specific gene delivery of FACN/pDNA nanocomplexes is achieved through folate receptor-mediated endocytosis. Taken together, these results demonstrate that FACN provides a promising carrier for cancer gene therapy.

scholarly journals Biological Characteristics and Carrier Functions of Pegylated Manganese Zinc Ferrite Nanoparticles

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Ting Guo ◽  
Fengxiao Dou ◽  
Mei Lin ◽  
Junxing Huang ◽  
Chenglin Zhou ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Zinc Ferrite ◽  
Transfection Efficiency ◽  
Ferrite Nanoparticles ◽  
Biological Characteristics ◽  
Manganese Zinc ◽  
Manganese Zinc Ferrite ◽  
Therapeutic Study

This study was performed to investigate the biocompatibility (BC), magnetothermal effect, and DNA binding biological characteristics of manganese zinc ferrite nanoparticles (Mn0.6Zn0.4Fe2O4-NPs (MZF-NPs)) coated with pegylated manganese (PEG-MZF-NPs). Their functions as gene transfer carrier for gene therapy and magnetic medium for tumor hyperthermia were also explored. The manganese zinc ferrite nanoparticles were synthesized through high temperature cracking, and their characterizations were discovered. Hemolysis test and MTT assay were performed to evaluate biocompatibility, and their self-heating effects in the alternating magnetic field were investigated. PEG-MZF-NPs with different concentrations were measured by using 7.0 T Micro-MR scanner (MRI) to calculate the T2 value and r2 relaxation rate of each sample. The CD44-shRNA plasmids were constructed, and their ability to bind PEG-MZF-NPs were examined. The DNA release from PEG-MZF-NP/DNA complex and protection of DNA from nuclease digestion were also detected. After CD44-shRNA-EGFP were transfected into the ovarian cancer SK-OV-3 cells by using PEG-MZF-NPs as carriers, the transfection efficiency was detected by a flow cytometer and expression of CD44 mRNA and protein in cells was detected using RT-PCR and Western blot, respectively. We successfully prepared PEG-MZF-NPs with favorable dispersity, magnetic responsiveness, and BC. Typically, the excellent magnetothermal effect can be used for a tumor magnetothermal therapeutic study. In vitro MRI showed the application potential for being magnetic resonance T2 relaxation contrast agents and the possibility to achieve goal of integration of targeting diagnosis and treatment. The CD44-shRNA plasmids have been successfully constructed and concluded that PEG-MZF-NPs may serve as gene transfer carriers for gene therapy.

Lentiviral vector transfection of chicken embryo primordial germ cells in vitro

2008 ◽  
Vol 5 (1) ◽  
pp. 1-5
Author(s):  
Ding Hong-Mei ◽  
Xu Shi-Yong ◽  
Shao Gen-Bao ◽  
Sun Yan ◽  
Wang Meng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Germ Cells ◽  
Lentiviral Vector ◽  
Transfection Efficiency ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Germline Stem Cells ◽  
Transfer Vectors

AbstractLentiviruses as gene transfer vectors have been used successfully to transfect mammal embryonic stem cells and germline stem cells, but this has not been attempted in avian primordial germ cells (PGCs). PGCs were isolated from the gonads of Isa-brown chicken embryos at stage 28 and co-cultured with gonadal stroma cells. A lentiviral vector pLenti-CMV-EGFP was constructed and the virus harvested by cotransfecting 293FT cells with the vector and packaging plasmids. Concentrated lentiviruses were used to transfect chicken PGCs, the transfection efficiency was up to 24.19%.

Functional Amphiphiles for Gene Delivery

MRS Bulletin ◽  
2005 ◽  
Vol 30 (9) ◽  
pp. 647-653 ◽  
Author(s):  
Philippe Barthélémy ◽  
Michel Camplo
Keyword(s):  
Gene Transfer ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Research Activity ◽  
Safety Issues ◽  
Significant Research

AbstractThe design of safe and efficient gene transfer vectors remains one of the key challenges in gene therapy. Despite their remarkable transfection efficiency, viral vectors suffer from known safety issues. Consequently, significant research activity has been undertaken to develop nonviral approaches to gene transfer during the last decade. Numerous academic and industrial research groups are investigating synthetic cationic vectors, such as cationic amphiphiles, with the objective of increasing the gene transfection activity. Within this area, the development of functional synthetic vectors that respond to local environmental effects have met with success. These synthetic vectors are based on mechanistic principles and represent a significant departure from earlier systems. Many of these systems for gene delivery in vitro and in vivo are discussed in this article.

scholarly journals Regulated cleavage of glycan strands by the murein hydrolase SagB in S. aureus involves a direct interaction with LyrA (SpdC)

2021 ◽  
Author(s):  
Stephanie Willing ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Protein Trafficking ◽  
Transmembrane Domain ◽  
Protein A ◽  
Direct Interaction ◽  
Staphylococcal Protein A ◽  
Lipid Ii ◽  
Molecular Ruler ◽  
Membrane Bound ◽  
Insight Into

LyrA (SpdC), a homologue of eukaryotic CAAX proteases that act on prenylated substrates, has been implicated in the assembly of several pathways of the envelope of Staphylococcus aureus. We described earlier the Lysostaphin resistance (Lyr) and Staphylococcal protein A display (Spd) phenotypes associated with loss of the lyrA (spdC) gene. However, a direct contribution to the assembly of pentaglycine crossbridges, the target of lysostaphin cleavage in S. aureus peptidoglycan, or of Staphylococcal protein A attachment to peptidoglycan could not be attributed directly to LyrA (SpdC). These two processes are catalyzed by the Fem factors and Sortase A, respectively. To gain insight into the function of LyrA (SpdC), here we use affinity chromatography and LC-MS/MS analysis and report that LyrA interacts with SagB. SagB cleaves glycan strands of peptidoglycan to achieve physiological length. Similar to sagB peptidoglycan, lyrA peptidoglycan contains extended glycan strands. Purified lyrA peptidoglycan can still be cleaved to physiological length by SagB in vitro. LyrA does not modify or cleave peptidoglycan, it also does not modify or stabilize SagB. The membrane bound domain of LyrA is sufficient to support SagB activity but predicted ‘CAAX enzyme’ catalytic residues in this domain are dispensable. We speculate that LyrA exerts its effect on bacterial prenyl substrates, specifically undecaprenol-bound peptidoglycan substrates of SagB, to help control glycan length. Such an activity also explains the Lyr and Spd phenotypes observed earlier. IMPORTANCE Peptidoglycan is assembled on the trans side of the plasma membrane from lipid II precursors into glycan chains that are crosslinked at stem peptides. In S. aureus, SagB, a membrane-associated N-acetylglucosaminidase, cleaves polymerized glycan chains to their physiological length. Deletion of sagB is associated with longer glycan strands in peptidoglycan, altered protein trafficking and secretion in the envelope, and aberrant excretion of cytosolic proteins. It is not clear whether SagB, with its single transmembrane segment, serves as the molecular ruler of glycan chains or whether other factors modulate its activity. Here, we show that LyrA (SpdC), a protein of the CAAX type II prenyl endopeptidase family, modulates SagB activity via interaction though its transmembrane domain.

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