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Saghir Akhtar - One of the best experts on this subject based on the ideXlab platform.

  • Toxicogenomics of non-viral vectors for gene therapy: a microarray study of Lipofectin- and oligofectamine-induced gene expression changes in human epithelial cells.
    Journal of Drug Targeting, 2003
    Co-Authors: Yadollah Omidi, Andrew John Hollins, Mustapha Benboubetra, Ross M. Drayton, Ibrahim F. Benter, Saghir Akhtar
    Abstract:

    Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturer's recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.

  • A c‐myc Phosphorothioate Oligonucleotide Protects U87‐MG Glioblastoma Cells from Toxicity at High Concentrations of Lipofectin and Streptolysin O
    Pharmacy and Pharmacology Communications, 1997
    Co-Authors: Judy M. Coulson, Saghir Akhtar
    Abstract:

    The pharmacological activity of an antisense oligodeoxynucleotide (ODN) targeted to c-myc (15mycAS) was evaluated in the glioblastoma-derived cell line U87-MG. ODNs with both phosphodiester (PO) and phosphorothioate (PS) backbones, and with 3′ hairpin sequences, were used with Lipofectin and streptolysin O as transfection reagents. At non-toxic concentrations of Lipofectin, cellular association was low and no evidence of an antisense mechanism was seen on evaluation of cell proliferation or c-Myc protein expression. At a high Lipofectin concentration (25 μg mL−1), which resulted in loss of more than 50% cell viability, ODN uptake was enhanced, but no reduction in c-Myc protein expression was observed with any of the 15mycAS ODNs. However, treatment with the antisense PS ODN (15mycAS PS), specifically resulted in protection of the viable cell number. This cytoprotective effect was concentration- and chemistry-dependent. Three 15mer control PS ODNs, the PO form of 15mycAS, and the hairpin stabilized PO and PS forms (15mycASHP2 PO and 15mycASHP2 PS, respectively) did not show cytoprotection. A similar protective effect of 15mycAS PS was also seen with the transfection agent streptolysin O. These data demonstrate a potential source of misinterpretation in antisense experiments and emphasize the need to consider possible interactions of ODNs with lipids or cell membranes in the design of effective antisense strategies.

  • a c myc phosphorothioate oligonucleotide protects u87 mg glioblastoma cells from toxicity at high concentrations of Lipofectin and streptolysin o
    Pharmacy and Pharmacology Communications, 1997
    Co-Authors: Judy M. Coulson, Saghir Akhtar
    Abstract:

    The pharmacological activity of an antisense oligodeoxynucleotide (ODN) targeted to c-myc (15mycAS) was evaluated in the glioblastoma-derived cell line U87-MG. ODNs with both phosphodiester (PO) and phosphorothioate (PS) backbones, and with 3′ hairpin sequences, were used with Lipofectin and streptolysin O as transfection reagents. At non-toxic concentrations of Lipofectin, cellular association was low and no evidence of an antisense mechanism was seen on evaluation of cell proliferation or c-Myc protein expression. At a high Lipofectin concentration (25 μg mL−1), which resulted in loss of more than 50% cell viability, ODN uptake was enhanced, but no reduction in c-Myc protein expression was observed with any of the 15mycAS ODNs. However, treatment with the antisense PS ODN (15mycAS PS), specifically resulted in protection of the viable cell number. This cytoprotective effect was concentration- and chemistry-dependent. Three 15mer control PS ODNs, the PO form of 15mycAS, and the hairpin stabilized PO and PS forms (15mycASHP2 PO and 15mycASHP2 PS, respectively) did not show cytoprotection. A similar protective effect of 15mycAS PS was also seen with the transfection agent streptolysin O. These data demonstrate a potential source of misinterpretation in antisense experiments and emphasize the need to consider possible interactions of ODNs with lipids or cell membranes in the design of effective antisense strategies.

Charles R Rinaldo - One of the best experts on this subject based on the ideXlab platform.

  • delivery of liposome encapsulated hiv type 1 proteins to human dendritic cells for stimulation of hiv type 1 specific memory cytotoxic t lymphocyte responses
    AIDS Research and Human Retroviruses, 1999
    Co-Authors: Lian Zheng, Xiaoli Huang, Luann Borowski, Cara C Wilson, Charles R Rinaldo
    Abstract:

    An important aspect of vaccine development involves delivery of antigens to antigen-presenting cells for the induction of potent antigen-specific T lymphocyte responses. We investigated the effect of a cationic liposome, Lipofectin, on delivery of whole proteins to human dendritic cells (DCs) derived from blood mononuclear cells by culture in interleukin 4 and granulocyte-monocyte colony-stimulating factor for stimulation of human immunodeficiency virus type 1 (HIV-1)-specific memory cytotoxic T lymphocyte (CTL) responses. Delivery of HIV-1 Gag, Pol, and Env proteins to DCs by Lipofectin stimulated greater anti-HIV-1 memory CTL responses in cells from HIV-1-infected subjects than those induced by DCs loaded with protein alone. The CTLs were CD8+ and HLA class I restricted. Antigen presentation was enhanced by chloroquine, but blocked by brefeldin A and peptide aldehyde inhibitors of proteasomes, indicating that the classic MHC class I cytosolic pathway was used for processing and presentation of HIV-1 pro...

A Mostageer - One of the best experts on this subject based on the ideXlab platform.

  • ١ Sperm-mediated gene transfer in poultry
    2015
    Co-Authors: Ea El-gendy, A. Y. Gad, A Mostageer
    Abstract:

    The cock spermatozoa were used in this study as vectors for gene transfer. The objectives of the study were to assess the efficacy of the sperm to uptake exogenous DNA in relation to sperm viability. Two trials were carried out. Trial 1 was achieved to assess the effects of semen dilution (4 µl diluent/1 µl semen), heat incubation (exposure of semen to 37°C for 30 minutes) and the addition of Lipofectin and their combinations on sperm characteristics. The sperm motility in the heat incubated or the diluted semen was 79 % and was significantly less than that estimated for the un-treated semen (90%). However, no significant effects of heat incubation or semen dilution were mostly observed in the percentages of live, dead and abnormal sperm. The combination of heat incubation and semen dilution resulted in more reduction in the sperm motility compared to that occurred by either one. The addition of Lipofectin to the diluted semen then heat incubated significantly detracted the motility and the percentage of live sperm, and increased the percentages of dead and abnormal sperm, and the effect was positively associated with Lipofectin concentration. Trial 2, was designed to assess the effects of Lipofectin (5%) addition on the fusion of DNA into the sperm and sperm viability. The exogenous DNA used was the plasmid pUC18, and two specifi

  • Sperm-mediated gene transfer in poultry 1. The relationship with cock sperm viability
    2007
    Co-Authors: Ea El-gendy, Ahmed Gad, A Mostageer
    Abstract:

    The cock spermatozoa were used in this study as vectors for gene transfer. The objectives of the study were to assess the efficacy of the sperm to uptake exogenous DNA in relation to sperm viability. Two trials were carried out. Trial 1 was achieved to assess the effects of semen dilution (4 µl diluent/1 µl semen), heat incubation (exposure of semen to 37°C for 30 minutes) and the addition of Lipofectin and their combinations on sperm characteristics. The sperm motility in the heat incubated or the diluted semen was 79% and was significantly less than that estimated for the untreated semen (90%). However, no significant effects of heat incubation or semen dilution were mostly observed in the percentages of live, dead and abnormal sperm. The combination of heat incubation and semen dilution resulted in more reduction in the sperm motility compared to that occurred by either one. The addition of Lipofectin to the diluted semen then heat incubated significantly detracted the motility and the percentage of live sperm, and increased the percentages of dead and abnormal sperm, and the effect was positively associated with Lipofectin concentration. Trial 2, was designed to assess the effects of Lipofectin (5%) addition on the fusion of DNA into the sperm and sperm viability. The exogenous DNA used was the plasmid pUC18, and two specific primers (forward and reverse) were used to recognize the existence of exogenous DNA in the sperm through the PCR analysis. It was observed that the plasmid DNA was successfully internalized into the sperm treated and un-treated with Lipofectin. However, the DNA fragment recognized in the sperm incubated with Lipofectin was apparently of higher yield. It was concluded that Lipofectin stabilizes and facilitates the fusion of DNA into the sperm. The sperm motility and percentage of live sperm reached to 56.7% and 65.1% in the diluted semen incubated with Lipofectin (5%)- DNA mixture, vs 75% and 90.6% in the diluted semen incubated with DNA only. This indicates that the addition of Lipofectin to the sperm, for some extent, depressed their viability and subsequently the availability for fertilization. Because of the necessity of using Lipofectin to enhance the fusion of DNA into the sperm, therefore it may be used at low concentration (5%, in this experiment).

Essam A. El-gendy - One of the best experts on this subject based on the ideXlab platform.

  • Visible Diode Laser Enhancement of Exotic DNA Uptake by Fowl Sperm
    Journal of Applied Biology and Biotechnology, 2015
    Co-Authors: Essam A. El-gendy, Mona M. Abdelaziz, Mohamed M. Abdelfattah, Mohamed S. Salama, Yhia M. Badr
    Abstract:

    An experiment was conducted to assess the use of low power laser irradiation and Lipofectin to enhance fowl sperm uptake of exogenous DNA. Semen samples of 10 roosters were collected and pooled. The pooled sample was diluted with a semen extender and then divided into 6 aliquots for 6 different treatments. The treatments included different combinations of the inclusion of exogenous DNA (bacterial plasmid pUC18), the exposure to low power laser irradiation and the transfection with Lipofectin (5%). Laser irradiation was by using visible diode laser (650 nm) at energy dose of 4 J/cm2. The recognition of the plasmid DNA in the sperm was by using two specific oligonucleotides (forward and reverse) to prime a 420-bp fragment on the pMB1 rep of the plasmid. The results indicated that low power laser irradiation enhanced the sperm uptake of the plasmid DNA. Also, Lipofectin enhanced the introduction of the plasmid DNA into the sperm, whether the semen was laser irradiated or not.

  • The development of transgenic chickens by sperm-mediated gene transfer
    African Journal of Biotechnology, 2013
    Co-Authors: Essam A. El-gendy, Marwa M. Ahmed, Shoukry M. El-tantawy
    Abstract:

    An experiment was carried out to develop transgenic chickens using sperm incubated with exogenous DNA. Two random bred lines of chickens were used as parental flocks, and the plasmid pUC18 was used as the exogene. Lipofectin was also used to facilitate the sperm cell uptake of the plasmid. Hens were inseminated with the semen that was subjected to different treatments and their eggs were hatched. Polymerase chain reaction (PCR) was performed on spermatozoa and blood genomes of the parental flocks and on blood genomes of the progeny. The plasmid DNA was recognized in spermatozoa of both lines in most of the treatments with different amplification rates. However, plasmid DNA was highly fused into the sperm cells when it was introduced in a complex with Lipofectin. Also, the plasmid DNA band was highly amplified in the progeny which resulted from sperm cells incubated with the plasmid and Lipofectin at 5% concentration. The results reveal the success of the development of F1 chickens by sperm-mediated gene transfer (SMGT). The positively detected SMGT-derived offspring formed 40.0 to 50.0% of the F1 generation.   Key words: Rooster spermatozoa, Lipofectin, sperm-mediated gene transfer-derived offspring, sperm-mediated gene transfer.

  • Fertilization Capacity of Rooster Spermatozoa in Response to the Modification in the Semen Composition
    International Journal of Poultry Science, 2012
    Co-Authors: Essam A. El-gendy, Marwa M. Ahmed, Shoukry M. El-tantawy, Shawki A. Ibrahim
    Abstract:

    2 Abstract: An experiment was carried out to study the changes in fertilization capacity of rooster sperms in response to the modification in the biochemical composition of the semen. Chickens of two lines (CE2 and CE4) were used. Seven treatments of semen were designed and included the incubation of sperm with the plasmid, with a mixture of the plasmid and Lipofectin at 2.5 or 5% concentration and the incubation of spermatozoa with Lipofectin and a semen extender (BPSE). The progenies were obtained from the insemination of hens by the semen of different treatments. Sperm motility was greatly influenced by th e treatments. Motility was significantly the highest in the control semen and averaged 92.42% and highly significantly declined to 52.08 and 58.75% in the semen samples treated with the plasmid, Lipofectin at 2.5 or 5% concentration and diluted with BPSE. The percentage of live sperm was not affected by the addition of the plasmid. The addition of the plasmid and Lipofectin or the dilutent BPSE resulted in a significant reduction in the percentage of live sperms. The percentage of live sperms was 59-62% when the plasmid, Lipofectin and BPSE were all together added to the semen samples. The percentages of dead and abnormally-shaped sperm reached to 26.88 and 17.13%, respectively, in the semen treated with plasmid, Lipofectin 5% and BPSE. Fertility averaged 88.22% in the eggs of hens inseminated with the control semen and significantly decreased to 42.14% when semen was incubated with the plasmid pUC18 and reached to 58.98% when semen was treated with plasmid, Lipofectin (5%) and BPSE.

Pi-wan Cheng - One of the best experts on this subject based on the ideXlab platform.

  • Transferrin-facilitated lipofection gene delivery strategy: characterization of the transfection complexes and intracellular trafficking.
    Human Gene Therapy, 2002
    Co-Authors: Nirmal Joshee, Dhundy R. Bastola, Pi-wan Cheng
    Abstract:

    We previously showed that mixing transferrin with a cationic liposome prior to the addition of DNA, greatly enhanced the lipofection efficiency. Here, we report characterization of the transfection complexes in formulations prepared with transferrin, Lipofectin, and DNA (pCMVlacZ) in various formulations. DNA in all the formulations that contain Lipofectin was resistant to DNase I treatment. Transfection experiments performed in Panc 1 cells showed that the standard formulation, which was prepared by adding DNA to a mixture of transferrin and Lipofectin, yielded highest transfection efficiency. There was no apparent difference in zeta potential among these formulations, but the most efficient formulation contained complexes with a mean diameter of three to four times that of liposome and the complexes in other gene delivery formulations. Transmission electron microscopic examination of the standard transfection complexes formulated using gold-labeled transferrin showed extended circular DNA decorated with...

  • Effects of epidermal growth factor, transferrin, and insulin on lipofection efficiency in human lung carcinoma cells.
    Cancer Gene Therapy, 2000
    Co-Authors: Katsunori Yanagihara, Helen Cheng, Pi-wan Cheng
    Abstract:

    Poor transfection efficiency is the major drawback of lipofection. We showed previously that addition of transferrin (TF) to Lipofectin enhanced the expression of a reporter gene in HeLa cells by 120-fold and achieved close to 100% transfection efficiency. The purpose of this study was to determine whether TF and other ligands could improve the efficiency of lipofection in lung carcinoma cells. Confluent A549, Calu3, and H292 cells were transfected for 18 hours with a plasmid DNA (pCMVlacZ) using Lipofectin plus TF, insulin, or epidermal growth factor as the vector. The transfected cells were assessed for transfection efficiency by β-galactosidase activity (light units/μg protein) and the percentage of blue cells following 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside staining. Lipofectin supplemented with epidermal growth factor yielded the largest enhancement of lipofection efficiency (≤23-fold over that by Lipofectin alone) in all three cell lines. Insulin significantly enhanced the lipofection efficiency in A549 and Calu3 cells but not in H292 cells, whereas TF showed significant lipofection efficiency-enhancing effect in Calu3 and H292 cells but not in A549 cells. The transfection efficiency correlated well with the amounts of DNA delivered to the nucleus as well as the amounts of the receptor. These results indicate that the gene delivery strategy employing ligand-facilitated lipofection can achieve high transfection efficiency in human lung carcinoma cells. In addition, enhancement of the expression of the receptor may be a possible strategy for increasing the efficiency of gene targeting.

  • Receptor ligand-facilitated gene transfer: enhancement of liposome-mediated gene transfer and expression by transferrin.
    Human Gene Therapy, 1996
    Co-Authors: Pi-wan Cheng
    Abstract:

    A high-efficiency, nonviral gene transfer protocol employing cationic liposome plus a receptor ligand is described. The delivery of the beta-galactosidase (beta-Gal) gene (pCMVlacZ) by Lipofectin plus transferrin can achieve 98-100% transfection of HeLa cells as compared to 3-4% by Lipofectin alone. A dose-dependent gene transfer was observed between 1 and 16 micrograms transferrin, and maximal transfection efficiency was obtained at > or = 16 micrograms transferrin. The expression of beta-Gal activity in 100% transfected cells decreased progressively with each passage and returned to the baseline value after six passages, indicating that the DNA delivered was only transiently expressed. The amount of DNA delivered to the cells by Lipofectin plus transferrin was approximately two times that obtained by Lipofectin, which in turn was two times that by transferrin or without Lipofectin and transferrin. In addition, DNA can form complexes with Lipofectin and transferrin. These results suggest that transferrin enhances gene transfer and expression in the presence of Lipofectin by further facilitating the entry of DNA into the cells through the Lipofectin-DNA-transferrin complex. The enhancement of liposome-mediated gene transfer efficiency and expression by transferrin varies with different cationic liposomes. The four different liposomes examined show the following relative transfection efficiency: transfectin > lipofectACE > > DC-cholesterol > > lipofectAMINE.