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

  • Characterization of Human Serum Albumin-Facilitated Lipofection Gene Delivery Strategy
    Journal of Cell Science & Therapy, 2011
    Co-Authors: Robert W Arpke, Pi-wan Cheng
    Abstract:

    We report the characterization of a facilitated Lipofection strategy, which employs a formulation prepared with human serum albumin, DMRIE-C and pCMV?. The transfection complexes in the Lipofection formulation containing albumin were characterized for size by light scattering, intracellular trafficking by confocal microscopy, and uptake mechanism by inhibitors. Supplementation of the formulation of DMRIE-C plus pCMV? with albumin enhances Lipofection efficiency 8-9 fold and the size of the complexes 2-2.5 fold as measured by light scattering. Analysis of intracellular trafficking of the transfection complexes by confocal microscopy reveals colocalization of DNA and albumin in the cytoplasm and the nucleus. Pretreatment of the cells with chlorpromazine or excess albumin, cytochalasin B or filipin complexes results in inhibition of the transfection efficiency by 20%, 55%, or none, respectively. The results suggest a moderate involvement of clathrin, a significant involvement of actinassociated macropinocytosis, and no involvement of caveolae in the uptake of the transfection complexes prepared with human serum albumin, DMRIE-C and pCMV?.

  • 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.

  • Lectin enhancement of the Lipofection efficiency in human lung carcinoma cells.
    Biochimica et biophysica acta, 1999
    Co-Authors: Katsunori Yanagihara, Pi-wan Cheng
    Abstract:

    Poor transfection efficiency of human lung carcinoma cells by Lipofection begs further development of more efficient gene delivery strategies. The purpose of this study was to determine whether lectins can improve the Lipofection efficiency in lung carcinoma cells. A549, Calu3, and H292 cells grown to 90% confluence were transfected for 18 h with a plasmid DNA containing a beta-galactosidase reporter gene (pCMVlacZ) using lipofectin plus a lectin as the vector. Ten different lectins, which exhibit a wide range of carbohydrate-binding specificities, were examined for their abilities to enhance the efficiency of Lipofection. The transfected cells were assessed for transfection efficiency by beta-galactosidase activity (units/microg protein) and % blue cells following X-Gal stain. Lipofectin supplemented with Griffonia simplicifolia-I (GS-I) yields largest enhancement of the Lipofection efficiency in A549 and Calu3 cells (5.3- and 28-fold, respectively). Maackia amurensis gives the largest enhancement (6.5-fold) of Lipofection efficiency in H292 cells. The transfection efficiency correlates with the amounts of DNA delivered to the nucleus. Binding of FITC-labeled GS-I and the enhancement of the Lipofection efficiency by GS-I were inhibited by alpha-methyl-D-galactopyranoside, indicating an alpha-galactoside-mediated gene transfer to lung carcinoma cells. We conclude that lectin-facilitated Lipofection is an efficient gene delivery strategy. Employment of cell type-specific lectins may allow for efficient cell type-specific 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.

Leaf Huang - One of the best experts on this subject based on the ideXlab platform.

  • overcoming the inhibitory effect of serum on Lipofection by increasing the charge ratio of cationic liposome to dna
    Gene Therapy, 1997
    Co-Authors: J P Yang, Leaf Huang
    Abstract:

    Since cationic liposome was first developed as a Lipofection reagent, a drawback has been noted in that the efficiency of Lipofection decreases dramatically after addition of serum to the Lipofection medium. This drawback hampers the application of cationic liposome for systematic delivery of genes. In the present studies, we found that the effect of serum on DC-chol liposome-mediated Lipofection is dependent on the charge ratio of liposome to DNA. Serum inhibited Lipofection activity of the lipoplex at low charge ratios, whereas it enhanced the Lipofection activity at high charge ratios. This phenomenon was observed using DOTAP/DOPE but not lipofectamine. Measurement of cellular association of DNA showed that serum could reduce the binding of lipoplex to cells at all tested charge ratios, i.e. 0-10.6. Removal of negatively charged proteins from serum by DEAE Sephacel column abolished the inhibitory effect of serum on Lipofection. The fraction contained only negatively charged serum proteins which strongly inhibited Lipofection at low charge ratios but not at higher charge ratios. Furthermore, preincubation of serum with positively charged polylysine, which neutralized negatively charged serum proteins, eliminated the inhibitory effect of serum on Lipofection. In summary, inactivation of cationic liposome by serum is due to negatively charged serum proteins and it can be overcome by increasing charge ratio of cationic liposome-DNA lipoplexes or by neutralizing the serum with polylysine.

  • factors influencing the drug sensitization of human tumor cells for in situ Lipofection
    Gene Therapy, 1996
    Co-Authors: Leaf Huang
    Abstract:

    : The cisplatin induced enhancement of in situ Lipofection was optimized by considering the factors that can increase the degree of sensitization. Two other anticancer drugs, mechlorethamine (nitrogen mustard) and taxol, enhanced CAT gene expression but the degree of sensitization was not as great as cisplatin. Besides human 2008 ovarian cancer cells we also found that human lung (A549) and head and neck cancer cells (SCC 25) were transiently sensitized by cisplatin. The transfectability of the two commercially available cationic liposomes, Lipofectin and LipofectAmine, was either weak or not consistent among tumors tested. In vivo transfection efficiency of 2008 cells was the highest at 1 microgram DNA per nmol or microgram liposome with all three cationic liposomes. In vitro transfection efficiency of 2008 cells at 1:1 (microgram of DNA:nmole of DC-chol/DOPE liposome) increased in a dose-dependent manner while at 1:10, an optimal ratio for in vitro Lipofection, rapidly decreased with an increase in dose. This result indicated that there was a correlation between in vivo and in vitro Lipofection at 1:1 ratio for delivering liposomal DNA. Most of the DNA injected into the tumor was concentrated in the tumor and in the skin above the tumor whether cisplatin was preinjected or liposomes were used as carriers.

  • Exposure of human ovarian carcinoma to cisplatin transiently sensitizes the tumor cells for liposome-mediated gene transfer.
    Proceedings of the National Academy of Sciences of the United States of America, 1994
    Co-Authors: Kyonghee Son, Leaf Huang
    Abstract:

    Abstract Human ovarian carcinoma cells (line 2008) grown as subcutaneous solid tumor in the severe combined immunodeficient mouse can be transfected by directly injecting a plasmid DNA-liposome complex into the tumor (in situ Lipofection). The level of reporter gene expression in the tumor cells was significantly elevated if the animal received a single i.p. injection of cisplatin 1 week before the Lipofection. Sensitization of the tumor for Lipofection peaked 1 week after cisplatin injection and declined thereafter. Cells exposed to low concentration of cisplatin in vitro for four to five doubling times also showed elevated sensitivity for Lipofection in vitro. Cisplatin was the only anticancer drug tested that exhibited this activity. These results suggest a sequential combinational gene therapy protocol with cisplatin for the ovarian carcinoma.

Scott L. Diamond - One of the best experts on this subject based on the ideXlab platform.

  • RNA interference screen to identify pathways that enhance or reduce nonviral gene transfer during Lipofection
    Molecular therapy : the journal of the American Society of Gene Therapy, 2008
    Co-Authors: Gregory A Barker, Scott L. Diamond
    Abstract:

    Some barriers to DNA Lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on Lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after Lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced Lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from Lipofection, with a quantitative PCR–validated knockdown of ∼76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.

  • Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germline transmission potency.
    Methods (San Diego Calif.), 2004
    Co-Authors: Qin Liu, Scott L. Diamond, Eric A. Pierce
    Abstract:

    Embryonic stem (ES) cells are an important tool in developmental biology, genomics, and transgenic methods, as well as in potential clinical applications such as gene therapy or tissue engineering. Electroporation is the standard transfection method for mouse ES (mES) cells because Lipofection is quite inefficient. It is also unclear if mES cells treated with cationic lipids maintain pluripotency. We have developed a simple Lipofection method for high efficiency transfection and stable transgene expression by employing the nonclassical nuclear localization signal M9 derived from the heterogeneous nuclear ribonucleoprotein A1. In contrast to using 20lg DNA for 10 � 10 6 cells via electroporation which resulted in 10–20 positive cells/mm 2 , M9-assisted Lipofection of 2 � 10 5 cells with 2lg DNA resulted in > 150 positive cells/mm 2 . Electroporation produced only 0.16% EGFP positive cells with fluorescence intensity (FI) > 1000 by FACS assay, while M9-Lipofection produced 36-fold more highly EGFP positive cells (5.75%) with FI > 1000. Using 2.5 � 10 6 ES cells and 6lg linearized DNA followed by selection with G418, electroporation yielded 17 EGFP expressing colonies, while M9-assisted Lipofection yielded 72 EGFP expressing colonies. The mES cells that stably expressed EGFP following M9-assisted Lipofection yielded > 66% chimeric mice (8 of 12) and contributed efficiently to the germline. In an example of gene targeting, a knock-in mouse was produced from an ES clone screened from 200 G418-resistant colonies generated via M9assisted Lipofection. To our knowledge, this is the first report of generation of transgenic or knock-in mice obtained from lipofected mES cells and this method may facilitate large scale genomic studies of ES developmental biology or large scale generation of mouse models of human disease. 2003 Elsevier Inc. All rights reserved.

  • Adenovirus or HA-2 fusogenic peptide-assisted Lipofection increases cytoplasmic levels of plasmid in nondividing endothelium with little enhancement of transgene expression.
    The journal of gene medicine, 2002
    Co-Authors: Ajit Subramanian, Kris Noel Dahl, Jingya Zhu, Scott L. Diamond
    Abstract:

    Background Adenovirus-assisted Lipofection has been reported to increase transfection efficiency through mechanisms potentially involving endosome escape and/or nuclear targeting activity. Similarly, transfection with the viral fusogenic peptide HA-2 of the influenza virus hemagglutinin can increase transfection efficiency. However, there are few studies examining the mechanism and intracellular trafficking of these viral and/or viral fusogenic peptide-assisted Lipofections. Methods and results Endosome escape was directly assayed with T7 RNA polymerase bound to plasmid (pTMβgal) expressing β-galactosidase under a T7 promoter to detect transcribable plasmid that escapes the endosomal compartment. Lipofection of pTMβgal with replication-deficient adenovirus (Ad5-null) at a multiplicity of infection (MOI) of 100 and 1000 increased cytoplasmic levels of transcribable plasmid by 24- and 117-fold, respectively, over Lipofection alone, without an effect on total plasmid uptake. However, Lipofection of pCMVβgal with Ad5-null at a MOI of 100 and 1000 increased transgene expression only seven- and eight-fold, respectively, over Lipofection alone. Thus, a 24-fold increase in endosome escape saturated expression from pCMVβgal and provided only a seven-fold benefit in nondividing cells, which was not significantly increased with further increases in endosome escape. A cationic form of HA-2 (HA-K4) also caused significant enhancements in endosome escape, as detected with the cytoplasmic transcription assay. However, HA-K4 enhancement of endosome escape did not correlate with transgene expression from pCMVβgal, consistent with the detection of HA-K4-mediated partitioning of plasmid to the insoluble fraction of the cell lysate. Conclusion These results indicate that enhancement of endosome escape in nondividing cells does not fully alleviate rate limits related to nuclear import of the plasmid. Copyright © 2001 John Wiley & Sons, Ltd.

  • Non-classical nuclear localization signal peptides for high efficiency Lipofection of primary neurons and neuronal cell lines.
    Neuroscience, 2002
    Co-Authors: J. Zhu, M. Maronski, Paul T. Kotzbauer, Virginia M.-y. Lee, Marc A. Dichter, Scott L. Diamond
    Abstract:

    Abstract Gene transfer into CNS is critical for potential therapeutic applications as well as for the study of the genetic basis of neural development and nerve function. Unfortunately, lipid-based gene transfer to CNS cells is extremely inefficient since the nucleus of these post-mitotic cells presents a significant barrier to transfection. We report the development of a simple and highly efficient Lipofection method for primary embryonic rat hippocampal neurons (up to 25% transfection) that exploits the M9 sequence of the non-classical nuclear localization signal of heterogeneous nuclear ribonucleoprotein A1 for targeting β 2 -karyopherin (transportin-1). M9-assistant Lipofection resulted in 20–100-fold enhancement of transfection over Lipofection alone for embryonic-derived retinal ganglion cells, rat pheochromocytoma (PC12) cells, embryonic rat ventral mesencephalon neurons, as well as the clinically relevant human NT2 cells or retinoic acid-differentiated NT2 neurons. This technique can facilitate the implementation of promoter construct experiments in post-mitotic cells, stable transformant generation, and dominant-negative mutant expression techniques in CNS cells.

Lan Xiao - One of the best experts on this subject based on the ideXlab platform.

  • Different Influences of Lipofection andElectrotransfection on In Vitro Gene Delivery toPrimary Cultured Cortex Neurons
    March 2016, 2016
    Co-Authors: Lan Xiao
    Abstract:

    Background: Many pain states are linked to central nervous system (CNS) diseases involving the dysfunction of dendritic arborization, making restoration a promising therapeutic strategy. Transfection of primary cortex neurons offers the possibility to study mechanisms which are important for the restoration of proper arborization. Its progress is, however, limited at present due to the lack of suitable gene transfer techniques. Objective: To obtain better insight into the transfection potential of currently used techniques, 2 non-viral transfection methods, Lipofection and gene electrotransfer (GET), were compared. Study Design: This is a comparison study performed on cultured cells. Methods: The transfection efficiency and neuronal viability, as well as the neuronal dendritic arborization after Lipofection or GET, were compared. Primary cultured cortex neurons were transfected with the pEGFP-N1 plasmid, either using Lipofectamine 2000 (2, 3, or 4μL) or with electroporation, with our previously optimized protocol (200V/25 ms). Results: Transfection efficiency and cell viability were inversely proportional for Lipofection. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for Lipofection. Although GET offered higher transfection efficiency, it could not induce complex dendritic arborization, which made it unsuitable for in vitro gene transfer into cortex neurons. Limitations: Limitations include species variability and translational applicability for CNS diseases and pain states related to potential toxicity. Conclusions: Based on these findings, Lipofection might be advantageous for in vitro application to primary cultured cortex neurons. Pain states, stress mediated pathogenesis, and certain CNS diseases might potentially utilize this important technique in the future as a therapeutic modality. Key words: Lipofection, gene electrotransfer, CNS diseases, pain states, dendritic arborization, transfection of primary cortex neurons

  • Different Influences of Lipofection and Electrotransfection on In Vitro Gene Delivery to Primary Cultured Cortex Neurons.
    Pain physician, 2016
    Co-Authors: Xui-si Zhang, Jing Huang, Cong-qing Zhan, Jing Chen, Alan D. Kaye, Lan Xiao
    Abstract:

    Background Many pain states are linked to central nervous system (CNS) diseases involving the dysfunction of dendritic arborization, making restoration a promising therapeutic strategy. Transfection of primary cortex neurons offers the possibility to study mechanisms which are important for the restoration of proper arborization. Its progress is, however, limited at present due to the lack of suitable gene transfer techniques. Objective To obtain better insight into the transfection potential of currently used techniques, 2 non-viral transfection methods, Lipofection and gene electrotransfer (GET), were compared. Study design This is a comparison study performed on cultured cells. Methods The transfection efficiency and neuronal viability, as well as the neuronal dendritic arborization after Lipofection or GET, were compared. Primary cultured cortex neurons were transfected with the pEGFP-N1 plasmid, either using Lipofectamine 2000 (2, 3, or 4µL) or with electroporation, with our previously optimized protocol (200V/25 ms). Results Transfection efficiency and cell viability were inversely proportional for Lipofection. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for Lipofection. Although GET offered higher transfection efficiency, it could not induce complex dendritic arborization, which made it unsuitable for in vitro gene transfer into cortex neurons. Limitations Limitations include species variability and translational applicability for CNS diseases and pain states related to potential toxicity. Conclusions Based on these findings, Lipofection might be advantageous for in vitro application to primary cultured cortex neurons. Pain states, stress mediated pathogenesis, and certain CNS diseases might potentially utilize this important technique in the future as a therapeutic modality.

Theodore Friedmann - One of the best experts on this subject based on the ideXlab platform.

  • Polybrene increases the efficiency of gene transfer by Lipofection
    Gene therapy, 1998
    Co-Authors: Akihiro Abe, Atsushi Miyanohara, Theodore Friedmann
    Abstract:

    Lipofection involves the introduction of foreign genetic information into mammalian cells through the use of lipophilic reagents that enhance cellular uptake of polynucleotides. Despite the use of currently optimized Lipofection conditions, including the use of serum-depleted media, the efficiency of gene transfer is often low. We show here that, in a variety of cell lines, polybrene markedly enhances the efficiency of Lipofection under standardized conditions and also compensates the serum-mediated inhibition of Lipofection. Although the degree of the polybrene effect depends on the nature of the cell line, these results indicate that individually optimized concentrations of polybrene can be useful for increasing the efficiency of lipofectin-mediated gene transfer in vitro.