Gene transfer into mammalian cells by rapid freezing

Summary In this paper, we describe a simple technique to introduce DNA into cells through cracks and/or pores in cell membranes caused by intracellular ice crystal formation induced by liquid nitrogen. We mixed mouse BALB 3T3 cells and pSV2-neo DNA and froze the cell suspension under various conditions to determine those optimum for the introduction of DNA into mammalian cells. We found that brief treatment with liquid nitrogen, which showed only moderate cell killing, resulted in the induction of G-418 resistant colonies. These results suggest that this new technique is useful for transfection of genes into mammalian cells. This work was supported by a Grant-in Aid from the Ministry of Health and Welfare for the Comprehensive 10-Year Strategy for Cancer Control, Japan.     

Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells.

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.     

Staining of human metaphase chromosomes with fluorescent conjugates of polylysine

The interaction of polylysine and partially substituted dansyl, fluorescein, and quinacrine conjugates of polylysine with cytological preparations of human metaphase chromosomes has been studied by fluorescence microscopy. The fluorescence intensity along chromosomes stained with the dansyl and fluorescein conjugates exhibits little variation, suggesting that regions capable of binding these polycations are nearly evenly distributed. In contrast, the quinacrine derivatives of polylysine stain the chromosomes in a banded fluorescence pattern resembling that observed following quinacrine or quinacrine mustard treatment.     

Efficient gene transfer by histidylated polylysine/pDNA complexes.

Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 +/- 5% of the epsilon-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.     

Distribution of microsomal triglyceride transfer protein within sub-endoplasmic reticulum regions in human hepatoma cells

Very low-density lipoprotein (VLDL) particles are formed in the endoplasmic reticulum (ER) through the association of lipids with apolipoprotein B (apoB). Microsomal triglyceride transfer protein (MTP), which transfers lipid molecules to nascent apoB, is essential for VLDL formation in ER. However, little is known of the distribution and interaction of MTP with apoB within ER. In this study, distribution patterns of apoB and MTP large subunit (lMTP) within ER were examined. Microsomes prepared from HuH-7 cells, a human hepatoma cell line, were further fractionated into rough ER (RER)-enriched subfractions (ER-I fraction) and smooth ER (SER)-enriched subfractions (ER-II fraction) by iodixanol density-gradient ultracentrifugation. ApoB was evenly distributed in the ER-I and the ER-II fractions, while 1.5 times more lMTP molecules were present in the ER-I fraction than in the ER-II fraction. lMTP and apoB were coprecipitated both in the ER-I and in the ER-II fractions by immunoprecipitation whenever anti-apoB or an anti-lMTP antibodies were used. ApoB-containing lipoprotein particles showed a lower density in the ER-II fraction than those in the ER-I fraction. From these results, it is suggested that MTP can function in both rough and smooth regions of ER in human hepatoma cells.     

Receptor-mediated oligodeoxynucleotides delivery by estradiol and folic acid polylysine conjugates

The lack of efficient and specific delivery to target cells still limits the potential application of antisense oligodeoxynucleotides as therapeutic agents in cancer disease. We have covalently linked a polylysine chain (10,000–20,000 mW) to compounds as folic acid, retinoic acid, transferrin, insulin and estradiol, to deliver c-myb antisense oligonucleotide into tumor cells. Using these complexes as carriers for the oligodeoxynucleotides can be achieved an increase in their uptake into target cells through a natural endocytosis pathway.     

Gene transfer by electroporation into intact scutellum cells of wheat embryos

Summary Gene transfer into intact cells was achieved by electroporating zygotic wheat embryos without any special pretreatment. Electroporation was tissue specific in so far as scutellum cells were found to be much more susceptible to gene transfer than other cell types of the embryo. The orientation of the embryos in the electroporation chamber also influenced the number of transformed scutellum cells; during electroporation, as in electrophoresis, the negatively charged plasmid DNA molecules seemed to move towards the positive electrode. Therefore, the embryos were arranged so that the scutella faced the negative electrode. The use of plasmids carrying either two chimeric anthocyanin regulatory genes or a chimeric gusA gene allowed clear identification of transformed cells in the scutellum. On some of the embryos, more than 100 transformed scutellum cells were found after electroporation with single electric pulses of 275 V/cm discharged from a 960-F capacitor and with 100 g DNA/ml electroporation buffer. Using the anthocyanin marker system, visibly transformed cells grew to produce red sectors.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MES 2-N-morpholinoethane sulfonic acid     

Gene transfer of human heme oxygenase into coronary endothelial cells potentially promotes angiogenesis

Heme oxygenase (HO-1) is a stress protein that has been suggested to participate in defense mechanisms against agents that induce oxidative injury such as hemoglobin/heme, hypoxia-ischemia and cytokines. Overexpression of HO-1 in endothelial cells (EC) might, therefore, protect against oxidative stress produced under these pathological conditions, by generation of CO, a vasodilator, and bilirubin, which has antioxidant properties that enhance blood vessel formation to counteract hypoxia-induced injury. A plasmid containing the cytomegalovirus promoter (pCMV) neomycin human HO-1 gene complexed to cationic liposomes, lipofectin, was used to transfect rabbit coronary microvessel EC. Cells transfected with human HO-1 gene demonstrated a twofold increase in HO activity and maintained a similar phenotype as in the nontransfected cells. Cell number in transfected cells with human HO-1 gene increased by about 45%, as compared to nontransfected or those transfected with control pCMV. Transfected and nontransfected EC revealed a similar response to basic fibroblast growth factor (bFGF) in capillary formation. However, transfected cells with the human HO-1 gene exhibited a twofold increase in blood vessel formation. The angiogenic response of EC to overexpression of HO-1 gene provides direct evidence that the inductive form of HO-1 following injury represents an important tissue adaptive mechanism for moderating the severity of cell damage produced in inflammatory reaction sites of hemorrhage, thrombosis and hypoxic-ischemia. Thus, HO-1 may participate in the regulation of EC activation, proliferation and angiogenesis. J. Cell. Biochem. 68:121–127, 1998. © 1998 Wiley-Liss, Inc.     

Direct Fmoc/tert-Bu solid phase synthesis of octamannosyl polylysine dendrimer-peptide conjugates

The mannose binding proteins on the surface of the dendritic cells are responsible for capture of pathogens in the early stages of immune response. Conjugation to mannose dendrimers is a rarely explored but potentially powerful strategy for enhancing immunogenicity of synthetic peptides relying on direct delivery to dendritic cells. We describe a general protocol for preparation of pure, monodisperse third-generation mannosylated poly-L-lysine dendrimer-peptide conjugates using direct, machine-assisted Fmoc/t-Bu solid phase peptide synthesis. The glycodendrons were elaborated onto the N- or C-terminus of sequences derived from HIV-1 gp41, SARS-CoV S2 protein, and Influenza Hemagglutinin (consisting of 15-44 residues). The products were obtained in a homogeneous state after cleavage from the resin, deprotection, and a single purification on semipreparative RP-HPLC.     

Gene transfer into eukaryotic cells using activated polyamidoamine dendrimers

The development of efficient methods to transfer genes into eukaryotic cells is important for molecular biotechnology. A number of different technologies to mediate gene transfer have been developed over the last 35 years, but most have drawbacks such as cytotoxicity, low efficiency and/or restricted applicability. Activated polyamidoamine (PAMAM)-dendrimers provide a new technology for gene transfer that offers significant advantages over classical methods. Reagents based on this technology provide high gene transfer efficiencies, minimal cytotoxicity, and can be used with a broad range of cell types. This technology could also be useful for in vivo gene transfer in gene therapy applications.     

Gene transfer into mammalian somatic cells in vivo.

Direct gene transfer into mammalian somatic tissues in vivo is a developing technology with potential application for human gene therapy. During the past 2 years, extensive progress and numerous breakthroughs have been made in this area of research. Genetically engineered retroviral vectors have been used successfully to infect live animals, effecting foreign gene expression in liver, blood vessels, and mammary tissues. Recombinant adenovirus and herpes simplex virus vectors have been utilized effectively for in vivo gene transfer into lung and brain tissues, respectively. Direct injection or particle bombardment of DNA has been demonstrated to provide a physical means for in situ gene transfer, while carrier-mediated DNA delivery techniques have been extended to target specific organs for gene expression. These technological developments in conjunction with the initiation of the NIH human gene therapy trials have marked a milestone in developing new medical treatments for various genetic diseases and cancer. Various in vivo gene transfer techniques should also provide new tools for basic research in molecular and developmental genetics.     

Gene transfer into hemopoietic stem cells using retroviral vectors

Gene transfer to hemopoietic cells offers a variety of new approaches to the experimental hematologist as well as potentially providing a means for correcting a number of genetic disorders of humans. From the experimental viewpoint, gene transfer utilizing retroviral vectors introduces new methods for analyzing hemopoietic cell lineages, and the effects of over-expression of genes affecting hemopoietic cell proliferation and differentiation. The unique properties of retroviral vectors and the optimized methods currently in use to infect hemopoietic cells are represented as a brief review of a rapidly expanding new field of experimental hematology.     

Efficient stable gene transfer into human cells by the Sleeping Beauty transposon vectors

Transposable elements can be considered as natural, non-viral gene delivery vehicles capable of efficient genomic insertion. The plasmid-based transposon system of Sleeping Beauty (SB) combines the advantages of viruses and naked DNA molecules. In contrast to plasmid vectors, transposons integrate through a precise, recombinase-mediated mechanism into chromosomes, providing long-term expression of the gene of interest in cells. The advantages of transposons in comparison to viral systems include their simplicity and improved safety/toxicity profiles. In addition, the hyperactive SB100X is the first plasmid-based delivery system that overcomes the efficacy of non-viral delivery. The transposon delivery system consists of the transposase and the integration cassette, recognized by the transposase. The plasmid-based transposon delivery system can be combined with any non-viral delivery method. Here we provide two detailed protocols to apply SB-mediated, non-viral gene transfer in cultured cells. In our first example, we use a lipid-based delivery method in combination with the transposon-based integration system in an easy-to-transfect (HeLa) cell line. Second, we show how to achieve 40–50% stable expression of a transgene in clinically relevant, hard-to-transfect cells (hematopoetic stem cells, HSCs) by nucleofection. The given protocols are adaptable to any vertebrate cells in culture.     

Gene transfer into mouse lyoma cells by electroporation in high electric fields.

Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.     

Gene transfer into chicken embryos by retrovirus vectors

While chickens have many properties that are advantageous for embryological studies, their genetic analysis has been restricted. However, by using retrovirus vector systems in combination with classical techniques of experimental developmental biology, it has recently become possible to analyze the function of genes involved in the development of this organism. Avian retrovirus vectors are unique in that they can be divided into two categories: replication-competent and replication-defective (replication-incompetent). By choosing the vectors correctly, there are many experimental applications of these vectors such as induction of constitutive (or regulated) gene expression in a restricted region of tissues, organs and embryos; cell lineage analysis; and formation of concentration gradients of morphogens in micromass cultures. In this paper, several retrovirus vectors available for the chicken will be introduced and their applications in developmental biology will be reviewed.