Encoding Enhanced Green Fluorescent Protein Research Articles

Ferrocene-containing cationic lipids for the delivery of DNA: Oxidation state determines transfection activity

The ability of two redox-active, ferrocene-containing cationic lipids [11-(ferrocenylundecyl)trimethylammonium bromide (FTMA) and bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA)] to transfect mammalian cells was investigated. This study sought to determine the range of conditions over which these lipids were capable of transfecting cells and whether the oxidation state of the ferrocenyl groups in these materials could be used to influence the extent of transfection. Experiments conducted in the COS-7 cell line demonstrated that reduced and oxidized FTMA were substantially cytotoxic and did not transfect cells. Subsequent experiments conducted using BFDMA and reporter plasmids encoding enhanced green fluorescent protein (EGFP) and firefly luciferase demonstrated that BFDMA was able to transfect cells. However, the extent of transfection depended significantly upon both the concentration of BFDMA and the oxidation state of the lipid. Quantitative characterization of cytotoxicity and gene expression demonstrated that a window of concentration existed over which reduced BFDMA was non-cytotoxic and yielded high levels of transfection, but over which electrochemically oxidized BFDMA yielded very low (background) levels of transfection. Characterization of lipoplexes using dynamic light scattering demonstrated that reduced and oxidized BFDMA formed small aggregates (ca. 90 to 250 nm) at concentrations of lipid ranging from 2 to 10 μM. Taken together, these results demonstrate that the oxidation state of BFDMA, which can be controlled electrochemically, can be used to control the extent of cell transfection. These results could form the basis of transfection procedures that exploit the redox behavior of ferrocene-containing lipids to achieve active spatial and temporal control over transfection using electrochemical methods.

193. Gene Transfection Using Inorganic Particle/PVA/DNA Complexes Prepared by Ultra High Pressure Technology

Various non-viral gene systems, such as naked DNA, lipoplexes, micelles and polyplexes, have been developed for effective and safe gene delivery into target cells. Although cationic compounds were employed as gene carriers due to the ability of complex formation with DNA electrostatically and effective gene transfer into cells, the intrinsic cytotoxicity of them is essential problem in non-viral gene delivery system. Therefore, we have tried the development of DNA complex with non-ionic, water soluble polymers via hydrogen bond using ultra high pressure (UHP) technology because the inter-, intra- molecular week hydrogen bonding interaction was empathized with high pressure process. Previously, polyvinyl alcohol (PVA) was utilized as the model hydrogen bonding polymers, and the PVA/ DNA complexes were obtained by UHP treatment. Although the PVA/DNA complexes were up-taken by cells, a little enhancement of gene expression was observed using them. In this study, we hypothesize that inorganic particles, such as calcium phosphate (CP), calcium carbonate and hydroxy apatite (HAp), promote the endosomal escape of transferred DNA because the inorganic particles are dissolved under low pH condition in endosome vesicles and then the rupture of endosome is induced by osmotic shock. We performed the development of inorganic particle/PVA/DNA complexes using UHP technology. Plasmid DNAs encoding enhanced green fluorescent protein (EGFP) gene or luciferase gene under CMV promoter were used. Nano-HAps having the average diameter of 50nm were synthesized by modified micro-emulsion method. Nano- HAp was dispersed ultrasonically in PVA solution and then mixed with DNA solution. CP/DNA complexes were prepared by general method and mixed with PVA solution. Their mixtures were treated under 10000 atmospheric pressures at 40 degree for 10min. By SEM observation, the irregular surface of inorganic particles/PVA/ DNA complexes was observed, indicating the encapsulation of inorganic particles in PVA/DNA particle. The nano-HAp/PVA/DNA complexes showed a higher transfection activity than DNA complexes with nano-HAp or PVA. With CP/PVA/DNA complexes, also, the transfection activity increased several fold than the PVA/ DNA complexes. These results indicate the utility of the inorganic particle/PVA/DNA complexes prepared by the UHP treatment for DNA delivery. Various non-viral gene systems, such as naked DNA, lipoplexes, micelles and polyplexes, have been developed for effective and safe gene delivery into target cells. Although cationic compounds were employed as gene carriers due to the ability of complex formation with DNA electrostatically and effective gene transfer into cells, the intrinsic cytotoxicity of them is essential problem in non-viral gene delivery system. Therefore, we have tried the development of DNA complex with non-ionic, water soluble polymers via hydrogen bond using ultra high pressure (UHP) technology because the inter-, intra- molecular week hydrogen bonding interaction was empathized with high pressure process. Previously, polyvinyl alcohol (PVA) was utilized as the model hydrogen bonding polymers, and the PVA/ DNA complexes were obtained by UHP treatment. Although the PVA/DNA complexes were up-taken by cells, a little enhancement of gene expression was observed using them. In this study, we hypothesize that inorganic particles, such as calcium phosphate (CP), calcium carbonate and hydroxy apatite (HAp), promote the endosomal escape of transferred DNA because the inorganic particles are dissolved under low pH condition in endosome vesicles and then the rupture of endosome is induced by osmotic shock. We performed the development of inorganic particle/PVA/DNA complexes using UHP technology. Plasmid DNAs encoding enhanced green fluorescent protein (EGFP) gene or luciferase gene under CMV promoter were used. Nano-HAps having the average diameter of 50nm were synthesized by modified micro-emulsion method. Nano- HAp was dispersed ultrasonically in PVA solution and then mixed with DNA solution. CP/DNA complexes were prepared by general method and mixed with PVA solution. Their mixtures were treated under 10000 atmospheric pressures at 40 degree for 10min. By SEM observation, the irregular surface of inorganic particles/PVA/ DNA complexes was observed, indicating the encapsulation of inorganic particles in PVA/DNA particle. The nano-HAp/PVA/DNA complexes showed a higher transfection activity than DNA complexes with nano-HAp or PVA. With CP/PVA/DNA complexes, also, the transfection activity increased several fold than the PVA/ DNA complexes. These results indicate the utility of the inorganic particle/PVA/DNA complexes prepared by the UHP treatment for DNA delivery.

Incorporation of quantum dots on virus in polycationic solution

Developing methods to label viruses with fluorescent moieties has its merits in elucidating viral infection mechanisms and exploring novel antiviral therapeutics. Fluorescent quantum dots (QDs), an emerging probe for biological imaging and medical diagnostics, were employed in this study to tag retrovirus encoding enhanced green fluorescent protein (EGFP) genes. Electrostatic repulsion forces generated from both negatively charged retrovirus and QDs were neutralized by cationic Polybrene, forming colloidal complexes of QDs-virus. By examining the level of EGFP expression in 3T3 fibroblast cells treated with QDs-tagged retroviruses for 24 hours, the infectivity of retrovirus incorporated with QDs was shown to be only slightly decreased. Moreover, the imaging of QDs can be detected in the cellular milieu. In summary, the mild method developed here makes QDs-tagged virus a potential imaging probe for direct tracking the infection process and monitoring distribution of viral particles in infected cells.

Dkk1 Transgenic Mice for the Study of Bone Lesions in Human Multiple Myeloma.

Purpose: Extracellular antagonists of the cellular WNT signalling pathway can be divided into two classes depending on the mechanism by which ligand-receptor interactions are inhibited. Members of the first class including SFRP (secreted frizzled-related proteins), WIF1 (WNT inhibitory factor 1) and CER1 (cerberus homolog 1) bind to WNT proteins in the extracellular milieu. Members of the second class including Dickkopf (DKK) proteins bind to the WNT receptor subunits LRP5/6. Reduced WNT signalling may play a role in the natural history of the bone disease that is a hallmark of multiple myeloma (MM). Gene expression profiling of MM plasma cells has shown that over-expression of the Dickkopf family member DKK1 is linked with abundance and size of osteolytic lesions (N Engl J Med 2003, 349:2483–94). The biological mechanism of DKK1-mediated bone destruction is not known.Experimental procedure: To elucidate the mechanism by which DKK1 facilitates osteolysis, we generated transgenic mice in which mouse Dkk1 is expressed under control of the mouse immunoglobulin Eα enhancer. This enhancer is located 3′ of the immunoglobulin heavy-chain locus and comprised of four individual elements that span ~30 kb of genomic DNA. These elements were subcloned into a contiguous stretch of DNA, which we refer to as “reconstituted” Eα enhancer or HS1234 (Int Immunol 2001, 13:1003–12). Here we appended HS1234 to a Dkk1 cDNA whose expression is controlled by a mouse immunoglobulin VH promoter inserted 5′ of the transcriptional start site of Dkk1. Because of an internal ribosome entry site (IRES) 3′ of Dkk1, the gene encoding enhanced green fluorescence protein (EGFP) is co-expressed with Dkk1; i.e., it is a reporter of Dkk1 expression. A scheme of the structure of the Eα-Dkk1 transgene is depicted in Figure 1. [Display omitted] Results: We generated 14 founder lines of Eα-Dkk1 transgenic mice that expressed Dkk1 and the EGFP-encoding gene by RT-PCR (Figure 2). [Display omitted] The mice contained a variable number of Eα-Dkk1 copies, ranging from 2–3 to more than 100. There was but a loose association of transgene expression (qPCR of Dkk1 mRNA levels) and copy number (qPCR of genomic Ea-Dkk1 fragments); i.e., the high-copy number mice exhibited higher average mRNA levels than the low-copy number mice, although there were outliers in each group. Consistent with Eα-dependent restriction of Dkk1 expression to B/plasma cells, Dkk1 mRNA was readily detectable in lymphoid tissues, such as spleen, mesenteric lymph node and bone marrow, but not in liver.Conclusion: The newly developed Eα-Dkk1 transgenics may provide a valuable model system to elucidate DKK1-dependent bone disease in human MM.

Rational attenuation of a morbillivirus by modulating the activity of the RNA-dependent RNA polymerase.

Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPV(KO)) can be rescued. A recombinant virus, rRPV(KO)L-RRegfpR, which grew at an indistinguishable rate and to an identical titer as rRPV(KO) in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 10(4) TCID(50) of the rRPV(KO) parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPV(KO)L-RRegfpR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo.

Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows

Porcine mammary epithelial cells (PMECs) were isolated from lactating sow mammary glands and cultured on a matrix gel. Primary culture cells expressed significant amounts of the specific marker cytokeratin as determined by immunohistochemistry, and exhibited mammary-specific functions, such as transcription of alpha-lactalbumin, beta-casein and beta-lactoglobulin genes. They also formed mammospheres when the medium was supplemented with lactogenic hormones. The PMECs were used to study gene transfer and expression in vitro. A gene encoding enhanced green fluorescent protein (EGFP) was used as a reporter and two constructs were investigated, pEGFP-N1 (a vector constructed with a CMV promoter followed by the EGFP gene) and pGB562/GFP (a mammary gland-specific expression vector with regulatory sequences from the goat beta-casein gene linked to EGFP). The efficiency of DNA transfer into the cultured PMECs was about 20-30%. GFP expression in the pGB562/GFP-transfected PMECs was markedly stimulated by prolactin supplements in the medium. The established PMECs maintained optimal gene expression from 1 to 20 passages and appeared to provide an efficient and convenient system for assessing the expression of transgenes containing mammary gland-specific promoters.

655. Compacted DNA Nanoparticles Effectively Transfect Brain Cells In Vitro and In Vivo

This study determined the feasibility of using DNA compacted into unimolecular nanoparticles as a gene therapy delivery system for post-mitotic brain cells. DNA compacted with polyethylene glycol (PEG)-substituted 30-mer lysine peptides has been shown to effectively transfect multiple types of non-dividing cells and tissues, including growth-arrested neuroblastoma cells, and may be a viable system for the delivery of neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), to dopaminergic neurons. This formulation of DNA nanoparticles is non-inflammatory and non-immunogenic in peripheral organs, and therefore may circumvent the problem of immunogenicity that is inherent with some viral vectors. In this study we examined whether specific nanoparticle formulations of single DNA molecules encoding enhanced green fluorescent protein (EGFP) are capable of transfecting brain cells in primary ventral midbrain cultures or after direct injection into the rat striatum. Ellipsoidal and rod-like formulations of DNA nanoparticles were prepared by using lysine peptides having either trifluoroacetate (TFA) or acetate, respectively, as the polycation counterion at the time of DNA mixing. Primary ventral midbrain cultures were maintained for 5 days. During the first 4 days, cultures were exposed to DNA compacted into the acetate rod nanoparticles [250 ng/well; 5.0 |[times]| 1010 particles]; fresh media containing nanoparticles was added to the cultures on a daily basis. On the fifth day in vitro (DIV 5), |[sim]|99% of the cells expressing either the neural marker NeuN or the glial marker glial fibrillary acidic protein (GFAP) were also EGFP+; similarly, most tyrosine hydroxylase-positive cells also co-expressed EGFP. In a second study, ellipsoidal and rod-like nanoparticles were injected directly into the striatum of young adult rats and 4-5 days later we observed EGFP expression in |[sim]|50% of both neurons and glia located in the brain tissue surrounding the injection site. There was no evidence of an inflammatory cell infiltrate at the injection site. In conclusion, the results from these in vitro and in vivo studies suggest that compacted DNA nanoparticles can be used as a non-viral gene therapy technique to efficiently transfect post-mitotic brain cells and induce transgene expression in both neurons and glia. This study was supported by NS42862 (NINDS/NIH) and DAMD 17-01-1-0786 (DOD).

Survival, Integration, and Axon Growth Support of Glia Transplanted into the Chronically Contused Spinal Cord

Due to an ever-growing population of individuals with chronic spinal cord injury, there is a need for experimental models to translate efficacious regenerative and reparative acute therapies to chronic injury application. The present study assessed the ability of fluid grafts of either Schwann cells (SCs) or olfactory ensheathing glia (OEG) to facilitate the growth of supraspinal and afferent axons and promote restitution of hind limb function after transplantation into a 2-month-old, moderate, thoracic (T8) contusion in the rat. The use of cultured glial cells, transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP), permitted long-term tracking of the cells following spinal cord transplantation to examine their survival, migration, and axonal association. At 3 months following grafting of 2 million SCs or OEG in 6 microl of DMEM/F12 medium into the injury site, stereological quantification of the three-dimensional reconstructed spinal cords revealed that an average of 17.1 +/- 6.8% of the SCs and 2.3 +/- 1.4% of the OEG survived from the number transplanted. In the OEG grafted spinal cord, a limited number of glia were unable to prevent central cavitation and were found in patches around the cavity rim. The transplanted SCs, however, formed a substantive graft within the injury site capable of supporting the ingrowth of numerous, densely packed neurofilament-positive axons. The SC grafts were able to support growth of both ascending calcitonin gene-related peptide (CGRP)-positive and supraspinal serotonergic axons and, although no biotinylated dextran amine (BDA)-traced corticospinal axons were present within the center of the grafts, the SC transplants significantly increased corticospinal axon numbers immediately rostral to the injury-graft site compared with injury-only controls. Moreover, SC grafted animals demonstrated modest, though significant, improvements in open field locomotion and exhibited less foot position errors (base of support and foot rotation). Whereas these results demonstrate that SC grafts survive, support axon growth, and can improve functional outcome after chronic contusive spinal cord injury, further development of OEG grafting procedures in this model and putative combination strategies with SC grafts need to be further explored to produce substantial improvements in axon growth and function.

Efficient gene transfer into murine embryonic stem cells by nucleofection

Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.

Targeted in vivo expression of proteins in the calyx of Held.

The calyx of Held serves as a model for synaptic transmission in the mammalian central nervous system. While offering unique access to the biophysics of presynaptic function, studies addressing the molecular mechanisms of neurotransmitter exocytosis in this model have been mainly limited to pharmacological interventions. To overcome this experimental limitation we used stereotaxic delivery of viral gene shuttles to rapidly and selectively manipulate protein composition in the calyx terminal in vivo. Sindbis or Semliki Forest viruses encoding enhanced green fluorescent protein (EGFP) were injected into the ventral cochlear nucleus (VCN) of rats (postnatal days 7-21) and yielded bright fluorescence in cells of the VCN, including globular bushy cells with their axon and calyx terminal. Fluorescence imaging and three dimensional reconstructions visualized developmental changes in calyx morphology. Small cytoplasmic and synaptic vesicle proteins were successfully overexpressed in the calyx. We extended two-photon microscopy to obtain simultaneous fluorescence and infrared scanning gradient contrast images, allowing for efficient patch-clamp recordings from EGFP-labelled calyces in acute brain slices (postnatal days 9-14). Recordings of spontaneous miniature excitatory postsynaptic currents and short-term depression in synapses overexpressing EGFP or synaptophysin-EGFP revealed normal synaptic function. Thus, Sindbis and Semliki Forest virus-directed overexpression of proteins in the calyx of Held provides a new avenue for molecular structure-function studies of mammalian central synapses.

A piggyBac transposon gene trap for the analysis of gene expression and function in Drosophila.

P-element-based gene and enhancer trap strategies have provided a wealth of information on the expression and function of genes in Drosophila melanogaster. Here we present a new vector that utilizes the simple insertion requirements of the piggyBac transposon, coupled to a splice acceptor (SA) site fused to the sequence encoding enhanced green fluorescent protein (EGFP) and a transcriptional terminator. Mobilization of the piggyBac splice site gene trap vector (PBss) was accomplished by heat-shock-induced expression of piggyBac transposase (PBase). We show that insertion of PBss into genes leads to fusions between the gene's mRNA and the PBss-encoded EGFP transcripts. As heterozygotes, these fusions report the normal pattern of expression of the trapped gene. As homozygotes, these fusions can inactivate the gene and lead to lethality. Molecular characterization of PBss insertion events shows that they are single copy, that they always occur at TTAA sequences, and that splicing utilizes the engineered splice site in PBss. In those instances where protein-EGFP fusions are predicted to occur, the subcellular localization of the wild-type protein can be inferred from the localization of the EGFP fusion protein. These experiments highlight the utility of the PBss system for expanding the functional genomics tools that are available in Drosophila.

Stable transgene expression in tumors and metastases after transduction with lentiviral vectors based on human immunodeficiency virus type 1.

The relatively low efficiency of target cell transduction and variations in the stability of transgene expression by retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are major impediments to the use of such vectors in cancer gene therapy approaches. The present study was designed to investigate the stability and efficiency of transgene expression in human lung and breast cancer cell lines transduced with vectors based on human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo in nude mouse models of metastasis. H460 lung carcinoma cells and MDA-MB-231 breast carcinoma cells were transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP) and beta-galactosidase (beta-Gal), respectively. Transduced H460 cells were administered to nude mice by either intravenous or subcutaneous injection and MDA-MB-231 cells were implanted orthotopically into the mammary fat pad of such mice to induce primary tumor and metastatic lung tumor formation. High-level EGFP expression was maintained in transduced H460 cells in metastatic lung nodules for up to 6 weeks and transgene expression in vitro persisted for at least 23 days after retrieval of EGFP-positive H460 cells from the lungs of tumor-bearing mice and subsequent cultivation in vitro. Likewise, beta-Gal expression levels in metastatic MDA-MB-231 cells in lungs remained high for up to 11 weeks. Southern blot analyses carried out with DNA from lung nodules showed that proviral DNAs in H460 cells were maintained stably over many cell generations and during subsequent reimplantation in vivo. However, molecular analyses revealed variations in transgene copy numbers and expression levels among individual lung clones. These results demonstrate the usefulness of HIV-1-based lentiviral vectors for sustained and stable transgene expression in human lung and breast cancer cell lines in vitro and in vivo.

Capturing and profiling adult hair follicle stem cells

The hair follicle bulge possesses putative epithelial stem cells. Characterization of these cells has been hampered by the inability to target bulge cells genetically. Here, we use a Keratin1-15 (Krt1-15, also known as K15) promoter to target mouse bulge cells with an inducible Cre recombinase construct or with the gene encoding enhanced green fluorescent protein (EGFP), which allow for lineage analysis and for isolation of the cells. We show that bulge cells in adult mice generate all epithelial cell types within the intact follicle and hair during normal hair follicle cycling. After isolation, adult Krt1-15-EGFP-positive cells reconstituted all components of the cutaneous epithelium and had a higher proliferative potential than Krt1-15-EGFP-negative cells. Genetic profiling of hair follicle stem cells revealed several known and unknown receptors and signaling pathways important for maintaining the stem cell phenotype. Ultimately, these findings provide potential targets for the treatment of hair loss and other disorders of skin and hair.

Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice.

We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained in a normothermic state or cooled to 25 degrees C. The aorta or both the pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase type VI (AC(VI)) with saline, sodium nitroprusside, acetylcholine, or serotonin. Clamping was maintained for 30 s (normothermia) or 2 min (25 degrees C) after adenovirus administration. Mice were killed 7 or 21 days later, and hearts were examined for EGFP expression. Compared with clamping the aorta alone and with no cooling, gene transfer was increased as follows: 1) 1.3-fold with hypothermia to extend dwell time; 2) 4.5-fold by clamping the aorta and the pulmonary artery; 3) 11.4-fold with nitroprusside administration; 4) 11.8-fold with serotonin addition, and 5) 14.3-fold with acetylcholine delivery. Gene expression remained substantial at 21 days, and no significant inflammatory response was seen. Efficacy of the method was tested by performing gene transfer of adenovirus encoding AC(VI). Fourteen days after gene transfer, hearts isolated from mice that received adenovirus encoding AC(VI) showed increased contractile function. Indirect intracoronary delivery of adenovirus vectors in mice is associated with efficient cardiac gene transfer and increased left ventricular function after AC(VI) gene transfer.

Effects of human locus control region elements HS2 and HS3 on human β-globin gene expression in transgenic mouse

The locus control region (LCR) is the most important cis -element in the regulation of β-globin gene expression. DNaseI-hypersensitive site (HS) 2 and HS3 are two significant components of β-LCR. To examine the effect of HS2, HS3, and HS2–HS3 (combination of HS2 and HS3) on the spatial and temporal expression of the human β-globin gene, we have produced transgenic mice with constructs, in which the gene encoding enhanced green fluorescent protein (EGFP) is driven by β-globin promoter and under the control of HS2, HS3, and HS2–HS3, respectively. The results showed that HS2 and HS3 each had the same enhancement activity in regulation of β-globin gene expression in transgenic mice. When HS2 and HS3 were in combination (HS2–HS3), the two cis -elements showed a marked synergy in regulating β-globin gene spatial and temporal expression as well as its expression level in transgenic mice although the EGFP expression varied largely among different transgenic mouse litters. The results also showed that HS2 was able to confer β-globin gene expression in embryonic yolk sac, fetal liver, and adult bone marrow, which was not developmentally stage-specific, while HS3 could confer the same β-globin gene expression in the adult. Thus, HS3 was different from HS2, the former being more important for specific expression of β-globin gene in the developmental stages and the switch of γ→β-globin genes. Our results indicate that the mechanism of γ→β switch could be best explained by the “divided model.”

Effects of human locus control region elements HS2 and HS3 on human β-globin gene expression in transgenic mouse

The locus control region (LCR) is the most important cis-element in the regulation of β-globin gene expression. DNaseI-hypersensitive site (HS) 2 and HS3 are two significant components of β-LCR. To examine the effect of HS2, HS3, and HS2–HS3 (combination of HS2 and HS3) on the spatial and temporal expression of the human β-globin gene, we have produced transgenic mice with constructs, in which the gene encoding enhanced green fluorescent protein (EGFP) is driven by β-globin promoter and under the control of HS2, HS3, and HS2–HS3, respectively. The results showed that HS2 and HS3 each had the same enhancement activity in regulation of β-globin gene expression in transgenic mice. When HS2 and HS3 were in combination (HS2–HS3), the two cis-elements showed a marked synergy in regulating β-globin gene spatial and temporal expression as well as its expression level in transgenic mice although the EGFP expression varied largely among different transgenic mouse litters. The results also showed that HS2 was able to confer β-globin gene expression in embryonic yolk sac, fetal liver, and adult bone marrow, which was not developmentally stage-specific, while HS3 could confer the same β-globin gene expression in the adult. Thus, HS3 was different from HS2, the former being more important for specific expression of β-globin gene in the developmental stages and the switch of γ→β-globin genes. Our results indicate that the mechanism of γ→β switch could be best explained by the “divided model.”

Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts

Miracidia (and adults) of Schistosoma mansoni which had been subjected to particle bombardment with a plasmid DNA encoding enhanced green fluorescent protein (EGFP) under control of the S. mansoni heat shock protein 70 (HSP70) promoter and termination elements were shown to express the reporter gene. Bombarded miracidia were able to penetrate and establish in Biomphalaria glabrata the intermediate host snail. Gold particles could be detected in the germ balls of parasites in paraffin-sections of snail tissue. The bombarded miracidia were able to develop normally and to transform into mother sporocysts. Reporter gene activity could be determined at 10 days post-infection by RT-PCR in snail tissues, but not by microscopy or Western blot which probably reflected sub-optimal expression levels of constructs. Our findings indicated that it is feasible to return transgenic miracidia to the life cycle, a crucial step for the establishment of a transgenesis system for schistosomes. Index Descriptors and Abbreviations: schistosome, miracidium, sporocyst, particle bombardment, gold particles, transgene, Biomphalaria glabrata; germ ball; EGFP, enhanced green fluorescent protein; RT-PCR, reverse transcription-polymerase chain reaction; gDNA, genomic DNA; PBS, phosphate buffered saline; TBS, Tris buffered saline; FCS, fetal calf serum; DEAE, diethylaminoethyldextran

Promoter interference mediated by the U3 region in early-generation HIV-1-derived lentivirus vectors can influence detection of transgene expression in a cell-type and species-specific manner.

In a previous study using an early-generation VSV-G-pseudotyped lentivirus vector encoding enhanced green fluorescent protein (EGFP) under the transcriptional control of a human cytomegalovirus (CMV) immediate-early promoter, we examined transduction efficiency in dissociated dorsal root ganglia (DRG) cultures. In cultures of murine origin, transgene expression was observed solely in the sensory neurons with the stromal cell population failing to show evidence of transduction. In contrast, efficient and sustained transduction of both sensory neurons and the stromal cell population was observed in cultures of human origin. Given the widespread use of murine models in preclinical gene therapy studies, in the current study we investigated the basis of this apparent neuron specificity of lentivirus-mediated transduction in murine DRG cultures. The interspecies differences persisted at high multiplicities of infection, and irrespective of whether lentiviral vector stocks were packaged in the presence or absence of human immunodeficiency virus type 1 (HIV-1) accessory proteins. Cell-type specificity of CMV promoter expression, tropism of the VSV-G envelope, and blocks to molecular transduction were also precluded as possible mechanisms, thereby implicating transcriptional repression of the internal heterologous promoter. This promoter interference effect was found to be mediated by cis-acting sequences upstream of the core promoter elements located in the U3 region of the proviral long terminal repeats (LTRs). Deletion of this region, as in late-generation self-inactivating (SIN) lentivirus vectors, relieves this effect. This provides a basis for reevaluating data produced using early-generation U3-bearing lentivirus vectors and for reconciling these with results obtained using more contemporary SIN lentivirus vectors carrying a U3 deletion.

Highly efficient lentiviral gene transfer in CD34+ and CD34+/38-/lin- cells from mobilized peripheral blood after cytokine prestimulation.

Because mobilized peripheral blood (mPB) represents an attractive source of cells for gene therapy, we investigated lentiviral gene transfer in CD34(+) cells and the stem/progenitor-cell-enriched CD34(+)/38(-)/lin(-) cell subset isolated from mPB. In this study, we used an optimized third-generation self-inactivating lentiviral vector containing both the central polypurine tract and the woodchuck hepatitis posttranscriptional regulatory element sequences and encoding enhanced green fluorescent protein (EGFP) under the control of the elongation factor lalpha promoter. This lentivector was first used to compare multiplicity of infection (MOI)-dependent gene transfer efficiency in cord blood (CB) versus mPB CD34(+)-derived cells, colony-forming cells (CFCs), and long-term culture-initiating cells (LTC-ICs). Results showed a difference in the percentage of transduced cells particularly significant at low MOIs. A plateau was reached where 15% and 25% of CB and mPB cells, respectively, remained refractory to lentiviral trans-duction. Effects of a cytokine prestimulation period (18 hours) with interleukin-3, stem cell factor, Flt-3 ligand, and thrombopoietin were then analyzed in total cells, CFCs, and LTC-ICs derived from mPB CD34(+) cells. Transduction levels in those conditions demonstrated a two- and fourfold increase in CFCs and LTC-ICs, respectively, compared with unstimulated (<3 hours) control cells. Moreover, using the same transduction protocol, we were able to efficiently transduce CD34(+)/38(-)/lin(-) cells isolated from mPB, with up to >85% of colonies derived from LTC-ICs expressing EGFP and gene transfer levels remaining stable for 10 weeks in liquid culture. We therefore demonstrate a highly efficient gene transfer in this therapeutically relevant target cell population.

DNA immunization via intramuscular and intradermal routes using a gene gun provides different magnitudes and durations on immune response

We investigated the antibody (Ab) and cytotoxic T lymphocyte (CTL) responses to gene gun (GG)-mediated DNA immunization via the intramuscular (i.m.) and intradermal (i.d.) routes. BALB/c mice were immunized five times at weekly intervals with plasmid DNA encoding enhanced green fluorescent protein (EGFP). EGFP production was rapidly detected in the target tissues after injection via either delivery route. There were significant differences in the magnitude and duration of the Ab and CTL responses according to the route employed. Intradermal injection elicited higher Ab and CTL responses to EGFP than i.m. injection 1 week after the last immunization. However, both immune responses were reduced rapidly 5 weeks after the last immunization via i.d. injection. In contrast, in mice injected via the i.m. routes, Ab and CTL responses 5 weeks after the last immunization remained at levels similar to those detected after 1 week. All mice generated a predominantly IgG1 Ab response via either route. These findings suggest that a combination of these two routes of DNA immunization would provide optimal conditions for induction of a broad immune response, and this information is expected to be very important for future applications of DNA vaccination.