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At least 37 records · Page 2Linked to original sources
PubMed1998

Improved survival of biolistically transfected mouse islet allografts expressing CTLA4-Ig or soluble Fas ligand.

BACKGROUND: Pancreatic islet transplantation is limited because of immune rejection of the transplanted tissue. Long-term survival of allogeneic pancreatic islet grafts in the absence of systemic immunosuppressive agents should be possible by transfecting the islets directly with DNA encoding immunoregulatory molecules. Localized production of these molecules should affect only the immune cells that come into the vicinity of the foreign tissue. We investigated whether local expression of human CTLA4-Ig or soluble human Fas ligand from biolistically transfected mouse islets would have a protective effect on allograft survival. METHODS: Isolated CBA (H2k) islets were biolistically transfected using the gene gun. The experimental groups were naked gold particles (n=6), empty vector DNA (n=5), DNA encoding human CTLA4-Ig (n=8), or soluble human Fas ligand (n=5). Secretion of the transfected gene product was confirmed by screening islet culture supernatants for protein production using a sandwich ELISA. The blasted islets were transplanted under the kidney capsule of alloxan-diabetic BALB/c (H2d) recipients. RESULTS: Control grafts survived for 23 days, on average. CTLA4-Ig-transfected islets showed a bimodal distribution: 50% of cases survived > or = 46 days and 50% were similar to the controls. In the soluble human Fas ligand group, 80% of grafts survived > or = 50 days. There was no correlation between graft survival times and pretransplant levels of protein production. CONCLUSION: Our results indicate that local production of human CTLA4-Ig or soluble human Fas ligand by biolistically transfected islets can promote allograft survival. This approach should be valuable as a potential immunoprotective therapeutic strategy in tissue transplantation.

Abatacept↗
PubMed1997

Transient expression of the beta-glucuronidase gene after biolistic transformation of the anaerobic fungus Neocallimastix frontalis.

The rumen anaerobic fungus Neocallimastix frontalis was biolistically transformed using plasmids containing the bacterial beta-glucuronidase gene (GUS) fused to the promoter sequences of the enolase gene from N. frontalis. Multiple copies of the plasmids were precipitated onto tungsten particles and delivered into zoosporangia and a mycelial mat by a helium-driven biolistic device. Transformants were detected by histochemical assay for beta-glucuronidase. It was found that the enolase promoter sequences tested were responsible for the transient expression of the beta-glucuronidase gene. This is the first study presenting results on the transformation of an anaerobic fungus.

Anaerobiosis↗
PubMed2002

Genetic transformation of two species of orchid by biolistic bombardment.

We report here the transformation of two species of orchid, Dendrobium phalaenopsis and D. nobile,by biolistic bombardment. Calli or protocorm-like bodies (PLBs) were used as target explants. Gold particles (1.0 microm) coated with plasmid DNA (pCAMBIA1301) encoding an intron-containing beta-glucuronidase gene (gus-int) and a hygromycin phosphotransferase (hpt) gene were introduced into the PLBs or calli using the Bio-Rad PDS-1000/He Biolistic Particle Delivery System. Calli and PLBs were then chopped up and pre-cultured in 1/2-strength MS medium supplemented with 0.4 M mannitol for a 1-h osmoticum treatment before bombardment. Immediately after bombardment, the calli and PLBs were transferred to 1/2-strength MS medium without mannitol for recovery. Putatively transformed plantlets were obtained by selection and regeneration on medium supplemented with 30 mg/l hygromycin. The highest efficiency of transformation was obtained when selection was conducted at 2 days post-bombardment. For D. phalaenopsis and D. nobile, respectively, about 12% and 2% of the bombarded calli or PLBs produced independent transgenic plants. Integration and expression of the transgenes were confirmed by Southern hybridization and Northern hybridization. No nontransformed plants were regenerated, indicating a tight selection scheme. However, separate incorporation of the gus gene and the hpt gene was observed, and in one transgenic line the gus gene was integrated into the genome of the transgenic plant, but not expressed.

Biolistics↗
PubMed2003

High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' (Vitis vinifera L.) containing npt-II and antimicrobial peptide genes.

A reliable and efficient system for transformation and regeneration of 'Chardonnay' (Vitis vinifera L.) plants via microprojectile bombardment was developed. Improvements over the previous biolistic transformation system included: (1) the use of gold particles for bombardment; (2) step-wise selection at 10 then 15 mg/l kanamycin; and (3) embryo induction at 27 degrees C. Embryogenic cell cultures were either bombarded with pBI426, which contains the reporter gene gus (uidA) coding for beta-glucuronidase (GUS), or were co-bombarded with pSAN237 carrying the npt-II (neomycin phosphotransferase II) selectable marker gene, and a second plasmid with an antimicrobial peptide gene. A large number of transient (7,883 +/- 1,928) and stable (46 +/- 32) blue spots per plate at 2 and 95 days after bombardment, respectively, were obtained according to GUS expression analyses. A total of 447 putative transgenic embryos was harvested from 84 bombarded plates. From these embryos, 242 (54%) were regenerated into plants within the first year of the experiment. Southern blot analyses confirmed integration of the transgenes into the grape genome. Co-transformation was tested with four separate antimicrobial constructs. The co-transformation frequency of unlinked genes was 48% as measured by polymerase chain reaction (PCR), and 56% as estimated by dot blot hybridization. Expression of the gus gene, and PCR and Southern blot analyses of npt-II and antimicrobial genes from regenerated plants document stable transformation of 'Chardonnay' and establish the parameters for highly-efficient biolistic transformation in V. vinifera.

Anti-Bacterial Agents↗
PubMed2004

A model of organotypic rat spinal slice culture and biolistic transfection to elucidate factors that drive the preprotachykinin-A promoter.

The tachykinin substance P (SP) is a neuropeptide that is expressed in some nociceptive primary sensory afferents and in discrete populations of spinal cord neurons. Expression of spinal SP and the preprotachykinin-A (PPT-A) gene that encodes SP exhibits plasticity in response to conditions such as peripheral inflammation but the mechanisms that regulate expression are poorly understood. We have developed a spinal cord organotypic culture system that is suitable for the analysis of PPT-A gene promoter activity following biolistic transfection of recombinant DNA constructs. Spinal cord organotypic slices showed good viability over a 7-day culture period. Immunostaining for phenotypic markers such as NeuN and beta-III tubulin demonstrated preservation of neurons and their structure, although there was evidence of axotomy-induced down-regulation of NeuN in certain neuronal populations. Neurokinin-1 receptor (NK-1R) immunostaining in laminae I and III was similar to that seen in acute slices. Biolistic transfection was used to introduce DNA constructs into neurons of these organotypic cultures. Following transfection with a construct in which expression of enhanced green fluorescent protein (EGFP) is controlled by the PPT-A promoter, we showed that induction of neuronal activity by administration of a forskolin analogue/high K(+) (10 microM/10 mM) for 24 h resulted in a fourfold increase in the number of EGFP-positive cells. Similarly, a twofold increase was obtained after treatment with the NK-1R-specific agonist [Sar(9),Met (O(2))(11)]-substance P (10 microM). These data demonstrate the usefulness of this model to study physiological and pharmacological factors relevant to nociceptive processing that can modulate PPT-A promoter activity.

Animals↗
PubMed2006

The use of biolistic inoculation of cassava mosaic begomoviruses in screening cassava for resistance to cassava mosaic disease.

Inoculation of cassava with infectious clones of cassava mosaic geminiviruses (Geminiviridae: Begomovirus) and total DNA extracts from plants infected with well-characterised viruses was evaluated using the Bio-Rad Helios Gene Gun System. Total DNA extracts from infected plants and cloned viruses were produced for coating gold particles and bombardment onto new cassava genotypes, 96/1089A, 96/1039, 96/0160, 96/0304 and three local landraces TME 117, TME 3 and TME 4. Cloned DNA of a Kenyan isolate of the recombinant variant of East African cassava mosaic virus (EACMV-UG2-[Ka]), was only infectious to TME 117 (7/10 plants), 3 weeks post-inoculation with mild infection symptoms in the newly developing leaves. Biolistic inoculation with a chimeric pseudorecombinant virus between DNA A and B components from EACMV-[Ke-Kilifi] and EACMV-UG2-[Ka], respectively, was infectious to TME 117, 96/1039 and 96/0304 and developed very severe and persistent symptoms. TME 3 and TME 4 also developed symptoms, 12 days post-inoculation (d.p.i.). Total DNA extracts of ACMV and EACMV-[Ke-Kilifi] resulted in serious infections with symptoms already evident, 10d.p.i. In general, biolistic inoculation trials with total DNA extracts resulted in a higher number of infected plants expressing symptoms at a much earlier stage (10-12d.p.i.) compared with trials inoculated with virus clones.

Begomovirus↗
PubMed2006

Biolistic inoculation of soybean plants with soybean dwarf virus.

Soybean dwarf virus (SbDV), a member of the genus Luteovirus, has been transmitted only by aphid vectors. The possibility of using a biolistic procedure of transmitting SbDV to soybean plants without relying on aphid vectors was investigated. Biolistic inoculation using the Helios Gene Gun System with RNA transcribed in vitro from a full-length cDNA clone of the DS strain of SbDV (pSV-DS) resulted in 1/12 to 3/13 soybean plants infected systemically. The infected soybean plants showed the characteristic symptoms of SbDV-DS within 6 weeks after inoculation and the accumulation of SbDV-specific RNA species such as genomic and subgenomic RNAs in the upper non-inoculated leaves. The progeny virus derived from RNA transcribed in vitro from pSV-DS could be transmitted by aphid vectors, as is the case with native SbDV-DS. This is the first report of direct inoculation of soybean plants with SbDV without using aphid vectors.

Biolistics↗
PubMed2003

Biolistic transfection.

Recent improvements in the biolistic technique and devices have increased its usefulness for transfection of neurons. With these recent advances, both dissociated and slice cultures can be transfected at reasonably high rates. This chapter focuses on the parameters that determine the successful biolistic transfection of neurons in both types of cultures.

Animals↗
PubMed1999

Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun.

DNA-transfer into postmitotic neurons or neuronal tissues has been a major problem in neurobiology. For this aim different methods have been used such as viral infection, microinjection, lipofection or calcium phosphate precipitation. However, using these techniques, very poor transfection efficiency was achieved except for virus-mediated gene transfer. Though viral infections are very efficient, this method is expensive and labor-intensive, especially when recombination is used to prepare viral vectors. Biolistic gene transfer of neurons represents another promising transfection technique. This technique was originally used to transfect plant cells and has been further developed for gene transfer into neurons or neuronal tissues. Up to now, only a few reports are available where successful biolistic gene transfer into neurons or neuronal tissues could be shown. Transfection efficiencies were only about 2%. Most of the previously published experiments were carried out under vacuum conditions using in-chamber gene gun types. Here we describe an improved method for efficient neuronal cell transfection using a hand-held gene gun. Expression vectors could be successfully transferred into dissociated cultured hippocampal neurons, PC12 cells, cultured cerebellar granule cells and cerebellar brain slices. In cerebellar granule cells and hippocampal neurons, transfection efficiencies of about 10% were reached.

Animals↗
PubMed2003

Preliminary attempts to biolistic inoculation of grapevine fanleaf virus.

Biolistics has been studied to inoculate grapevine fanleaf virus (GFLV), a Nepovirus, to its natural woody host, Vitis sp., and its herbaceous host, Chenopodium quinoa. At first, bombardment conditions for in vitro and greenhouse grown plants were set using the uidA reporter gene. The infectious feature of the cartridges was then evaluated by studying infection of C. quinoa plants. Systemic infection was obtained with either GFLV particles or RNA extracts in experimental conditions which gave also the highest transient uidA gene expression. Concerning grapevine, our results indicate that extrapolation to this plant is difficult. In only 1 out of 8 independent bombardment experiments done with GFLV and 41B, we were able to detect the virus in freshly bombarded leaves. Similarly, later after bombardment, Pol mRNAs were detected once, at days 7 and 14 only. Incubating the plants in darkness, as suggested in the literature, or using Rupestris Saint Georges, an indicator for GFLV presence, did not yield any improvement. Finely, our observations suggest that detection of GFLV in bombarded grapevine tissues by immunological or molecular techniques remains a limiting factor, probably due to an excess of inhibitory compounds released during the biolistic process.

Base Sequence↗
PubMed2001

The biolistic method as a tool for testing the differential activity of putative silkmoth chorion gene promoters.

Bombyx mori unpaired early chorion gene copies 6F6.1,.2 and.3 are exceptions to the typical organization and distribution pattern of known early ErA/ErB, middle A/B and late HcA/HcB divergently transcribed gene pairs. Contrary to such pairs, the boundaries of the 6F6 regulatory sequences are not easily defined; moreover, they share common sequence elements with the regulatory sequences of middle and late genes. In order to perform a functional study of the tissue and temporal specificity of the 6F6 putative promoter region, we decided to apply biolistics. In the present work, use of a region from the 6F6.2 5' untranslated sequence, spanning nucleotides -138 to the cap site, gave an expected expression pattern of a lacZ reporter gene. Temporal specificity was further verified by control experiments using the cloned intergenic sequence of the late gene pair HcA/B.12, which resulted in lacZ expression in late choriogenic follicles. At present, despite the recent successful germinal transgenesis of Bombyx mori, the biolistic transient expression system seems to be the most rapid technique to pursue the functional study of the promoter region of early chorion genes, including the three unconventional early 6F6 genes.

Animals↗
PubMed2003

Generation of rye (Secale cereale L.) plants with low transgene copy number after biolistic gene transfer and production of instantly marker-free transgenic rye.

Rye is extremely recalcitrant to tissue culture and genetic transformation. We describe the efficient and reproducible production of stably expressing transgenic rye plants after biolistic gene transfer to callus tissue derived from immature embryos. Key factors in the production of transgenic rye plants include the identification of biolistic gene transfer parameters and a selection protocol, which does not affect its regeneration ability. The bar gene was used as a selectable marker and selection was performed by spraying the regenerated shoots with 0.05% Basta solution without any previous selection of tissue cultures. Based on Southern blot analysis, a total of 21 transgenic rye plants with independent transgene integration patterns were produced. A low transgene copy number was observed in most transgenic plants and 40% of the plants had a single transgene copy insert. The high frequency of single transgene copy inserts might be a consequence of the selection system, which is based on the identification of stably expressing transgenic plantlets rather than stably expressing tissue cultures. All transgenic rye lines with single transgene inserts showed stable transgene expression in sexual progenies, but indications of transcriptional and post-transcriptional gene silencing were observed in few transgenic lines with multiple transgene inserts. Tissue culture-based selection was not necessary for the generation of transgenic rye. The identification of 17 transgenic rye plants without using any selectable marker gene by PCR amplification of transgene sequences is also demonstrated. Instant generation of selectable marker-free transgenic rye avoids a negative impact of selective agents on the transgenic tissue cultures, responds to public concerns on the safety of selectable markers and will support multiple transformation cycles for transgene pyramiding.

Aminobutyrates↗
PubMed1997

Eradication of tumor growth via biolistic transformation with allogeneic MHC genes.

Particle acceleration-mediated biolistics transformation has been rapidly adopted as a versatile technology for gene delivery since its original application as a physical method for delivery of foreign genes into higher plants. We have designed a versatile hand-held gene gun device for the genetic immunization of animals in vivo. The gene gun is driven by air blast and therefore does not require helium gas or vacuum for its function. In this report, we have employed this gene gun device to introduce allogeneic H-2Kb DNA directly into tumor nodules of K36 tumor-bearing AKR/J mice. Expression of the exogenous H-2Kb gene led to the regression of well-established K36 tumors. This tumor regression was correlated with the generation of potent secondary CTL responses against the K36 tumor cells following expression of the exogenous H-2Kb gene in the K36 tumor cells. Furthermore, it was observed that H-2Kb stimulator cells could stimulate the generation of both allogeneic and tumor-specific secondary CTL responses whereas mitomycin-C treated tumor cells could only stimulate tumor-specific secondary CTL responses. This approach of introducing allogeneic MHC genes directly into cutaneous tumor lesions via biolistics transformation of tumor cells in vivo can potentially be developed into an effective method for cancer gene therapy.

Animals↗
PubMed2001

Biolistic transformation of Arabidopsis root hairs: a novel technique to facilitate map-based cloning.

The final stage of map-based gene isolation is complementation of the mutant phenotype with wild-type DNA to determine the exact location of the gene of interest. This usually involves Agrobacterium tumefaciens-mediated transformation, which is reliable and produces stable transformants. However, the process of Agrobacterium transformation may take up to three months to complete. If the mutant phenotype can be seen in a single cell, and the wild-type copy of the gene can act cell autonomously, then complementation of the whole plant is not strictly necessary. We have developed a technique for the biolistic transformation of Arabidopsis thaliana root hairs, and used this to test large insert clones for complementation of two recessive mutant phenotypes, a procedure that takes less than a day. Our results show that biolistic transformation can be used with transient assays to conduct rapid tests for complementation by large insert clones.

Agrobacterium tumefaciens↗
PubMed2004

Homologous gene targeting in Caenorhabditis elegans by biolistic transformation.

Targeted homologous recombination is a powerful approach for genome manipulation that is widely used for gene alteration and knockouts in mouse and yeast. In Caenorhabditis elegans, several methods of target-selected mutagenesis have been implemented but none of them provides the opportunity of introducing exact predefined changes into the genome. Although anecdotal cases of homologous gene targeting in C.elegans have been reported, no practical technique of gene targeting has been developed so far. In this work we demonstrate that transformation of C.elegans by microparticle bombardment (biolistic transformation) can result in homologous recombination between introduced DNA and the chromosomal locus. We describe a scaled up version of biolistic transformation that can be used as a method for homologous gene targeting in the worm.

Animals↗
PubMed2002

Extending chestnut blight hypovirus host range within diaporthales by biolistic delivery of viral cDNA.

Biolistic bombardment was used to successfully transform three phytopathogenic fungal species with an infectious cDNA clone of the prototypic hypovirus, CHV1-EP713, a genetic element responsible for the virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica. The fungal species included two strains each of C. parasitica and Valsa ceratosperma, as well as one strain of Phomopsis G-type (teleomorph Diaporthe Nitschke); all are members of the order Diaporthales but classified into three different genera. A subset of transformants for each of the fungal species contained CHV1-EP713 dsRNA derived from chromosomally integrated viral cDNA. As has been reported for CHV1-EP713 infection of the natural host C parasitica, biolistic introduction of CHV1-EP713 into the new fungal hosts V ceratosperma and Phomopsis G-type resulted in altered colony morphology and, more importantly, reduced virulence. These results suggest a potential for hypoviruses as biological control agents in plant-infecting fungal pathogens other than the chestnut blight fungus and closely related species. In addition, the particle delivery technique offers a convenient means of transmitting hypoviruses to potential host fungi that provides new avenues for fundamental mycovirus research and may have practical applications for conferring hypovirulence directly on infected plants in the field.

Ascomycota↗
PubMed1997

Expression of CTLA4-Ig by biolistically transfected mouse islets promotes islet allograft survival.

BACKGROUND: Localized delivery of immunosuppressive molecules, limited to the graft site, may allow transplantation of tissue in the absence of systemic immunosuppressive agents. We tested whether purified mouse islets that had been engineered to produce human CTLA4-Ig locally at the graft site could survive in allogeneic recipients receiving no systemic immunosuppression. METHODS: CBA (H2(k)) islets were subjected to biolistic (gene gun) transfection with a cDNA encoding human CTLA4-Ig under control of the human cytomegalovirus immediate early promoter. After 40-48 hr of culture, the transfected islets (500 per recipient) were transplanted beneath the renal capsule of alloxan-induced diabetic BALB/c (H2(d)) recipients. RESULTS: Control grafts (n=10) consisting of islets biolistically transfected with the expression plasmid alone (i.e., no gene inserted) survived for 12.8+/-3.6 (mean +/- SD) days. In contrast, islets transfected with CTLA4-Ig (n=12) survived 66.8+/-61.5 days (P=0.01), with 50% demonstrating functional survival until follow-up was concluded at 50 (n=2), 130 (n=2), or 165 (n=2) days. Immunohistochemistry on grafts that survived long term showed well-granulated, insulin-positive islets lying adjacent to, but not infiltrated by, dense aggregates of mononuclear cells. CONCLUSIONS: Transfection of allogeneic mouse islets with human CTLA4-Ig results in prolonged allograft survival. Although on histology mononuclear cells are present in the area of the transfected graft, they do not appear to infiltrate or destroy the islet graft.

Abatacept↗
PubMed2006

Biolistic delivery of Ca2+ dyes into plant and algal cells.

In eukaryotes, changes in cytosolic Ca2+ concentrations ([Ca2+]cyt) are associated with a number of environmental and developmental stimuli. However, measuring [Ca2+]cyt changes in single plant or algal cells is often problematic. Although a wide range of Ca2+-sensitive fluorescent dyes is available, they are often difficult to introduce into plant cells. Micro-injection is the most robust method for dye loading, but is time-consuming, technically demanding, and unsuitable in many cell types. To overcome these problems, we have adapted biolistic techniques to load Ca2+-sensitive dyes into guard cells of the flowering plant, Commelina communis, cells of the green alga Chlamydomonas reinhardtii, and zygotes of the brown alga, Fucus serratus. Using this approach, we have been able to monitor [Ca2+]cyt changes in response to various stimuli, including a novel [Ca2+]cyt response in C. reinhardtii. The method allows the use of free acid and dextran-conjugated dyes. Biolistic loading of differentiated plant cells is easier, quicker, and more widely applicable than micro-injection, and should broaden the study of plant signal transduction.

Animals↗