Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

Background

Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles.

Results

Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that < 10% of nuclei were damaged in nanoparticle-transfected samples, compared to > 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles.

Conclusions

We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.     

Regulation of the distribution of carotenoid droplets in goldfish xanthophores and possible implication to secretory processes

In goldfish xanthophores, the formation of pigment aggregate requires: 1) that a pigment organelle (carotenoid droplet) protein p57 be in the unphosphorylated state; 2) that self-association of pigment organelles occur in a microtubule-independent manner; and 3) that pigment organelles via p57 associate with microtubules. In the fully aggregated state, the pigment organelles are completely stationary. Pigment dispersion is initiated by activation of a cAMP-dependent protein kinase, which phosphorylates p57 and allows pigment dispersion via an active process dependent on F-actin and a cytosolic factor. This factor is not an ATPase, and its function is unknown. However, its abundance in different tissues parallels secretory activity of the tissues, suggesting a similarity between secretion and pigment dispersion in xanthophores. The identity of the motor for pigment dispersion is unclear. Experimental results show that pigment organelles isolated from cells with dispersed pigment have associated actin and ATPase activity comparable to myosin ATPase. This ATPase is probably an organelle protein of relative molecular mass approximately 72,000, and unlikely to be an ion pump. Isolated pigment organelles without associated actin have 5x lower ATPase activity. Whether this organelle ATPase is the motor for pigment dispersion is under investigation. The process of pigment aggregation is poorly understood, with conflicting results for and against the involvement of intermediate filaments.     

Assessing horizontal transfer of nifHDK genes in eubacteria: nucleotide sequence of nifK from Frankia strain HFPCcI3

The structural genes for nitrogenase, nifK, nifD, and nifH, are crucial for nitrogen fixation. Previous phylogenetic analysis of the amino acid sequence of nifH suggested that this gene had been horizontally transferred from a proteobacterium to the gram-positive/cyanobacterial clade, although the confounding effects of paralogous comparisons made interpretation of the data difficult. An additional test of nif gene horizontal transfer using nifD was made, but the NifD phylogeny lacked resolution. Here nif gene phylogeny is addressed with a phylogenetic analysis of a third and longer nif gene, nifK. As part of the study, the nifK gene of the key taxon Frankia was sequenced. Parsimony and some distance analyses of the nifK amino acid sequences provide support for vertical descent of nifK, but other distance trees provide support for the lateral transfer of the gene. Bootstrap support was found for both hypotheses in all trees; the nifK data do not definitively favor one or the other hypothesis. A parsimony analysis of NifH provides support for horizontal transfer in accord with previous reports, although bootstrap analysis also shows some support for vertical descent of the orthologous nifH genes. A wider sampling of taxa and more sophisticated methods of phylogenetic inference are needed to understand the evolution of nif genes. The nif genes may also be powerful phylogenetic tools. If nifK evolved by vertical descent, it provides strong evidence that the cyanobacteria and proteobacteria are sister groups to the exclusion of the firmicutes, whereas 16S rRNA sequences are unable to resolve the relationships of these three major eubacterial lineages.      

Fate of L-(3,5- 3H) tyrosine in cell-free extracts and tissue cultures of melanoma cells: a new assay method for tyrosinase in living cells

On incubation of l-[3,5-³H]tyrosine (1 mm) with a cell-free extract of cultured melanoma cells (cell line C₂M) in the presence of dopa (0.1 mm), tritium was released as water at a steady rate for about 15 hr, or until 20% of the radioactivity had been converted to water. Some radioactivity was also found in dopa and melanin, but no appreciable amount in any other compounds. A cell-free extract of amelanotic cultured cells (cell line C₂W) did not produce tritiated water or melanin.     

Caerulein secretion by dermal glands in xenopus laevis

Since gastrin and its related peptides are secreted by a minority population of widely dispersed cells in mamamalian tissues it has, in the past, been difficult to study the subcellular aspects of their secretion. From published reports (1, 2) it seemed possible that a satisfactory system for such studies might be provided by the skin of certain amphibians such as Xenopus laevis since in these tissues high concentrations of peptides such as caerulein exist, and there is some indication (3) that this, or a similar gastrin-like peptide, may be a dermal gland secretory product. We have therefore explored this possibility by studying the structure, secretory process, and secretory product of the most prominent non mucous type of gland in the skin of X. laevis. These studies clearly demonstrate that most, if not all, of the caerulein in which the skin is contained in secretion granules within the dermal glands and that its release can be specifically evoked by adrenergic stimulation. The release process by a holocrine mechanism expels all of the stored secretion onto the skin surface and thus for biosynthetic studies it should now be possible to synchronize the processes which lead to the replenishment of the peptide.     

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

Actin-dependent carotenoid droplet dispersion in permeabilized cultured goldfish xanthophores

Organelle translocations are essential cellular processes. Although much progress has been made with regards to microtubule-dependent organelle translocations, little is known about actin-dependent organelle translocation(s) except cytoplasmic streaming in Nitella. On the other hand, there is indirect evidence that actin-dependent organelle translocation may be involved in secretion. We now present evidence that the dispersion of the pigment organelles carotenoid droplets in goldfish xanthophores is apparently actin dependent and that this process may be related to secretory processes. We show that, in digitonin-permeabilized goldfish xanthophores, the pigment organelles can be induced to disperse by a combination of cAMP, ATP, and xanthophore cytosol. This induced dispersion is inhibited by DNase I, phalloidin, or anti-actin, but not by anti-tubulin or anti-intermediate filament proteins, suggesting a dependence on F-actin. Since the dispersion of carotenoid droplets and secretion both involve outward translocation of organelles, we tested the possibility that cytosols of secretory tissues have similar activity. Such activity was indeed found in different tissues, apparently in parallel with the secretory activity of the tissues, suggesting that pigment dispersion in xanthophores and some secretory processes may share a common component.     

Rearrangements of pterinosomes and cytoskeleton accompanying pigment dispersion in goldfish xanthophores

The cytoskeleton of goldfish xanthophores contains an abundance of unique dense structures (400 nm in diameter) that are absent in goldfish nonpigment cells and are probably remnants of pterinosomes. No major difference in protein composition between xanthophores and nonpigment cells (without these structures) was found that could account for these structures. In xanthophores, these structures are foci of radiating filaments. The addition or withdrawal of ACTH causes a radical rearrangement of the xanthophore cytoskeleton accompanying redistribution of carotenoid droplets, namely, the virtual exclusion of these dense bodies with associated filaments from the space occupied by the carotenoid droplet aggregate vs. a relatively even cytoplasmic distribution of these structures when the carotenoid droplets are dispersed. These changes in cytoskeletal morphology are not accompanied by any major changes in the protein or phosphoprotein composition of the cytoskeleton.     

Duration of ischaemia determines matrix metalloproteinase-2 activation in the reperfused rabbit heart

It has been hypothesised that activation of matrix metalloproteinase-2 (MMP-2) contributes to reversible myocardial dysfunction (stunning) following short-term ischaemia and reperfusion. Gelatin zymography was used to measure release of both pro-MMP-2 (72 kDa) and MMP-2 (62 kDa), into the coronary effluent from isolated, perfused rabbit hearts during 90 min aerobic perfusion (control), or low-flow ischaemia (15 or 60 min at 1 mL/min), followed by 60 min reperfusion. In controls, pro-MMP-2 was detected in the coronary effluent throughout the first 30 min of aerobic perfusion, but MMP-2 was not detected. In contrast, MMP-2 was detected in the coronary effluent during reperfusion after both 15 and 60 min ischaemia. However, while left ventricular systolic function was impaired after both 15 min and 60 min ischaemia, a significant increase in the release of MMP-2 was only detected in hearts following 60 min ischaemia. The dissociation between mechanical function and MMP-2 levels suggest that MMP-2 does not contribute to myocardial stunning in this model, but may contribute to myocardial dysfunction following prolonged ischaemia.