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.     

Quantitative characterization of the filiform mechanosensory hair array on the cricket cercus

Background

Crickets and other orthopteran insects sense air currents with a pair of abdominal appendages resembling antennae, called cerci. Each cercus in the common house cricket Acheta domesticus is approximately 1 cm long, and is covered with 500 to 750 filiform mechanosensory hairs. The distribution of the hairs on the cerci, as well as the global patterns of their movement vectors, have been characterized semi-quantitatively in studies over the last 40 years, and have been shown to be very stereotypical across different animals in this species. Although the cercal sensory system has been the focus of many studies in the areas of neuroethology, development, biomechanics, sensory function and neural coding, there has not yet been a quantitative study of the functional morphology of the receptor array of this important model system.

Methodology/Principal Findings

We present a quantitative characterization of the structural characteristics and functional morphology of the cercal filiform hair array. We demonstrate that the excitatory direction along each hair''s movement plane can be identified by features of its socket that are visible at the light-microscopic level, and that the length of the hair associated with each socket can also be estimated accurately from a structural parameter of the socket. We characterize the length and directionality of all hairs on the basal half of a sample of three cerci, and present statistical analyses of the distributions.

Conclusions/Significance

The inter-animal variation of several global organizational features is low, consistent with constraints imposed by functional effectiveness and/or developmental processes. Contrary to previous reports, however, we show that the filiform hairs are not re-identifiable in the strict sense.     

Functional characterization and evolution of PTH/PTHrP receptors: insights from the chicken

ABSTRACT: BACKGROUND: The parathyroid hormone (PTH)-family consists of a group of structurally related factors that regulate calcium and bone homeostasis and are also involved in development of organs such as the heart, mammary gland and immune system. They interact with specific members of family 2 B1 G-protein coupled receptors (GPCRs), which have been characterised in teleosts and mammals. Two PTH/PTHrP receptors, PTH1R and PTH2R exist in mammals and in teleost fish a further receptor PTH3R has also been identified. Recently in chicken, PTHfamily members involved in calcium transport were characterized and specific PTHRs are suggested to exist although they have not yet been isolated or functionally characterized. The aim of this study is to further explore the evolution and function of the vertebrate PTH/PTHrP system through the isolation, phylogenetic analysis and functional characterization of the chicken receptors. RESULTS: Two PTHRs were isolated in chicken and sequence comparison and phylogenetic analysis indicate that the chicken receptors correspond to PTH1R and PTH3R, which emerged prior to the teleost/tetrapod divergence since they are present in cartilaginous fish. The vertebrate PTH2R receptor and its ligand TIP39 have been lost from bird genomes. Chicken PTH1R and PTH3R have a divergent and widespread tissue expression and are also evident in very early embryonic stages of development. Receptor stimulation studies using HEK293 cells stably expressing the chicken PTH1R and PTH3R and monitoring cAMP production revealed they are activated by chicken 1-34 N-terminal PTH-family peptides in a dose dependent manner. PTH-L and PTHrP were the most effective peptides in activating PTH1R (EC50 = 7.7 nM and EC50 = 22.7 nM, respectively). In contrast, PTH-L (100 nM) produced a small cAMP accumulation on activation of PTH3R but PTHrP and PTH (EC50 = 2.5 nM and EC50 = 22.1 nM, respectively) readily activated the receptor. PTHrP also stimulated intracellular Ca2+ accumulation on activation of PTH1R but not PTH3R. CONCLUSION: Two PTHR homologues of the vertebrate PTH1R and PTH3R were isolated and functionally characterized in chicken. Their distinct pattern of expression during embryo development and in adult tissues, together with their ligand preference, suggests that they have acquired specific functions, which have contributed to their maintenance in the genome. PTH2R and its activating ligand, TIP39, are absent from bird genomes. Nonetheless identification of putative PTH2R and TIP39 in the genome of an ancient agnathan, lamprey, suggests the PTH/PTHrP ligand and receptor family was already present in an early basal paraphyletic group of vertebrates and during the vertebrate radiation diverged via gene/genome duplication and deletion events. Knowledge of the role PTH/PTHrP system in early vertebrates will help to establish evolution of function.     

A new decapod crustacean faunule from the Middle Jurassic of north‐west France

Abstract: Decapod crustacean material collected recently from the lower Callovian (Middle Jurassic) in Maine‐et‐Loire (north‐west France) comprises two new species of prosopid and one new species of tanidromitid crabs, of the genera Nodoprosopon and Tanidromites, respectively. Also represented in this faunule is a probable paguroid anomuran, in the form of isolated chelae here assigned to the genus Orhomalus, as well as appendicular remains of unknown affinity; some of the latter might belong to prosopid crabs. These anomurans and brachyurans co‐occur with a diverse benthic fauna in limestones with abundant iron ooids; their main interest lies in the fact that they add valuable data to the rather poor record of Middle Jurassic decapod crustaceans.     

Rational design of complex phenotype via network models

We demonstrate a modeling and computational framework that allows for rapid screening of thousands of potential network designs for particular dynamic behavior. To illustrate this capability we consider the problem of hysteresis, a prerequisite for construction of robust bistable switches and hence a cornerstone for construction of more complex synthetic circuits. We evaluate and rank most three node networks according to their ability to robustly exhibit hysteresis where robustness is measured with respect to parameters over multiple dynamic phenotypes. Focusing on the highest ranked networks, we demonstrate how additional robustness and design constraints can be applied. We compare our results to more traditional methods based on specific parameterization of ordinary differential equation models and demonstrate a strong qualitative match at a small fraction of the computational cost.     

Hantavirus Transmission in Sylvan and Peridomestic Environments

We developed a compartmental model for hantavirus infection in deer mice (Peromyscus maniculatus) with the goal of comparing relative importance of direct and indirect transmission in sylvan and peridomestic environments. A direct transmission occurs when the infection is mediated by the contact of an infected and an uninfected mouse, while an indirect transmission occurs when the infection is mediated by the contact of an uninfected mouse with, for instance, infected soil. Based on population dynamics data and estimates of hantavirus decay in the two types of environments, our model predicts that direct transmission dominates in the sylvan environment, while both pathways are important in peridomestic environments. The model allows us to compute a basic reproduction number R ₀, which indicates whether the virus will be endemic or eradicated from the mouse population, in both an autonomous and a time-periodic model. Our analysis can be used to evaluate various eradication strategies.     

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.     

A STUDY OF THE FINE STRUCTURE OF THE EPIDERMIS OF RANA PIPIENS

The epidermis of adult Rana pipiens has been studied by electron microscopy and histological and histochemical methods. It was found that the epidermis is engaged in the production of both keratin and mucus. The basal cells are mainly filled with tonofilaments, whereas the cells located in the mid-portion of the epidermis contain both tonofilaments and mucous granules. Golgi vesicles and endoplasmic reticulum are found in relative abundance in the mucus-producing cells and seem to be involved in the production of mucous granules. The mucus seen was partly retained within the cells and partly secreted into the intercellular spaces. The outermost keratinized cells contain mainly filaments and a few remnants of cell constituents.     

A linkage group with FRA16B (the fragile site at 16q22.1)

Summary Polymorphic DNA markers located in bands 16q13, 16q21 and 16q22 were examined for recombination with FRA16B, the fragile site at 16q22.100. A tight linkage cluster D16S10-FRA16B-D16S4-HP was established. There were no recombinants between D16S10 and D16S4, which flank FRA16B. The markers D16S10 and D16S4 are in close proximity on the genetic map and delineate a small chromosomal segment, which contains the distamycin A-inducible fragile site.