Paul Gaynor

Cloned Cattle Derived from a Novel Zona-Free Embryo Reconstruction System

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well.

Couplet alignment and improved electrofusion by dielectrophoresis for a zona-free high-throughput cloned embryo production system.

Mammalian cloning by somatic nuclear transfer has great potential for developing medical applications such as biopharmaceuticals and generation of tissues for transplantation. For agricultural applications, it allows the rapid dissemination of genetic gain in livestock breeding. The maximisation of that potential requires improvements to overall cloning technology, especially with respect to increasing cloning efficiency and throughput rates in cloned embryo production. A zona-free embryo reconstruction system was developed to increase cloning throughput and ease of operation. Central to this system is a modified electrofusion procedure for nuclear transfer. Cytoplast-donor cell couplets were placed in a custom-designed ‘parallel plate’ electrode chamber. A 1 MHz sinusoidal AC dielectrophoresis alignment electric field of 6–10 kVm−1 was applied for 5–10 s. The couplets were then fused using 2×10 μs rectangular DC-field pulses (150–200 kVm−1), followed by application of the AC field (6–10 kVm−1) for another 5–10 s. Fusion was performed in hypoosmolar buffer (210 mOsm). Automated alignment of up to 20 couplets at a time has been achieved, resulting in greatly improved fusion throughput rates (2.5-fold increase) and improved fusion yields (1.3-fold increase), compared with commonly followed zona-intact protocols.

Dust anchoring characteristics of electret fibres with respect to Der p 1 allergen carrying particles.

The avoidance of house dust mite allergens is a major area of interest and essentially requires a significant removal of these allergens from the immediately respirable air. Electrostatic attraction and anchoring of particulate matter using electret polymers is commonly used for air filtration purposes. This effect is investigated for its possible use in domestic allergen avoidance. Polypropylene electret, heat-treated electret and non-electret, and wool and nylon fibre samples were soiled with house dust known to contain Der p 1 allergen. These samples were vacuumed at three air face velocities. The proportions of released and anchored dust were calculated. Released dust was collected and analysed for Der p 1 concentration and compared to stock dust values. Results showed that compared to uncharged fibres at least 95% more dust remained anchored in the electret fibres. Also, overall Der p 1 release was reduced by more than 49%. Der p 1 allergen concentrations in the collected dust were relatively constant for all the fibres tested, indicating no selective attraction or repulsion of Der p 1 allergen carrying particles in the experimental dust. The consistently high dust anchoring ability of the electret fibres could be used in many domestic products that are known to harbour particulate allergens, to reduce their release and inhalation.

Physical modelling of electroporation in close cell-to-cell proximity environments.

Many applications of electroporation, especially those utilizing electrofusion and in-vivo electroporation, involve cell environments that include close cell-to-cell proximity and a wide range of target cell size. It is important to understand how this kind of environment may alter optimum electroporation electrical parameters for any given application. A physical, electrically equivalent model of biological cell electroporation, based on aqueous solution filled thin latex rubber membrane spheroids, was used to investigate membrane permeabilization behaviour where there is both close cell-to-cell proximity and different cell radii. Cell model arrangements were pulsed using either a 50 micros or 10 micros, 1/e decay time constant dc capacitive discharge electric field, with peak amplitudes of 160-500 kV m(-1). Results indicate that, compared to cells in isolation, electroporation initiates at substantially decreased applied electric field magnitudes in regions of close cell-to-cell proximity where the external media conductivity is lower than the cell interior conductivity, and the membrane is maximally polarized. Additionally, the use of shorter time constant, higher peak magnitude pulse parameters should reduce the relative difference in threshold membrane permeabilization in regions of close cell-to-cell proximity for cells of different size so that the degree of electroporation is more uniform for variable size and shape target cell populations.

Electrostatic charge characteristics of Der P1 allergen carrying particles and the house dust mite, Dermatophagoides Pteronyssinus.

Control of the house dust mite allergen has received considerable attention owing to its importance in some allergic diseases. One aspect of dust mites and their allergen-carrying faecal particles that has not been reported on, which may have allergen control applications, is the electrostatic charge they carry in the natural environment. To promote tribo-electric charging, household dust containing dust mite allergen and live house dust mites are separately agitated while in contact with either polypropylene, nylon or earthed metal. The charged dust and mites are subsequently subjected to electrostatic separation and collection. Results for concentrations of the house dust mite allergen, Der p1, indicate that, when subjected to nylon, Der p1 carrier particles appear to be predominantly positively charged. Similarly, when subjected to polypropylene, Der p1 carrier particles also appear to be positively charged. Reduction of excess free charge by agitation against earthed metal does not appear to affect the observed charging characteristics, indicating that the positive charge may be bound or inherent in the Der p1 carrier particles. In contrast, house dust mites exposed to nylon appear to be generally charging negative, whereas mites exposed to polypropylene appear to be charging positive. The observed electrostatic characteristics of the mites and Der p1 carrying particles will be useful in the future development of electrostatic allergen control methods.

A high voltage amplifier for use in medical applications of electroporation

The bio-physical effect known as electroporation or electropermeabilization is becoming more widely used in medical treatment, especially for chemotherapy and gene therapy. The conventional electrical apparatus used is based on DC voltage pulses which may not be optimal. Indications are that AC pulses may provide significant improvements. A high voltage linear amplifier design incorporating vacuum tube valves is presented that could facilitate AC pulse experimentation. Valves are used as they most readily suit the desired electrical specifications of an output voltage of 4kV/sub p/, a bandwidth of 500 Hz-5 MHz, a voltage gain of 52 dB, and an output impedance of <100 /spl Omega/.

High voltage and frequency bipolar pulse generator design for electroporation-based cancer therapy

This paper describes a novel design of a high voltage, high frequency, bipolar pulse generator, for experimental use in electrochemotherapy and irreversible electroporation, using RF MOSFETs (DE375-102N12A) in a cascaded multilevel inverter topology. Its pulsing regime is fully programmable and is con-trolled by an FPGA. A systematic development approach was followed. The UCC37321 gate driver and the HCPL-2400 opto-coupler were selected. Damping resistors and RCD snubbers were employed to mitigate excessive ringing in the output circuit. Practical EMC reduction guidelines were followed. The pulse generator operated satisfactorily, producing a typical cascaded 5-level output of 1250 V peak at 1 MHz into a load of 80 ohm in parallel with 235 pF. Higher room temperature caused a slight “indent” into the output pulses.

A micropit for biological cell positioning

Dielectrophoresis (DEP) may be used to trap or to move biological cells. Automated position control of cells has been proposed to eliminate operator dependent experimental parameter variations and increase throughput of labour intensive laboratory procedures such as couplet fusion for mammalian cloning or plant hybridisation, and cell sorting. However, the use of DEP in laboratory procedures is often precluded due to the relatively high complexity of microfabricated apparatus. A simple two layer microchip was fabricated and demonstrated to position cells. Thin film interdigitated titanium electrodes (300 nm thick, 250 ¿m wide and 250 ¿m apart) were deposited on a solid borosilicate glass substrate. They were coated with a film of insulating photosensitive resist AZ1518. Circular holes (¿micropits¿) measuring 5, 10, 20, and 40 ¿m in diameter were fabricated above the electrodes. Saccharomyces cerevisiae (bakers yeast) cells were dispensed onto the microchip. Positive and negative dielectrophoresis were used to position cells resting on the surface of the micropit array. After patterning with positive DEP, a rinse was used to create small islands of cells. The experimental results were accompanied by a 2D numerical simulation of the micropit device to determine the influence of pit height and diameter on the DEP force.

Electrostatic Targeting for Allergen Removal and Pest Control Applications

Today. in the United Kingdom, approximately one in seven infants are diagnosed with asthma in the first six months of life. There has been a dramatic increase in recent years, and asthma-related drugs now account for approximately 11% of the total prescription costs to the National Health in the UK (Department of Health, Asthma and Epidemiology, HMSO, 1995). In addition to medication, preventative measures include attempts to exclude the allergen from the domestic environment. This requires careful control of the most common asthma allergen, Der p1,which is conveyed by the waste products produced by the house dust mite (Dermatophagoides pteronyssinus). Acaricides may be used to kill the dust mites,but these have to be combined with allergen denaturing agents if eradication of the allergen is sought. Mechanical barriers such as mattress and pillow covers offer some protection from contact with the allergen, but often with a varying degree of success. High levels of allergen carriers are also known to exist in carpeting, and this can lead to a particularly undesirable environment for infants who frequently crawl with their faces close to, and sometimes in contact with, the carpet pile. Inhalation of allergen is usually unavoidable in this situation. Research has indicated that the use of electret fibres can dramatically enhance the anchoring of allergen carriers in the carpet pile. Electrostatic adhesion of the carriers to the electret fibres ensures that for normal traffic conditions, such as walking, crawling, playing etc, much less dust is released from the carpet; thus reducing the probability of inhalation. Dispensing domestic products in aerosol form has undoubtedly been a commercial success; offering considerable consumer advantages in applications ranging from paints, polishes and insecticides. The targeting of products in aerosol form may not, however, be optimised with a high proportion of the product often missing its target. It is well known that electrostatic spraying can enhance targeting, but this technology is normally limited to industrial processes such as paint and powder coating. A novel and unique aerosol package has been developed which charges the aerosol on delivery in a controlled manner. The Smartseeker-(-Smartseeker is a trademark of Reckitt Benckiser) insect seeking technology is now incorporated into a commercial aerosol insecticide, and is the worlds first electrostatically optimised aerosol can which requires no active energy source. Flying insect knock-down is enhanced by up to 34% with this technology.

Multiple-Cylindrical Electrode System for Rotational Electric Field Generation in Particle Rotation Applications

Lab-on-a-chip micro-devices utilizing electric field-mediated particle movement provide advantages over current cell rotation techniques due to the flexibility in configuring micro-electrodes. Recent technological advances in micro-milling, three-dimensional (3D) printing and photolithography have facilitated fabrication of complex micro-electrode shapes. Using the finite-element method to simulate and optimize electric field induced particle movement systems can save time and cost by simplifying the analysis of electric fields within complex 3D structures. Here we investigated different 3D electrode structures to obtain and analyse rotational electric field vectors. Finite-element analysis was conducted by an electric current stationary solver based on charge relaxation theory. High-resolution data were obtained for three-, four-, six- and eight-cylindrical electrode arrangements to characterize the rotational fields. The results show that increasing the number of electrodes within a fixed circular boundary provides larger regions of constant amplitude rotational electric field. This is a very important finding in practice, as larger rotational regions with constant electric field amplitude make placement of cells into these regions, where cell rotation occurs, a simple task – enhancing flexibility in cell manipulation. Rotation of biological particles over the extended region would be useful for biotechnology applications which require guiding cells to a desired location, such as automation of nuclear transfer cloning.