A helium burst biolistic device adapted to penetrate fragile insect tissues

Abstract

To compensate for the extremely low penetration efficiency of the original PDS/1000-He Bio Rad biolistic device and the deleterious blast effect, design modifications have been made to the launching module. These modifications were evaluated on Bombyx mori embryos and fragile tissues, such as oocytes and imaginal wing disks. The original floppy macrocarrier was replaced by a rigid macrocarrier to avoid the effects of the helium blast. The efficiency of the gene gun bombardment was reinforced by the addition of a focusing nozzle. The reduced blast effect allowed us to carry out high-pressure shootings to small organs with improved penetration. This system allowed potentially all the internal embryonic tissues to be transfected with optimal survival rates. The new module was effective on tissues that are difficult to transfect, such as the epithelial wing disk that is covered by a peripodial membrane, and the ovarian follicle cells that lie under the ovariole cell membrane. The new macrocarrier allowed both an aqueous delivery of particles and an ethanolic dry delivery. No significant differences were noted between these two modes of delivery. The major improvement is the possibility of high pressure shooting correlated with appreciable penetration and a weak blast effect.

Figure 1.

Mechanical modifications on the Bio Rad PDS/1000 He biolistic device shooting module. A: Schematic illustration of the problems encountered with the original shooting module. B: Decentering of the shootings with the original shooting module. C: Schematic illustration of our mechanical modifications. D: Focusing the shootings with our shooting module. E: Plan of our macrocarrier mould (left) and the shooting module (right).

Figure 2.

LacZ gene expression in embryonic tissues after bombardment with the pBRJZ densonucleovirus vector by our shooting module. A: LacZ gene expression in all potential parts of the embryo. B: Expression is often observed in the brain with the pBRJZ vector (arrow). C: Histological section of the area shown by the arrow in B. D: Detail of the brain LacZ gene expression framed in C. E: Expression in the posterior silk gland. F: Histological section of an expressing silk gland. G: LacZ expression in the gonad (probably not in the germ cell). H: LacZ expression in the gut and integument.

Figure 3.

LacZ gene expression in a soft organ after gene bombardment with our shooting module. A and B: Expression in epithelial cells of imaginal wing disks of fifth instar larva. C and D: Expression in ovarian follicular cells of seven-day-old polyvoltine pupae.