Micro–Targeting: High Efficiency Gene Transfer Using a Novel Approach for the Acceleration of Micro–Projectiles

Abstract

We have constructed a novel micro-projectile accelerating system for efficient gene transfer into cells in situ that avoids binding DNA to micro-projectiles and keeps the DNA in solution. Further, instead of a macro-projectile (or the equivalent), it accelerates the particles in a Bernoulli air stream. The micro-targeting approach directs highly dispersed particles to sites with diameters as little as 0.15 mm, allowing precise aiming to restricted tissues. The system is physically flexible and should therefore be adaptable to different tissues and species. Transient expression of the Escherichia coli beta-glucuronidase gene in immature wheat embryo scutella was obtained at a frequency of up to 3% of the treated cells in the surface layer. In tobacco SR1, we achieved many transgenic plants, and the efficiency of stable transformation with the neomycin phosphotransferase (NPTII) gene was approximately 10(-3) per exposed cell.

Biolistic-mediated transient gene expression in shoot apical meristems of the prickly-pear (Opuntia ficus-indica)

“…Particle bombardment -We used a Biolistic PDS-1000He instrument from Bio-Rad Laboratories, and the conditions for bombardment were those described (Sauter et al 1991). The tungsten particles in which the transforming DNA was coated had a diameter of 1.3 µm, the distance between the flying disk and the target tissue was 7.5 cm and the shooting pressure was adjusted to 1200 psi.…”

From the Concept of Totipotency to Biofortified Cereals

“…Christof Sautter invented microtargeting technology, enabling precise biolistic treatment of native shoot meristems (9,13,27,28,35,42,63,(81)(82)(83)(84) reaching the cell layer of the meristem (LII), which forms the macro-and microspores and thus transfers genetic changes to the offspring. With Monika Clausen, he developed a fungusresistant wheat (13), which kept him busy long beyond my retirement and led to him becoming the focal point of anti-GMO activists, who repeatedly destroyed his test fields.…”

Transient gene expression in shoot apical meristems of sugarbeet seedlings after particle bombardment

“…Uniformly-sized gold particles (batches of 0.8, 1.2, 1.8, and 2.25 fun particles) made according to Sautter et al (1991), and Aldrich gold (Aldrich-Chemie, Steinheim, Germany; particles 0.7-4.9 ^m in diameter, average of 1.7 ^m), were stored in 40% polyethylene glycol (mol. wt.…”

“…For bombardment experiments, the microtargeting apparatus developed by Sautter et al (1991) was used. A 20 nl aliquot of a particle/DNA suspension is injected into a Pitot's tube and atomized and propelled by a pressure pulse of compressed nitrogen.…”

“…For this, the microtargeting apparatus developed by Sautter et al (1991) was chosen. Using this device, the transient expression of marker genes occurs in meristems of wheat immature embryos , seedlings (Bilang et al, 1993) and inflorescences , and stable transformation occurs in tobacco (Sautter et al, 1991). This technique has various advantages especially for the bombardment of small targets which are difficult to isolate.…”