Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-22
2002-06-18
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S459000, C435S470000
Reexamination Certificate
active
06406852
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a process for delivery of biological materials into living cells and tissues of plants and animals. More particularly, this invention relates to a process wherein, the biomolecules to be delivered into cells are coated on tiny inert particles, called microprojectiles, which are then bombarded on the target cells or tissues. The microprojectiles are coated with biological materials, like nucleic acids and delivered into cells, using a microprojectile gun, also called as ‘gene gun’ or ‘biolistic gun’. Commonly used microprojectiles are gold and tungsten particles. The improved process claimed here gives highly efficient, more uniform and reproducible delivery of microprojectiles. As a result, it gives several fold higher expression of the nucleic acid delivered into cells.
BACKGROUND OF INVENTION
Accelerated metal particles coated with nucleic acids are extensively used for introducing genes into intact plants and animals, tissues, cells and organelles (Sanford et al., 1993 Sanford J. C., Smith F. C. and Russell, J. A. 1993 Optimising the biolistic process for different biological applications. Methods in Enzymology, 217, 485-510). A variety of particle guns that deliver microprojectiles into living cells have been described in earlier inventions (U.S. Pat. Nos. 5,100,792, 5,179,022, 5,204,253 and 5,865,796). Though several different types of particles have been discussed, including high density metals like gold, tungsten, platinum, ferrite etc., and low density synthetic polymeric particles (U.S. Pat. No. 5,783,567), the former group has been more successful apparently because these can be delivered with higher kinetic energy and can therefore, go deeper into the target tissue. Gold particles have been more commonly used because gold is more inert than others, i.e., it does not interfere with biological processes and the gold particles have more uniform surface, desirable for minimum cell damage.
The particles are coated with the biological material to be delivered into living cells. The microprojecticles, coated with the biological material are then propelled at high speed, using one of a variety of particle guns made by certain research groups or available commercially.
Accelerated metal particles penetrate several layers deep into tissue, depending upon the velocity and momentum at which these strike the target tissue. The acceleration to particles can be provided by mechanical impulse, centripetal force, electric discharge (U.S. Pat. No. 5,100,792), firing explosives (U.S. Pat. No. 5,179,022) or compressed gas (U.S. Pat. No. 5,865,796) or any other process. The most commonly used and commercially available apparatus for acceleration of microprojectiles involves acceleration using gas shock (U.S. Pat. Nos. 5,204,253 and 5,865,796) and is available commercially from M/S Bio-Rad Laboratories, Hercules, Calif. 94547, USA.
Examples of biological substances that can be coated onto or impregnated into inert particles include biological stain such as fluorescent or radioactive probes, viruses, organelles, proteins, hormones and nucleic, acids. In certain cases delivery of molecules into cells and tissues under liquid pressure has also been claimed (U.S. Pat. No. 5,766,901).
The effectiveness of the delivery of particles is most conveniently measured by examining the expression of a reporter gene delivered in form of DNA coated on such particles. One of the very commonly used reporter genes is uidA (Jefferson, R. A. and Wilson, K. J. 1991. The GUS gene fusion system In: S. Gelvin and R. Schilperoort Ed. Plant Molecular Biology, Kluwer Academic Publishers, Dordrecht, B 14, pp. 1-33) which codes for an enzyme called glucuronidase. Once the gene is inside a cell and if the cell is viable, the gene makes the enzyme glucuronidase. Following bombardment of uidA gene containing particles, if a plant leaf is treated with a chromogenic substrate 5-bromo-4-chloro-3 indoyl-&bgr;-D-glucuronic acid, also called X-gluc, it is converted into a blue product due to activity of glucuronidase. The blue spots appear on the bombarded leaf and can be counted by treating the leaf with X-gluc, one to several days after bombardment. Therefore, the number of spots indicate the number of foci within target tissue where particles get delivered without causing significant damage to biological function of cells. Hence, expression of glucuronidase is indicative of delivery of the microprojectile particles in a manner that did not cause damage to biological function of the cell. Therefore, efficiency of biologically effective delivery of microprojectile particles can be measured by counting under microscope, blue spots on the leaf surface for example, in case of plants (or any other tissue) that are formed due to expression of glucuronidase. This method is called as ‘histochemical method’ since it allows seeing the activity by color. It is not quantitative but qualitative since it tells the presence of activity but does not quantitatively measure the level of glucuronidase activity. Details of this method, as applied to gold and tungsten coated particles are also given in Ratnayaka and Oard (1995, I. J. S. Ratnayaka and J. H. Oard 1995 A rapid method to monitor DNA precipitation onto microcarriers before particle bombardment, Plant Cell Reports, 14, 794-798).
A second more sensitive method to measure the activity of &bgr;-glucuronidase after microprojectile bombardment is by quantitatively measuring the formation of a fluorescent product. In this method, a non fluorescent substrate called 4-methlyumbelliferyl-&bgr;-D-glucuronide, also called MUG, is converted by glucuronidase into a fluorescent product called MU. Hence, the increase in fluorescence per mg protein in leaf gives quantitative expression of gene delivery in a manner that did not damage biological function of the target, cells. The less the expression, the less successful the delivery of DNA coated on microprojectiles. The fluorometeric as well as histochemical GUS assays are also described in Kloti et al (1999, A. Kloti, C. Henrich and others in Plant Molecular Biology, 40, 249-266). Both of these standard methods were employed in this invention to illustrate an amazingly high improvement of results obtained by the bombardment protocol claimed by us.
While coating microprojectiles with DNA the procedure recommended by Bio-Rad Laboratories in their catalogue or its minor variations are commonly used. This method is based on the original method developed by Sanford et al. (1993, J. C. Sanford, F. D. Smith and J. A. Russell, 1993 optimising the biolistic process for different biological applications, Meth. Enzymol. 217, 483-509). In this method, 3 mg gold particles (1 &mgr;m diameter particles for example, supplied by, Bio-Rad Laboratories, USA) are placed in a microcentrifuge tube and vortexed for 3 min in 0.5 ml 70% ethanol (v/v). The suspension is held at room temperature for 10 min, centrifuged (15000 rpm) for 5 sec and decanted. The pellet of particles is washed three times with 500 &mgr;l sterile distilled water. Between two washings, the suspension is mixed thoroughly by vortexing for 1 min, the particles are allowed to settle on bench for 1 min and then centrifuged for 5 sec. Finally, the washed particles are suspended in 50 &mgr;l of 50% glycerol. The suspended particles are then coated with DNA by adding 5 &mgr;l DNA (1 &mgr;g/&mgr;l in water), 50 &mgr;l CaCl
2
(2.5 M) and 20 &mgr;l spermidine (0.1 M stock) in that order, vortexed for 3 min, held at room temperature for 5 min, and finally the coated particles are pelleted by pulse centrifugation. The preparation is then washed with 150 &mgr;l of 70% ethanol, followed by absolute ethanol before they are suspended in 48 &mgr;l ethanol. The coated particles are then vortexed for 1 to 2 seconds before placing 8 &mgr;l aliquots on macrocarrier for bombardment on a target tissue, like tobacco leaf. The above commercially recommended procedure given by Bio-Rad Laboratories Ltd. Along with the Helium Drive PDS-1000/He system Biolistic Gun is stated
Sawant Samir V.
Tuli Rakesh
Council of Scientific and Industrial Research
Ketter James
Ladas & Parry
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