Aerosol beam microinjector

• US 5,240,842 A
• Filed: 06/19/1992
• Issued: 08/31/1993
• Est. Priority Date: 07/11/1989
• Status: Expired due to Fees

• Current Assignee: Biotechnology Research And Development Corporation (Dow, Inc.)
• Original Assignee: Biotechnology Research And Development Corporation (Dow, Inc.)
• Inventors: Mets, Laurens J.
• Primary Examiner(s): Weimar, Elizabeth C.
• Assistant Examiner(s): MOODY, PATRICIA
• Application Number:
  US07/902,478
• Time in Patent Office: 438 Days
• Field of Search: 435/172.1, 435/172.3, 935/52, 935/53, 935/85
• US Class Current: 435/470
• CPC Class Codes: C12M 35/00 Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87
• CPC Class Codes: C12M 35/04 Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

The present invention uses aerosol beam technology to accelerate either wet or dry aerosol particles to speeds enabling the particles to penetrate living cells. Aerosol particles suspended in an inert gas are accelerated to a very high velocity during the jet expansion of the gas as it passes from a region of higher gas pressure to a region of lower gas pressure through a small orifice. The accelerated particles are positioned to impact a preferred target, for example, a plant or animal cell or bacterial culture. When the droplets include DNA or other macromolecules, the macromolecules are introduced into the cells. The particles are constructed as droplets of a sufficiently small size so that the cells survive the penetration. Once introduced into the target cell the macromolecules can elicit biological effects. Because the method of introduction is a physical one, the biological barriers that restrict the application of other DNA transfer methods to a few plant species and a few cell types are not present. In addition, the method and apparatus of the present invention permit the treatment of a large number of cells in the course of any single treatment. Thus, the inventive method and apparatus should be applicable to a wide range of plant species and cell types that have proved in the past to be quite impervious to standard methods of genetic engineering.

Claims

1. A method of introducing nucleic acids into a living cell, the method comprising the steps of:

a) producing aerosol droplets comprising said nucleic acids;

b) adjusting the diameter of the droplets by drying them;

then

c) accelerating said droplets to a speed enabling them to penetrate and enter into said cell upon impact therewith; and

d) impacting said living cell with said accelerated aerosol droplets.

2. The method of claim 1 wherein said nucleic acids are DNA.

3. A method of introducing exogenous DNA into a living cell, the method including the steps of:

a) solubilizing said exogenous DNA to form a solution;

b) producing aerosol droplets from said solution;

c) adjusting the diameter of the droplets by drying the droplets to a diameter of a size range from above 0.1 to below 20 microns; and

d) accelerating said droplets to a speed enabling said droplets to penetrate and enter into said living cell upon impact therewith, said droplets being accelerated by passing from an area of high gas pressure through an aperture having a diameter of about 100 to 300 microns and into an area of low gas pressure, said area of low gas pressure having an atmospheric pressure of about 0.01 atmospheres and said area of high gas pressure having an atmospheric pressure of about 1 atmosphere, said living cell being positioned in said area of low gas pressure such that it is impacted by a plurality of the accelerated droplets; and

e) impacting said living cell with said accelerated droplets, said droplets penetrating and entering into said cell.

4. The method of claim 3 wherein the cell is a plant cell.

5. A method of introducing a nucleic acid into a living cell, the method comprising:

a) producing aerosol droplets comprising said nucleic acid;

b) subjecting said droplets to a pressure differential such that said droplets are accelerated to a supersonic speed enabling them to penetrate and enter said cell upon impact therewith; and

c) impacting said living cell with said accelerated droplets.

6. The method of claim 5 wherein the pressure differential ranges from ambient pressure to a substantial vacuum.

7. The method of claim 6 wherein the ambient pressure is about 1 atm and the substantial vacuum is about 0.01 atm.

8. The method of claim 5 wherein the nucleic acid is DNA.