Particle Bombardment

Miss.R.UmaDept. of BiotechnologyFAPM, WUSL

Biological Instrumentation

Introduction

Transformation is  the genetic  alteration  of  a cell resulting  from  the  direct  uptake,  incorporation and expression  of  exogenous genetic  material from  its  surroundings  and  taken up  through  the cell membrane.

Transformation occurs,

Naturally - some bacteria

Artificial - other cells 

Insertion of Exogenous genetic material

1. Transformation

2. Conjugation (horizontal gene transfer)

- transfer of genetic material between two bacterial cells in direct contact by pilli

3. Transduction

- injection of foreign DNA by a bacteriophage virus into the host bacterium

Transfection

Introduction of foreign DNA into eukaryotic cells.

(Transformation in plant and animal cells)

Artificial Gene Transfer

Mechanism for gene transfer

1. Agrobacterium mediated transformation

2. Particle bombardment

3. Electroporation

4. Viral transformation (transduction)

5. Microinjection

6. Lipofection

7. Calcium phosphate transfection

Agrobacterium mediated Plant Transformation

Agrobacterium tumefaciens

Crown gall disease (Tumour formation)

Micro-injection

The host cell is immobilized by applying a mild suction with a blunt pipette and a foreign gene is then injected with a micro-injection needle.

Electroporation System

Osmotic shock

Electric field is applied, the ions move according to their charge. Pathways are formed across the cell membrane allowing DNA to enter. Then the electric field is deactivated, the membrane heals.

Particle Bombardment

Biolistic Particle Delivery System

(Bioballistic method)

Background

Biolistic transfection is a physical means of transfecting cells via bombardment of living tissue with high velocity DNA coated gold particles.

eg: - DNA coated bullets to the final shooting of the

organotypic slice cultures using a gene gun

A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information

The payload is an elemental particle of a heavy metal coated with plasmid DNA

Particle bombardment

Particles  of  gold  or  tungsten  are  coated  with  DNA and then shot into young cells or embryos

Some  genetic  material  will  stay  in  the  cells  and transform them

In particle mediated gene transfer, in general transfected cells result when the bullet comes to rest in the nucleus

This  device  is  able  to  transform  almost  any  type  of cell,  including  plants,  and  is  not  limited  to  genetic material  of  the  nucleus:  it  can  also  transform organelles, including plastids

Instrument

Gene gun

John C Sanford

Inventor (1983 – 1986)

Invented by John C Sanford, Ed Wolf and Nelson Allen at Cornell, and Ted Klein of DuPont

Used a modified Crosman air pistol

Large onions cells were bombarded with tungsten particles coated with a marker gene

Genetic transformation was then proven by expressed gene

Advantages - Gene gun transfection

Is, Fast Efficient Easy means of transfecting Less labour intensive Useful for fluorescently labeling Small subset of cells

Mechanism

1. Helium pressure & vacuum circuits in the biolistic system effectively accelerate the microcarriers into the target cells

2. In certain conditions, DNA/RNA become “sticky,” adhering to biologically inert particles such as metal atoms

3. The chamber door is closed & the vacuum is applied4. Activating the Fire switch allows helium to flow into the

gas acceleration tube at a rate regulated by the helium metering valve and monitored by the helium pressure gauge

5. The gas is held until the burst pressure of the rupture disk is reached

6. This generates a helium shock wave into the bombardment chamber

7. The shock wave hits the microcarrier launch assembly and propels a plastic macrocarrier holding DNA-coated microcarriers toward the target cells.

8. By accelerating this DNA-particle complex in a partial vacuum and placing the target tissue within the acceleration path, DNA is introduced

9. A stopping screen placed between the macrocarrier assembly and the cells retains the plastic disk, while allowing the coated microprojectiles to pass through

10. The cells that take up the desired DNA, identified through the use of a marker gene, are then cultured to replicate the gene and possibly cloned

Particle Bombardment system

Gene Gun

adhering to biologically inert particles such as metal particles

Tungsten or Gold (~ 1µm)

Rupture Disc

burst diaphragm non-reclosing pressure relief membrane made up of Polyvinyl nylon/Polycarbonate allowing for precise pressure-based control

of particle application to a sample

Macrocarrier

Carries microprojectiles till the shock wave hits Polycarbonate membrane Move till the stopping screen

Stopping screen

Metal wire mesh Acting as seive/Perforatted plate Stops the macrocarrier Allows the DNA-microprojectiles No harm to the velocity of the particles

How to do Plant Transformation Using Gene Gun1. The target of a gene gun is often a callus of undifferentiated plant cells 

growing on gel medium in a petri dish2. After the gold particles have impacted the dish, the gel and callus are 

largely disrupted3. Some cells were not obliterated in the impact, and have successfully 

enveloped a DNA coated tungsten particle, whose DNA eventually migrates to and integrates into a plant chromosome

4. Cells from the entire petri dish can be re-collected and selected for successful integration and expression of new DNA using modern biochemical techniques, as tandem selectable gene and northern blots

5. Selected single cells from the callus can be treated with a series of plant hormones, such as auxins and gibberellins, and each may divide and differentiate into the organized, specialized, tissue cells of an entire plant. 

6. This capability of total re-generation is called totipotency7. The new plant that originated from a successfully shot cell may have new 

genetic (heritable) traits

Advantages

1. Biological projectiles such as E.coli, yeast & phage complexed with tungsten, used as particles with some success

2. Most plants can be transformed , useful for inserting genes into plant cells such as pesticide or herbicide resistance

3. Fast technique4. Easy technique 5. Useful for either transient or stable transformation6. Reproducible7. Stable integrated transgenic strains can be isolated directly

Disadvantage

1. In plant transformation efficiency is lower to Agrobacterium - transformation

2. As being relatively inefficient as relatively few numbers of cells are stably transformed

3. Irreparably damage the plant tissue4. Expensive equipment cost5. Takes long time to generate transgenics6. Need supply of pricey particles7. Material intensive

Efficiency of transformation

Delivery of a sufficient number of DNA-coated particles Quantity of DNA coats the metal particles Type of metal particle Temperature Amount of target cells Type/species of cells Regeneration ability of target cells Length of the flight path of particle Speed of particles fragile tissues can not be bombarded at the

same high speed as those which have more resistance to foreign particle

Applications - Animals

1. Popular in the field of neurobiology since post-mitotic neurons are notoriously difficult to transfect and assessing fine morphology of single neurons in intact brain slices

2. Used to deliver DNA vaccines3. The delivery of plasmids into rat neurons, is also used as a pharmacological

precursor in studying the effects of neurodegenerative diseases as Alzheimer's Disease

4. Popular in an edible vaccine production technique, the nano-gold particles coated with plant genetic material under the high vacuum pressurized chamber are transformed into suitable plant tissues

5. A common tool for labeling subsets of cells in cultured tissue6. In addition to being able to transfect cells with DNA plasmids coding for

fluorescent proteins7. Can be adapted to deliver a wide variety of vital dyes to cells

Applications - Plants

1. Resistant plants

Procedure in short

1. Helium pressure and vacuum circuits in the biolistic system effectively accelerate the microcarriers into the target cells.

2. After all the materials are in place, the chamber door is closed and a vacuum is applied.

3. Activating the Fire switch allows helium to flow into the gas acceleration tube at a rate regulated by the helium metering valve and monitored by the helium pressure gauge.

4. The gas is held until the burst pressure of the rupture disk is reached. 5. This generates a helium shock wave into the bombardment chamber. 6. The shock wave hits the microcarrier launch assembly and propels a plastic

macrocarrier holding DNA-coated microcarriers toward the target cells. 7. A stopping screen placed between the macrocarrier assembly and the cells

retains the plastic disk, while allowing the coated microprojectiles to pass through and transform the target cells.