Vous êtes sur la page 1sur 52

Particle Bombardment

Biological Instrumentation
Miss.R.Uma Dept. of Biotechnology FAPM, WUSL

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
Artificial

- some bacteria
- other cells

Insertion of Exogenous genetic material


1.
2.

Transformation
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. 2. 3. 4. 5. 6. 7.

Agrobacterium mediated transformation Particle bombardment Electroporation Viral transformation (transduction) Microinjection Lipofection 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.

2.

3. 4.

5.

Helium pressure & vacuum circuits in the biolistic system effectively accelerate the microcarriers into the target cells In certain conditions, DNA/RNA become sticky, adhering to biologically inert particles such as metal atoms The chamber door is closed & the vacuum is applied 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 The gas is held until the burst pressure of the rupture disk is reached

6. 7.

8.

9.

10.

This generates a helium shock wave into the bombardment chamber The shock wave hits the microcarrier launch assembly and propels a plastic macrocarrier holding DNA-coated microcarriers toward the target cells. By accelerating this DNA-particle complex in a partial vacuum and placing the target tissue within the acceleration path, DNA is introduced A stopping screen placed between the macrocarrier assembly and the cells retains the plastic disk, while allowing the coated microprojectiles to pass through 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 (~ 1m)

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 Gun


1. 2. 3.

4.

5.

6. 7.

The target of a gene gun is often a callus of undifferentiated plant cells growing on gel medium in a petri dish After the gold particles have impacted the dish, the gel and callus are largely disrupted 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 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 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. This capability of total re-generation is called totipotency The new plant that originated from a successfully shot cell may have new genetic (heritable) traits

Advantages
1.

2.

3.
4. 5.

6.
7.

Biological projectiles such as E.coli, yeast & phage complexed with tungsten, used as particles with some success Most plants can be transformed , useful for inserting genes into plant cells such as pesticide or herbicide resistance Fast technique Easy technique Useful for either transient or stable transformation Reproducible Stable integrated transgenic strains can be isolated directly

Disadvantage
1.

2.

3. 4. 5. 6. 7.

In plant transformation efficiency is lower to Agrobacterium - transformation As being relatively inefficient as relatively few numbers of cells are stably transformed Irreparably damage the plant tissue Expensive equipment cost Takes long time to generate transgenics Need supply of pricey particles 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.

2. 3.

4.

5. 6.

7.

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 Used to deliver DNA vaccines 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 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 A common tool for labeling subsets of cells in cultured tissue In addition to being able to transfect cells with DNA plasmids coding for fluorescent proteins Can be adapted to deliver a wide variety of vital dyes to cells

Applications - Plants
1.

Resistant plants

Procedure in short
1.

2.
3.

4. 5. 6. 7.

Helium pressure and vacuum circuits in the biolistic system effectively accelerate the microcarriers into the target cells. After all the materials are in place, the chamber door is closed and a vacuum is applied. 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. The gas is held until the burst pressure of the rupture disk is reached. This generates a helium shock wave into the bombardment chamber. The shock wave hits the microcarrier launch assembly and propels a plastic macrocarrier holding DNA-coated microcarriers toward the target cells. 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.

Vous aimerez peut-être aussi