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The Methods of Production and Subsequent Use of Transgenic Organisms

 Background (Purves et al., 1998):
A transgenic organism is , in its most basic sense, an organism to which a new gene has been added.  The first efforts at creating a transgenic organism were achieved using bacterium as hosts.  Bacteria are good candidates for being transgenic organisms because their circular pieces of DNA, plasmids, are good methods of importing new DNA.  Yeast cells are frequently used for creating transgenic organisms because they are easy to grow and have an extremely small genome while also possessing many of the characteristics of a eukaryote.  Plant cells are frequently used due to their totipotency.  It is possible to produce an entirely new plant with the desired genetic material from a single cell.

Methods:
+Vectors of Importing New Genes
Plasmids:
A plasmid is a piece of self-replicating circular DNA usually found in bacteria that unravels when it is cut with a restriction enzyme and has sticky ends on each side that will accommodate a new DNA segment.  In order for this to be possible, each side of the plasmid is cut with the same restriction enzyme and the foreign DNA is also given the same treatment.  DNA ligase is also added to fill the gap.  The antibiotic resistance gene of a plasmid is often used as a genetic marker for creating transgenic organisms.  In disrupting an antibiotic resistance gene with foreign DNA, it becomes vulnerable to that antibiotic and can then be destroyed by that antibiotic but not the other type of antibiotic protected against by another antibiotic resistance gene in the plasmid.  This can be exploited in a petri dish situation to verify which plasmid has taken up the DNA as needed by bombarding them with different types of antibiotics. This determines which plasmids have taken up the foreign DNA and which ones have not due to their susceptibility to said antibiotics.  A detailed view of the procedure can be seen in the image below.

Plasmid
Image permission pending from Brannon and Margaret Carter at http://crystal.uah.edu/~carter/index.html
Figure 1. Inserting a DNA sample to make a recombinant plasmid for the production of transgenic bacteria.
Viruses:
The DNA base pairs that cause a cell to lyse and die are deleted and the wanted foreign DNA is inserted into the virus capsule to cause modification of a cell through normal lytic infection processes.  The image below is an example of a viral vector in gene therapy

viral vector
Figure 2
Image permission pending from Dr. Huard at http://www.physsportsmed.com/issues/2000/02_00/huard.htm

 Artificial Chromosomes (Purves et al., 1998):These contain sequenced centromeres and telomeres as well as an origin of replication.  They are about 10,000 base pairs in size and can accommodate 50,000 to 1.5 million base pairs of inserted new DNA.  They are for all intents and purposes, fake but functional chromosomes.  One of such artificial chromosomes is depicted below in this image.

artificial chromosome
Figure 3
Pending permission from George Stamatis at http://www.cwru.edu/menu/research/synchrom.htm

 Plasmid Vectors for Plants (Purves et al., 1998):
Ti plasmid (T DNA), produces copies of itself in chromosomes in infected plant cells with the crown gall disease, T DNA has restriction enzyme sites and new DNA can be spliced in.  Cells from the gall can then be grafted into a new medium and a plant can be grown with the needed genetic material.  The image below shows the generation of these galls containing the desired genetic material in plants with the virus Agrobacterium tumefaciens.

crown gall
Figure 4
The use of Ti DNA to produce a crown gall from which cells are transplanted and grown into new plants with recombinant DNA.
  Image permission pending from Mrs. Hendricks at http://elmo.shore.ctc.edu/biotech/

Insertions(Purves et al., 1998):
Electroporation-
The cells for the incoming DNA vector are electrocuted and membranes rendered permeable so DNA can enter from environs.
Injection-
A fine pipette is used to insert DNA into a cell, usually a larger cell than usual.

Figure 5- A pipette is used to inject genetic material into a large cell to create a transgenic organism.  *Image permission pending from the UCSD Cancer Center.*

Lipofection-
DNA coated with phospholipids to form liposomes, they fuse to the cell membrane and allow DNA to enter target cell.

Figure 6- An Electron Micrograph of the Liposomes used in Lipofection as a method of creating a transgenic organism.  Image permission pending from Dr. Cheetham at www.carleton.ca/~jcheetha/







Particle Bombardment-
Tiny particles of Au or W coated with DNA are shot into cells.

Applications:
Protein Production-
An expression vector with protein producing DNA is imported into a cell through the use of plasmids into a bacterial cell.  This causes the bacterial cell to then produce said protein.  This method can be used in medicine to farm proteins on a wide-scale such as Insulin, Erythyroprotein, or Tissue Plasminogen Activator.  An image of this can be seen below:
 
 

Protein Harvesting

Figure 5

The creation of an expression vector with which proteins can be grown.
Image permission pending from Brannon and Margaret Carter at http://crystal.uah.edu/~carter/index.html

 Agriculture (Purves et al., 1998):
Insecticides-
have been changed with Ti vector to produce strains such as B. thuringiensis that is toxic to insect larvae
Herbicide-
adding an enzyme into plant chloroplasts that breaks down glysophate, which is the active ingredient of round-up
Nutrition-
add promoter signals for expression in developing rice grains, cause enzymes that convert precursors into Vitamin A, rice on its own cannot do this and it makes it more nutritionally valuable

"Pharming":
Some enzymes can be farmed from livestock in their milk by implanting an enzymatic gene near the genes in an expression vector that produce a normal milk protein in a developing livestock embryo so that the desired enzyme is produced in the milk of the livestock offspring.  More information on this technique and a diagram can be seen at http://www.biotechnology.gov.au/biotechnologyOnline/human/production/h_prod_pharm_img.htm.

I close with perhaps one of the most tantalizing and famous of photographs of a transgenic organism.  It is a photograph of a tobacco plant that has been genetically engineered with a fluorescent gene that causes it to glow...

transgenic plant
Image Permission Pending from Dr. Dana Krempels at http://fig.cox.miami.edu/Faculty/Dana/dana.html

Figure 6
The transgenic organism accompanied by a chime drawing of luciferase, the molecule responsible for the glowing effect.

References:
1) Purves, W.K., Orians, G.H., Heller, H.C., Sadava, D. (1998). Life. Sunderland, Massachusetts: Sinauer Associates, Inc.
 2) Unknown. Production of proteins by pharming.        <http://www.biotechnology.gov.au/biotechnologyOnline/human/production/h_prod_pharm_img.htm.> accessed 2003 February 12th.
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