methods of introducing DNA into living cells طرق مختلفة لادخال الدنا الي الخلايا الحية
Cells have
membranes that prevent DNA from simply diffusing in or out. This is the initial
barrier that scientists must overcome in order to insert foreign DNA into a
cell. The four ways of accomplishing this goal are transduction, transformation, transfection and
injection. But before
these four methods are employed, scientists must prepare the foreign DNA by
cutting it into smaller pieces or by using restriction enzymes to cut the DNA
and insert the desired portion into a bacterial plasmid. Plasmids are circular
pieces of DNA that can be passed between bacteria and viruses.
Viral Transduction
Transduction is the insertion of foreign DNA
into a cell via a virus. Viruses are made of a protein coat that houses DNA
within. Viruses can bind to living cells and inject their DNA. Or, viruses can
push into the host as a membrane-bound vesicle, before releasing their DNA
inside the host. The use of recombinant DNA technology can insert foreign DNA
into host cells that are then purposefully infected with viruses. When the
virus produces more of itself in the host cell, it also packages copies of the
foreign DNA into the new viruses. When these new viruses burst out of the host
cell, they are now carriers of the foreign DNA and can be used to introduce
this DNA into other host cells.
Transformation and Transfection
Transformation is a way that bacterial cells
pick up pieces of DNA from their environment ( the uptake of DNA by bacterial
cells ) . Exactly how this happens is unknown, but what is known is that
exposing the bacteria to calcium chloride followed by heat will cause it to
take up pieces of DNA. Another way to introduce foreign DNA into bacteria is to
transform or transduce them with the DNA and then allow them to mate. Bacterial
mating is called conjugation, and occurs when two bacteria exchange DNA through
a tube that connects them. Transfection can also be done on eukaryotic cells,
though the exact way in which this happens is also unknown. In eukaryotes,
mixing foreign DNA with calcium phosphate creates particles that fuse with the
host cell’s membrane. It is believed that the host may engulf the particle, a
process called endocytosis.
In this image, a gene from bacterial cell 1 is moved from
bacterial cell 1 to bacterial cell 2. This process of bacterial cell 2 taking
up new genetic material is called transformation.
Agrobacterium
Agrobacterium are bacteria that cause tumors,
called crown galls, in plants. Agrobacteria are drawn to plants that have been
wounded or chopped up, because sugar spills out from the wound, which the
bacteria can sense. Agrobacterium have a plasmid, called the Ti plasmid, that
contains genes that perform the transfection of plant cells. The Ti plasmid has
a region called the T-DNA, which is cut out of the plasmid and carried by
bacterial proteins out of the bacteria into the plant cell. Insertion of
foreign DNA into the T-DNA region by restriction enzymes is one way that
scientists insert genes into plants.
Injection
A common way of introducing foreign DNA into
plants is to physically inject the DNA with a gene gun. The concept of the gene
gun is to coat microscopic particles of gold or tungsten with the foreign DNA.
These particles are then loaded into the gun, which contains pressurized helium
gas. A release of the gas propels the DNA-coated particles out of the gun like
bullets. These particles penetrate the cell walls of plants and release the
foreign DNA, which is now part of the plant cell. Gene guns can be used
directly on the leaf of a plant or on plant cells that have been isolated from
ground-up plant tissue.
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