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Gene Transfer Methods
Vivek Prasad
Professor
Department of Botany
University of Lucknow
Lucknow
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Gene Transfer Methods
One of the key objectives of recombinant DNA technology is to genetically modify
organisms so that they begin to exhibit to desired traits. However, even before that stage,
recDNA has to be constructed and the gene of interest analyzed. In the process, the recDNA
constructed has to be introduced into a living cell for amplification.
The most frequently used methods of gene transfer (transformation) are chemically
assisted transformations of protoplasts, electroporation, bombardment of plant material with
DNA-coated microprojectiles, and specifically for plants, by using the bacterium
Agrobacterium tumefaciens and its resident T plasmid.
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Chemically-assisted transformation:
Protoplasts take up DNA from surrounding medium that gets stably integrated into the
genome in a proportion of transfected cells. The most commonly used chemical is
polyethylene glycol (PEG). Protoplast transformations present problems, due to the inability
of the host species to regenerate from protoplasts, independent replication of the DNA
inserted in this way, and random integration into any plant chromosome through non-
homologous recombination.
Bacterial Transformation:
E. coli is the most commonly transformed bacterium in the lab, despite the fact that
they are not transformable in nature. Hence, they have to be made competent to take up DNA
artificially. This requires growing E. coli LB broth, and treating them with MgCl - CaCl
2 2
solution initially, followed by M CaCl alone. This renders them capable to take up DNA
2
however, their membrane becomes fragile, and hence they have to either be transformed
o
immediately, or stored at -70 C. Transformation of competent bacterial cells is generally
carried out by the heat shock method where the competent cells and DNA are taken together,
o
kept on ice for 30 min, and then kept at 42 C in water bath for 90 sec, followed by an
immediate transfer to the ice-bath. This cold-hot-cold treatment (heat shock) causes the DNA
to enter the cell. Subsequently, more LB is added to the transformed mix, kept in a water bath
o o
at 37 C for 1 h, and then plated. Incubation at 37 C will show colonies in approx 12-16 h if
the transformation has been successful.
Electroporation:
This is another, arguably a more elegant way, of transformation, where the DNA
passes through electropores, generated in the membrane on exposure to an electrical pulse.
The pore formation is very rapid, about 1 µs. these electropores then reseal. Factors affecting
electroporation are temperature, electrical field, topological form of DNA, and host cell
factors.
Microprojectile bombardment (Biolistics):
A third method is by using DNA coated gold or tungsten spheres, 0.4 – 1.2 µm
-1
diameter (microprojectiles) that are accelerated to 300-600 msec with a particle gun. The
particle gun may use gunpowder, compressed air or compressed helium as the thrust. The
projectiles hit a stopping plate, and the microprojectiles are released at high velocity that
causes them to penetrate cells. The transforming DNA integrates randomly into plant DNA.
The advantages of the method are the introduction of DNA into many cell types, including
monocot plant tissue. Linear DNA is more efficiently integrated than circular DNA.
Microprojectile method can also introduce DNA into chloroplasts and mitochondria.
Agrobacterium-mediated Gene Transfer:
A highly favoured method for making transgenic plants is by using Agrobacterium
tumefaciens that causes Crown Gall disease in a wide range of plants. Crown gall tissue, in
infected plants, is the result of an oncogenic transformation, whose ability of continued
division is retained even after the infecting Agrobacterium perishes. This is because of a
plasmid, the T plasmid. This is a 200 kbp plasmid that has 2 regions of interest to recDNA
i
technology. One is the 35 kbp Vir region that houses the virulence genes, and the other is the
10 kbp T-DNA, the part that actually transforms the host. The simple concept is that the
foreign gene is hooked on to the T-DNA part of the T plasmid, it will be naturally introduced
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into the host and integrated as well. But such a system will also lead to disease, hence a
disarmed Ti plasmid has to be constructed. In this case, the tumour inducing genes are
removed, and replaced by a cloning vector3such as pBR 322. The vir genes are responsible
for the integration of the T-DNA into the plant genome, hence the part that now gets
introduced has the foreign gene of interest but no oncogenic properties.
The Ti plasmid based vectors fall into 2 major categories: co-integrate and binary.
The co-integrate vector involves co-integration, in A. tumefaciens, between
homologous regions on disarmed Ti plasmid and an intermediate E. coli cloning vector, such
as the pBR322, which contains a selectable marker gene that will function in plant cells as
well for identification. In the co-integrate system, vir genes are carried on the same plasmid
as the insert.
The T plasmid based binary vector is based on plasmids capable of replicating both in
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E. coli and A. tumefaciens, and that contain the T-DNA borders for identification by the Vir
proteins. Border sequences are on either side of the multiple cloning site (MCS) to allow
insertion of the gene of interest and markers for selection. This vector system thus consists of
two plasmids: one carrying the MCS, and the other carrying the Vir genes to function in trans.
The recombinant plasmid is transferred to A. tumefaciens carrying helper Ti plasmid
with Vir genes, the plant cells then grown in the presence of A. tumefaciens to promote the
transfer of recombinant T-DNA into the plant genome. Transformed plant cells are then
selected, and grown through tissue culture.
References:
Aneja, KR, Jain, Pranay, Aneja, R, 2008, A Textbook of Basic and Applied Microbiology,
New Age International Publishers, New Delhi, 773 pp.
Gupta, PK, 2016, Plant Biotechnology, Rastogi Publications, Meerut, 676 pp.
Primrose, SB, Twyman, RM, 2006, Principles of Gene Manipulation and Genomics,
Blackwell Publishing, Oxford, UK, 644 pp. 644.
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