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Gene-transfer

methods

SUBMITTED TO: SUBMITTED BY:


MR.HARSH MANISHA AHIR
LECTURER B.TECH BIOTECH
SECTION-A
ROLL NO.-09
REG NO.-1040070111
ACKNOWLEDGEMENT

This is to acknowledge all those without whom my project would have not been
completed.I would like to thanks my biotechnology lecturer, Mr Harsh as he had been of
great help while making this term paper. He has devoted his previous time towards this
term paper. He has also provided us a great knowledge regarding this which has been
great importance in its fulfillment.

Although range wide of information in this term paper has been taken from the internet,
books and magazines are also consulted.
INTRODUCTION
The uptake of foreign DNA or transgenes by plant cells is called transformation.A
variety of techniques have been used to introduce by plant cells ;these can be grouped
into the following two broad categories: (1)indirect-gene transfers and (2)direct gene
transfers. The type of plant cells employed for transformation would largely depend on
the objectives of the study.For studies on gene regulation ,etc., (i) the cells needs to be
competent to take up DNA and allow the expression of trangene. (ii) But for the
production of trangenic tissues, the cells,in addition, must be merestematic. Finally, (iii)
trangenic plants can be produced only when the cells are also capable of regenerating
complete plantlets.
In case of plants,stable tranformation may be either nonintegrative, or integrative. In non-
integrative stable transformation, the transgene is maintained stably in an
extrachromosomal state. But such transformation are not passed from one generation to
next. On the other hand, integrative stable transformation results when the transgene
becomes integrated into the plant genome; these intrgratios are heritable.
Gene transfer methods in plants

To achieve genetic transformation in plants, we need the construction of a vector (genetic


vehicle) which transports the genes of interest, flanked by the necessary controlling
sequences i.e. promoter and terminator, and deliver the genes into the host plant. The two
kinds of gene transfer methods in plants are:

Vector-mediated or indirect gene transfer

Among the various vectors used in plant transformation, the Ti plasmid of


Agrobacterium tumefaciens has been widely used. This bacteria is known as “natural
genetic engineer” of plants because these bacteria have natural ability to transfer T-DNA
of their plasmids into plant genome upon infection of cells at the wound site and cause an
unorganized growth of a cell mass known as crown gall. Ti plasmids are used as gene
vectors for delivering useful foreign genes into target plant cells and tissues. The foreign
gene is cloned in the T-DNA region of Ti-plasmid in place of unwanted sequences.
To transform plants, leaf discs (in case of dicots) or embryogenic callus (in case of
monocots) are collected and infected with Agrobacterium carrying recombinant
disarmed Ti-plasmid vector. The infected tissue is then cultured (co-cultivation) on shoot
regeneration medium for 2-3 days during which time the transfer of T-DNA along with
foreign genes takes place. After this, the transformed tissues (leaf discs/calli) are
transferred onto selection cum plant regeneration medium supplemented with usually
lethal concentration of an antibiotic to selectively eliminate non-transformed tissues.
After 3-5 weeks, the regenerated shoots (from leaf discs) are transferred to root-inducing
medium, and after another 3-4 weeks, complete plants are transferred to soil following
the hardening (acclimatization) of regenerated plants. The molecular techniques like PCR
and southern hybridization are used to detect the presence of foreign genes in the
transgenic plants.

Vectorless or direct gene transfer

In the direct gene transfer methods, the foreign gene of interest is delivered into the host
plant cell without the help of a vector. The methods used for direct gene transfer in plants
are:

Chemical mediated gene transfer e.g. chemicals like polyethylene glycol (PEG) and
dextran sulphate induce DNA uptake into plant protoplasts.Calcium phosphate is also
used to transfer DNA into cultured cells.

Microinjection where the DNA is directly injected into plant protoplasts or cells
(specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0
micrometerdiameter) glass needle or micropipette. This method of gene transfer is used
to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo.

Electroporation involves a pulse of high voltage applied to protoplasts/cells/ tissues to


make transient (temporary) pores in the plasma membrane which facilitates the uptake of
foreign DNA.
The cells are placed in a solution containing DNA and subjected to electrical shocks to
cause holes in the membranes. The foreign DNA fragments enter through the holes into
the cytoplasm and then to nucleus.
Particle gun/Particle bombardment - In this method, the foreign DNA containing the
genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3
micrometers) and bombarded onto the target tissue or cells using a particle gun (also
called as gene gun/shot gun/microprojectile gun).The microprojectile bombardment
method was initially named as biolistics by its inventor Sanford (1988). Two types of
plant tissue are commonly used for particle bombardment- Primary explants and the
proliferating embryonic tissues.
Transformation - This method is used for introducing foreign DNA into bacterial cells
e.g. E. Coli. The transformation frequency (the fraction of cell population that can be
transferred) is very good in this method. E.g. the uptake of plasmid DNA by E. coli is
carried out in ice cold CaCl2 (0-50C) followed by heat shock treatment at 37-450C for
about 90 sec. The transformation efficiency refers to the number of transformants per
microgram of added DNA. The CaCl2 breaks the cell wall at certain regions and binds
the DNA to the cell surface.
Conjuction - It is a natural microbial recombination process and is used as a method for
gene transfer. In conjuction, two live bacteria come together and the single stranded DNA
is transferred via cytoplasmic bridges from the donor bacteria to the recipient bacteria.

Liposome mediated gene transfer or Lipofection - Liposomes are circular lipid


molecules with an aqueous interior that can carry nucleic acids. Liposomes encapsulate
the DNA fragments and then adher to the cell membranes and fuse with them to transfer
DNA fragments. Thus, the DNA enters the cell and then to the nucleus. Lipofection is a
very efficient technique used to transfer genes in bacterial, animal and plant cells.

Selection of transformed cells from untransformed cells

The selection of transformed plant cells from untransformed cells is an important step in
the plant genetic engineering. For this, a marker gene (e.g. for antibiotic resistance) is
introduced into the plant along with the transgene followed by the selection of an
appropriate selection medium (containing the antibiotic). The segregation and stability of
the transgene integration and expression in the subsequent generations can be studied by
genetic and molecular analyses (Northern, Southern, Western blot, PCR).

Gene transfer into muscle by electroporation in vivo


Among the nonviral techniques for gene transfer in vivo, the direct injection of plasmid
DNA into muscle is simple, inexpensive, and safe. Applications of this method have been
limited by the relatively low expression levels of the transferred gene. We investigated
the applicability of in vivo electroporation for gene transfer into muscle, using plasmid
DNA expressing interleukin-5 (IL-5) as the vector. The tibialis anterior muscles of mice
were injected with the plasmid DNA, and then a pair of electrode needles were inserted
into the DNA injection site to deliver electric pulses. Five days later, the serum IL-5
levels were assayed. Mice that did not receive electroporation had serum levels of 0.2
ng/ml. Electroporation enhanced the levels to over 20 ng/ml. Histochemical analysis of
muscles injected with a lacZ expression plasmid showed that in vivo electroporation
increased both the number of muscle fibers taking up plasmid DNA and the copy number
of plasmids introduced into the cells. These results demonstrate that gene transfer into
muscle by electroporation in vivo is more efficient than simple intramuscular DNA
injection.

In animals
The art and science of producing genetically engineered animals have advanced rapidly
in the past few years. One challenge in creating transgenic animals is to ensure that the
transgene turns on at the right time and in the right tissue. To be functional, the integrated
gene must be expressed and regulated appropriately. Thus, the gene to be transferred
must be accompanied by the appropriate promoter and regulatory sequences. Some genes
require an enhancer that may be located far from the promoter.
GENE TRANSFER METHOD

Using a soil bacterium as a gene transporter

Binary vectors: two from one. To prevent the T-DNA introduced into the Ti plasmid from
spreading uncontrollably, binary vectors were developed.

Normally, the Ti plasmid also contains ‘virulence genes’ (vir genes), which are necessary for
the transfer of the T-DNA. In the case of the binary vectors, the vir genes are removed and the
desired DNA can be integrated between the borders. These ‘disarmed’ binary plasmids have
several
ADVANTAGES:
• They are easy to propagate in laboratory bacteria like e.g. E. coli .

• A binary plasmid is also much smaller than a normal Ti plasmid, so technically


easier to handle.

The vir genes needed for transfer to the plant are arranged on a second plasmid, from which
the T-DNA and both borders are removed. With a binary vector, the target gene (T-DNA) and
virulence gene, which are naturally joined on one plasmid ring, are split between two
plasmids. One transfers the target gene, while the other helps with the transformation through
the activity of the vir genes, without the information for the transfer itself being integrate into
the plant as well.

• First step: Integration of foreign DNA (green) into a Ti plasmid without virulence
genes (vir genes). This vector is propagated in E.coli bacteria.

• Second step: The two plasmids are joined together in an Agrobacterium strain.

• Third step: The T-DNA is transferred to plant cells. The Agrobacteria and plant leaf
pieces are cultivated together, followed by regeneration. The untransformed cells are
selected with the help of the marker gene.

Diagram: Agrobacterium-mediated transformation using binary vectors

Binary vectors: room for improvement. Binary vectors have a few

DISADVANTAGES also:
• They are not very stable and are still too big.

The T-DNA sometimes contains unnecessary DNA sequences that are transferred as
well.

They are not suitable for co-transformation – the transfer and integration of two
independent genes.

The left border is ‘leaky’. It has been known for a long time now that the transfer and
integration of T-DNA starts at the RB (right border) and ends at the LB (left border).
However, the LB appears to be ‘leaky’, since sequences outside the T-DNA are often
transferred as well, sometimes even the whole plasmid. Depending on the extent and
type of the transferred sequences beyond the ‘leaky’ LB, the release or breeding of this
kind of transgenic plant is problematic, since not all transferred genes/DNA sequences
are desirable (e.g. certain antibiotic resistance genes).

Results: Several biosafety research projects have focused on optimizing binary


vectors.

Stability and size: It was possible to approximately halve the size of the vector
(smaller than 5 kb). Work is still being carried out on the suitable Agrobacterium
strain.

Removal of unwanted DNA sequences on the T-DNA:

It was possible to remove DNA sequences that do not have to be located on the plasmid.
For this to work, these so-called partition genes must be present in the genome of the
Agrobacterium instead. The integration of these genes into the Agrobacterium genome
has, however, proved difficult and was not completed within the term of the project.

Suitability for cotransformation: Cotransformation – the transfer and


integration of two independent genes – appears to be possible with Arabidopsis
(thale cress), if a vector is used that contains two independent T-DNA sections.
In another project with oilseed rape and tobacco, an Agrobacterium strain with
two plasmids appeared the most promising. By contrast, with barley, co-
transformation was most successful when two Agrobacterium strains were
used, each bearing one plasmid with the target gene/marker gene.

‘Leaky’ left border: Vectors were developed which were designed to achieve
more accurate integration of the T-DNA. For this, the termination sequence
(‘stop signal’) used as the left border of the T-DNA was employed twice or
even four times. So far, thirty plants transformed with this vector have been
studied. DNA sequences from beyond the left border had been transferred in
only one of these plants. There was not time to carry out segregation analyses
for statistical confirmation before the end of the project.
Bibliography-

1. EXPANDING HORIZONS OF BIOTECHNOLOGY:BY:-B.D. SINGH

2. www.biobasics.gc.ca./english/view

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