Genetic transformation involves inserting foreign DNA into a cell's genome. There are several methods of genetic transformation, including bacterial transformation using viruses or bacteria, retroviral transformation using retroviruses, and Agrobacterium-mediated plant transformation. These techniques are used for applications such as producing recombinant proteins, creating transgenic animals and plants, and developing gene therapies and vaccines.

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Genetic transformation systems

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Genetic Transformation System
Introduction
Transformation means changing/alterations and genetic means in genome. Genetic
transformation is the changes in the cell result in direct uptake and exogenic material
incorporation from surrounding environment through the cell membrane. Those techniques in
which each cell having specific genetic material are changing by inserting foreign DNA in its
genome.
Till (/2014) 80 species of bacteria are known to having proficiency of transformation .for
transformation to take place calls or bacteria should have the state of complete competence
which exist in nature for limited time depending on environmental conditions
.Environmental conditions like starvation or cell density.(These can be artificially induced in
the lab).
History:
1st evidence of transformation of bacteria was given by Fredrecik Griffith in 1928.He made
the strains of streptococcus pneumonia virulent by exposing the heat killed virulent strains.
He hypothesized that heat killed strains are cause of making harmless strains virulent.
In 1970 Marton and Akiko said that E.coli treatment with calcium chloride prompt the DNA
from bacteriophage without use of helper phage. In 1972 Stanley and Annie showed that
calcium chloride is also effective for plasmid DNA transformation.
Transformation including electroporation is induced in 1980 which is an efficient in in-vitro
transformation and bacterial strain transformation.
 In 1970 Ti plasmid discovered.
 In 1980 Biolistic particle delivery system.
 1982 transgenic animals and plants produce, 1st transgenic mouse produce. A gene
for a rat growth hormone was inserted in to a mouse embryo.
There are 2 methods of transformation (Direct and Indirect method)
Plant Transformation:
Genetic engineering techniques:
 Microbial vector
 Micro projectile Bombardment
 Electroporation
 Microinjection
 Transposable elements
Techniques other than genetic engineering:
 Simple selection
 Crossing
 Interspecies crossing
 Embryo Rescue

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 Somatic Hybridization
 Somacolonal variations
 Cell selection
Bacterial Transformation (Indirect method):
a) Exogenous genetic material transformation:
(Technique in which DNA is taken from external environment and insert in to the genome.
important in recombinant DNA technology.it involves the use of plasmids. External sources
mean from the dead bacterial cells.
b) Conjugation:
(A technique that involves merging of DNA from other bacteria, two bacterial contact leads
to exchange of genetic material.one bacterial cell act as donor and other act as recipient. Gene
transfer occur)
c) Transduction:
(This process occurs with the help of Bacteriophage .bacteriophage act as a vector.
Bacteriophage carries its own genome and a DNA fragment which is taken from bacteria
which it recently effect.it bacteria survive virus attack then recombination occur)
Animal Transformation Method:
Transformation in animals is take place in 3 ways.
 Biological means:
 Viral mediated.
 Bacterial mediated.
Agrobacterium bacterium is beneficial in over expression of recombinant protein in plants.
More stable and require more less of lengthy steps which are require for regeneration.
 Physical means (Direct method)
 Gene gun
 Electroporation
 Chemical mean (Direct method)
 Liposome mediated gene transformation
 Stem cell mediated gene transformation.
Gene transformation in bacteria
In bacteria, asexual reproduction is common they multiply like mitotic cell division and
multiply within short time. But in gene transformation system bacterial genome can be
transformed by two main methods.
 Conjugation
 Transduction
Conjugation:
This process discovered in 1946 by Joshua Lederberg and Edward Tatum.

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In this process two bacterial cells merge and genetic material from one bacterial cell is
transfered to other. One cell is act as donor and other act as recipient. The ability of donating
genetic material depends upon F factor or fertility factor. F factor gene produces pilus which
is small thin tube which attaches with the recipient bacterial cell and transfer gene in the form
of plasmid. Conjugation give recipient cell an advantage but it makes the recipient cell
infertile due to lack of F factor.
 Conjugation occurs in prokaryotes otherwise it is energy-driven phenomenon.
 Conjugation helps in transformation of antibiotic resistance genes
 We can use it in insulin production.
 Plasmids are also transfer by this method.
Fig: conjugation
Transduction:
Transformation of foreign DNA into bacterial cell genome by virus is known as
transduction. This method is first explained by Joshua Lederberg and Norton Zinder in
1952.
Bacteriophage mediated transformation
This is done by bacteriophage having foreign DNA. This method is widely used and helped
us in formation in many products the foremost example is insulin. Many antibiotics,
hormones and other proteins are formed by this method. In this method bacteriophages
having gene of interest incorporated foreign DNA into host genome that is actually a bacterial
cell. This complete cycle can understand by following steps.

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Steps involve:
Fig; cycle describes whole process of transformation
Bacteriophages:
In general or general transduction occurs when the bacteriophages attaches to the bacterial
cell and incorporates its DNA and enzymes necessary for packing of homogenized proteins.
DNA of virus incorporates naturally into host genome and replicating product produces viral
proteins that packed and form new phage virus. This new prophage causes lysis of bacterial
cell, which is ready to infect other bacterial cells. Before lysis viral genome gets accidentally
some part of bacterial genome that is rDNA. This DNA enclosed into capsid and transfered
into next bacterial cell. Bacterial cell then gets this recombinant DNA and replicates through
lysogenic cycle. In this cycle multiple copies of rDNA are formed.
In specialized or specific transduction, bacteriophages attaches to the host cell incorporates
its DNA that attaches with host genome but special enzymes cut this DNA at specific site
having some part of bacterial genome that can be our gene of interest. This excised DNA
goes through lytic cycle and infects other cells. When it transmitted to other cells the gene
attaches with prophage DNA is replicated and we take our product when cell lysis occurs.
Phages normally do cell lysis when feasible conditions are available and large amount of
specific product is formed.
 Bacteriophages gives our desire product in short time
 More efficient
 Commercially used for production of insulin
isolation ofgene
of interest
clonningof gene
of interest
insertionof our
gene into
bacteriophage
formationof
host colony
insertionof
bacteriophages
into host colony
lysogenic cycle /
lytic cycle
desire product
(isolation)

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Fig: generalized transduction Fig: specialized transduction
Viral mediated gene transformation
Retroviral gene transformation:
There are two strains of HIV.
 Ecotrophic
 Amphotropic
Ecotrophic:
Those strains of HIV that only infect mimetic cells (mice cells)

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Amphotropic:
Those strain that effect other than murinic cells.
Life Cycle of HIV:
It involves 6 basic steps
 Fusion /attachment
 Reverse transcription
 Integration
 Replication
 Assembling
 Budding
Fig; HIV structure
 Fusion:
HIV cell binds with the receptors of the CD4+ cells and the single stranded RNA along with
the three enzymes pass through the cell leaving behind the capsid.
 Reverse transcription:
The machinery inside the cell consists of two active sites. First is polymerase active site while
the other is ribonuclease active site. The single stranded RNA passes through the polymerase
active site and then through the ribonuclease active site where a template strand will form of
the RNA which will again pass through the polymerase active site where it will be converted
to double stranded DNA.
 Integration:
Integrase enzyme facilitates the transfer of the DNA to viral genome and help in integration
of DNA with the viral genome by cutting the DNA and producing sticky ends which help in
binding with the viral genome.
 Replication:
As the process of integration is done, HIV begins to use the machinery of the CD4+ cell and
starts the process of replication by making copies of the viral genome and produce large viral
polypeptide chains, these chains act as the building blocks of HIV.

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 Assembling:
 Protease breaks these long chains and converts them into smaller subunits (capsids,
spikes etc.).then the assembling of all the structural and functional units of the virus
takes place on command of psi(primary sequence initiator)
Fig: retroviral mediated gene transformation
 Budding
Newly formed HIV cells push themselves towards the membrane of the CD4+ cell and bud
out. A new HIV cell is thus released in the cytoplasm.
.

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Agrobacterium bacterium mediated genetic transformation
It is an indirect method of genetic transformation in plants. Agrobacterium tumefaciens cause
crown gall disease and Agrobacterium rhyzogen cause hairy root disease. Agrobacterium
species are responsible for genetic transformation in plants. It contain the Ti (tumor inducing)
plasmid which contain the T-DNA (transferred DNA).Ti plasmid has three main region. First
is T DNA region which is present between left and right border. Second is Virulence region
and third region is opine catabolism.
T DNA region:
This region contain opine synthesis and tumor inducing gene (oncogene).
Virulence region:
This region has many Virulence genes. Vir A function is to recognize the Acitosyringone
which are released by wounds of plants. Vir E function is gene transfer protein.VirD1 gene
is used for cleavage of super coiled stranded of substrate and it also assists the VirD2
gene.VirC is used to transfer of DNA. Energy provide to DNA is performed by VirB11
which exhibit the ATPase activity. Vir B function is used to Transfer T DNA to plant
genome. Vir D4 formed the conjugal tube between plant and bacteria cells for the purpose of
transfer of T DNA.
Opine catabolism:
Bacteria take up the opine as a nutrient.
Chromosome gene:
The initial bindings are done to plant genome by
chvA, chvB pscA and it is responsible for the
polysaccharide for bacterial surface. PscA
play a major role in transportation of t DNA.
Explanation:
The wound plant releases the Acitosyringone which are recognized by VirA. Border
sequence recognized by VirD2 and single stranded copy of T DNA are produced by directing
the DNA polymerase Vir d2 is covalently attached to 5` end of T DNA copy. and the
remaining portion of T DNA is covered by molecules of VirE2 and a complex is formed
which are rod shaped and are transferred through wounds of plants.Vir D2 have a nuclear
targeting theme in the Amino acid sequence and it transfer the T-complex from nuclear pore
to nucleus in which the T DNA is finally integrated to targeted cells.
Formation of single strand copy of T DNA:

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Fig:tDNA copy formation
Mechanism:
The T DNA has a gene which is tumor inducing. We can eliminate the Tumor inducing gene
from TDNA and a gene of interest is added so that we can get the desired products. The steps
are as follows.
1. Gene of interest is inserted in agrobacterium.
2. Cells of plants are transformed by modifying the T DNA which has a novel gene.
3. Plants are regenerated from the transformed cells.
Limitations:
1. There is an obstacle which is size of the plasmid and it is overcome by constructing a
small size vector form which a gene of interest is added.
2. It only affects dicots plants not monocot plants because it does not produce
Acitosyringone.
Stem cell Mediated Gene Transformation
It is the method of gene transformation and creating genetic animals. As we know stem cells
are undifferentiated cells which can be differentiate into all types but this is not the case.
Stem cells actually have ability to renew it and to divide through mitosis not meiosis. These
are actually somatic cells. These cells can be differentiated into blood circulatory system,
Hepatocytes, capillary system. Stem cells are present in multicellular system.
Types of stem cells:
Embryonic Stem cell:
It is progenitor stem cell .They are not specific stem cells and they can be differentiate into
any system.

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Adult stem cell:
It is the type of stem cell and it cannot be differentiate into any type .In case of injury they are
able to regenerate. They are rich in liver, bone marrow, lymphocytes skeletal tissues, Adipose
Tissue, Blood circulatory system these entire region contain stem cells.
Explanation:
Instead of taking Adult stem cell we use embryonic stem cells because they can be transfer
into any system. Blastula is the stage having stem cells .From the start of the blastula each
blastula has almost 200 to 300 blastocysts and these blastocysts from blastula stem cells are
separated. Through process of Electroporation physical mean we transfer gene of interest to
stem cells. Under the influence of electric impulse the membranes are actually permeated and
become porous and genome of interest can be transferred. We have to culture it in vivo.
Electroporation is effective means even from chemical means. Basic purpose of culturing is
to increase the concentration of our stem cells. Stem cell technology is actually used to make
clone either it is part of culture cloning or reproductive cloning .Now these stem cells are
transferred through Micro injectors to blastula step. Implant blastula into uterus of Foster
mother of mice model. Then it give birth of chimeric mouse. This is actually transgenic
animal. This is further mutated the normal female mice.
Purpose and Uses:
 Its purpose is to enhance the total clone .They will all contain gene of interest.
 We are increasing milk production, meat production and livestock production. All is
due to this technique .We applied our gene of interest .Now this technique is more
important in Health biotechnology.
 This technique has border application in biotechnology, we use microinjectors, gene
gun.
 This technology is used in cancer treatment.
Applications of biological Means of Gene Transformation
i. Viral mediated gene transfer is used for cancer treatment .Cancer growth can be
altered by gene therapy vector. These vectors carry single growth inhibitory gene that
take on immune response against the tumor. These are used in Cancer treatment.
These vectors show Anti clinical activity in the tumor. Multi-gene techniques may
also be used to attack cancer cell several pathways. It also stretches the use of
cytotoxic gene transfer which is combining with immunotherapy.
ii. Retroviral gene transfer is used in making Transgenic Animals. These are used as
vectors to transfer genetic material to the cell. It will produce a wide variety of species
.It is used to transfer gene to host cell and vector are used as vehicle for this process
of transformation. Vectors are carrier molecules used to transfer our gene of intrest.
iii. Bacteriophage is used for insulin production. These are used to treat pathogenic
bacterial infections. These are used for making medicines as well as dentistry,
veterinary science and agriculture.
iv. Agrobacterium is used in Agroinfiltration. It is the method for the production of
recombinant proteins in plant cell. Agrobacterium is important pathogen used in plant
cells. It is used as a tool for biological transformation of plants It is used to improve
crop plants for farmers.

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v. Viral mediated gene transfer is used in gene therapy as well as making vaccines.
Adenovirus is actively used as vaccine. These are used to protect viral infections such
as Malaria. Gene therapy is used to cure genetic disorders.
vi. Agrobacterium is used in genetic engineering for the improvement of plant .In it we
use modified Ti or Ri plasmids.
vii. Agrobacterium also use in Recombinant DNA technology. It is use as vector for
sugarcane, cotton, corn , soya bean etc.
Conclusion
Transformation is a technique in which individual cell having specific genetic material is
changed by inserting foreign DNA in its genome. Bacterial Transformation is by viral and
bacterial mediated. Bacterial mediated is beneficial over expression of recombination through
bacteriophages and conjugation success rate is high and many commercial products are
formed. Retroviral mediated transformation method uses retroviruses which help in
transferring the genetic material inside the host cell. However as a result organisms are
produced which have cells and tissues of unique and diverse genetic makeup. Agrobacterium
mediated gene transformation widely used in crop production. Biological means of
transformation are most reliable mean of transformation.