Advanced Techniques and Applications in Genetic Engineering: A Report

This report provides a detailed overview of genetic engineering, encompassing its history, techniques, and applications. It begins with an introduction to genetic engineering, defining the process of altering an organism's genes to achieve desired characteristics, particularly in the creation of genetically modified organisms (GMOs), primarily in bacteria and plants. The report then delves into the history of genetic engineering, highlighting key discoveries and milestones from the early 20th century to the sequencing of the human genome. The core of the report focuses on the techniques of genetic engineering, differentiating between genome editing methods like homologous recombination, meganucleases, and CRISPR, and genetic transformation techniques. The report also describes indirect and direct gene transfer methods, including Agrobacterium-mediated gene transfer and various direct methods such as electroporation, particle bombardment, microinjection, liposome-mediated transformation, and chemical gene transfer. The report provides figures to illustrate key concepts and methods. The report concludes by summarizing the key aspects of genetic engineering and its significance.

Table of Content
s
Introduction:................................................................................................................................................3
What is genetic engineering?.......................................................................................................................3
History of genetic engineering:...................................................................................................................3
Techniques of genetic engineering:.............................................................................................................4
Genome editing:......................................................................................................................................5
Homologous recombination:...................................................................................................................5
Meganucleases and ZNF.........................................................................................................................5
TALEN and CRISPER............................................................................................................................5
Genetic transformation techniques:.............................................................................................................6
Indirect method:......................................................................................................................................7
Agrobacterium mediated gene transfer:...............................................................................................7
Direct gene transfer method:.....................................................................................................................10
Physical gene transfer method:..............................................................................................................10
Electroporation:.................................................................................................................................10
Particle bombardment:.......................................................................................................................11
Microinjection:..................................................................................................................................11
Liposome mediated transformation:..................................................................................................12
Silicon carbide mediated transformation:..........................................................................................12
Chemical gene transfer method:............................................................................................................13
Polyethylene Glycol mediated transfer:.............................................................................................13
DEAE dextran mediated transfer:......................................................................................................13
Calcium phosphate co-operation mediated transfer:..........................................................................13
Conclusion:...............................................................................................................................................13
References:................................................................................................................................................14

Table of figures:
Figure 1: Genetic engineering in plants.......................................................................................................4
Figure 2: Gene transfer method in plant......................................................................................................7
Figure 3: Indirect gene transfer...................................................................................................................9
Figure 4: Electroporation...........................................................................................................................10
Figure 5: particle bombardment gun.........................................................................................................11
Figure 6: microinjection.............................................................................................................................11
Figure 7: Liposomes mediated transformation..........................................................................................12

Introduction:
Genetic engineering is a technique in which desired characteristics are introduced into an
organism in order to make it novel organisms. The main purpose of the genetic engineering is the
production of GMO’s (Genetically Modified Organisms). This technique mostly applied on
bacteria and plants. Its applications include production of genetically modified crops having
good yield and gene therapy to eliminate the diseases. The importance of this process is going up
day by day [1].
What is genetic engineering?
“Genetic engineering is a process in which gene of the organism is altered or change with the
organism in order to get the desired characteristics or phenotype of that organism and as a result
a novel organism will be created” [2].
This process is also known with two other names; one is genetic modification and the second one
is genetic manipulation. This process is called Genetic Modification because the characteristics
of that organism changed and it became novel or better one organism. The reason of this process
being called Genetic Manipulation is that, the genes or genetic makeup of the organism is being
manipulated with other. Lot of organisms have been using this technique that are following:
 Bacteria
 Animals such as fish, sheep, insects etc.
 Plants (cotton, rice, soybean, maize, etc.)
 Viruses [3].
History of genetic engineering:
About 1000 years ago, people harvest their crops and cultivate the food from crops and animals.
In 1942, corn was firstly introduced into the world that lead to development in the agriculture.
Through this way, the concept of selective breeding was introduced into the world. The

discoveries that were made in 1900s paved the way for genetic engineering. In 1922, Robbin and
Kolte cultured root and stem tips.
In 1950, Barbara McClinton explains about the transposons. Transposons are the DNA sections
that move from one place or location of chromosome to another and this process is known as
jumping gene. She said that, this all jumping led to alteration of DNA.
When the DNA was discovered by Watson and Crick in 1953 then , a scientist with the name of
Kornberg made the DNA in test tube for the very first time in 1958 by extracting the DNA
polymerase from bacteria and then assembled the nucleotides of the DNA.
1n 1962, Osamu isolated the GFP (green florescence protein) from the jelly fish and introduced
it into a plasmid. In 1967, discovery of the DNA ligase was made. In 1968, restriction
endonucleases were discovered and purified in 1970 [4].
In 1973, genetic modification was done by Herbert and Stanley. A mouse is the organism that
was first genetically modified by Rudolf Jaensch, in 1974 [5].
In 1971, Takebe developed the first plant from the protoplast.
In 1982, Mary Dell created the first genetic engineered tobacco plat. In this process, a gene
resistant to bacterium was introduced into the tobacco plant to make it resistant to bacteria. This
achievement paved the way for the scientists to manipulate the genetic makeup of organisms [6].
In 1994, first genetically modified food i.e. tomato was brought into the market. This was done
to lower the ripening of tomato but the scientists didn’t get the results that they wanted. In 1996,
a sheep was cloned and named as dolly. In 2003, human genome was sequenced [7].
Techniques of genetic engineering:
The process of genetic engineering is usually
taking place with the help of other techniques or in
simple words, other techniques are also used in the
genetic engineering. If its method is discussed in
simple word, then according to this method first of
all, a gene is selected whose characteristics have to
be show in another organism. Then isolate the gene Figure 1: Genetic engineering in plants

and then insert it into plant and then grows that seeds. Then it will turn out into a genetic
engineered plant having desired phenotype or characteristics and as a result, it will become novel
plant.
In genetic engineering, a foreign gene is inserted by gene targeting through different techniques
and these techniques provide safe transgenic crops and gene integration.
Genome editing:
It is a type of genetic engineering, in which insertion, deletion or replacement of DNA into the
genome of organisms take place. In the genome, double stranded breaks are produced by using
following techniques:
 Homologous recombination
 MEGANUCLEASES
 Zinc Finger Nucleases
 CRISPR
 Transcription Activator Like Effector Nucleases
Among all of these, most commonly used techniques are two; one is TALEN and second is
CRISPR.
Homologous recombination:
It is a technique that plays important role in maintenance of chromosome. It protects the DNA
from damage. Damage can be double stranded break or inter stranded crosslinks. There are three
ways to insert the foreign gene that include; single strand annealing, end joining and illegitimate
recombination. Breaks are made at the target site to introduce the gene.
Meganucleases and ZNF
Meganucleases:
In 1998, this technique was performed on mammalian cells. Another name of them is endo
deoxyribonucleases, because they perform their function as restriction enzymes. They are
highly specific and act at specific sites and are highly specific then restriction enzymes. They are
specific because of their acting on specific site rather then disturbing the whole genome.
Zinc-finger nucleases:

In 1996, ZNF were used with Fok1 nucleases. Like the meganucleases, they also act on specific
site and can be used to cut or breakdown the DNA at specific site and add nucleotides at the
specific site. They can bind with sequence that has least or no ability with the particular
sequence.
TALEN and CRISPER
TALEN:
It is same as the ZFN. TALEN stands for transcription activator like effector. It is more effective
than other nucleases. 1n 2003, Xanthomas that are the plant pathogen, start the transcription
process upon binding to the gene of plant cell and result is in form of infection. Its construction
is tough due to the presence or sequence in repeat form.
CRISPR:
It was discovered in 2011. With the help of CRISPR/Cas, bacteria defend themselves by cleaving
the DNA of virus and incorporating this into their own genome. Then DNA convert or
transcribe into RNA and that RNA combines with the protein and produce breaks in the genome
of virus. So, with help of this gene editing is possible. It can produce cuts or break DNA at
specific site and that can be repaired by DNA repair enzymes [8].
Genetic transformation techniques:
As the term ‘transformation’ is indicating the transfer or incorporation of gene into host cell. So,
genetic transformation technique is defied as the whole process of transfer and incorporation of
gene into the host cell. According to molecular biology, it is the transfer of gene from external
material into the specific cell by direct method or indirect method.
In this material, genetic material either it is in form of DNA or RNA is introduced into the host
cell either by artificial or by natural process. If we discuss about the plant, then there will be
transfer of DNA into the totipotent which as a result will generate a whole new plant taking the
desired characteristics and will become a novel plant. Transformation technique has different
methods but in case of plant genetic transformation, two conditions are required which includes
suitable and proper induction method. The transformation should be into those parts of plant
which have the tendency to generate a whole plant.

Genetic engineering techniques are broadly classified into two:
 Indirect gene transfer
 Direct gene transfer
Figure 2: Gene transfer method in plant
Indirect method:
As the name indicating, there is not direct transfer of gene into plant. In this method a vector act
like a messenger that transfer the gene to plant after inoculating the required gene to it. In this
method, agrobacteria cat as a second messenger. That’s why it is known as agrobacterium-
mediated transformation. Another name for it, is plant gene vector. According to this term,
vectors that are having potential to transfer the gene from an organism to the plant.
Agrobacterium mediated gene transfer:
Agrobacterium is the bacteria that is present naturally in environment or surrounding. It is a
gram-negative bacterium that is naturally found in soil. In 1983, there was successful plant

transformation by the use of the agrobacteria. The two main species of this bacterium are
following:
 A. Rhizogenes that have the ability to cause hairy root disease in plant.
 A. Tumiefacines that have the ability to cause gall disease in plant.
Crown gall disease:
It is the disease that is due to entry of A. Tumiefacines into the plant cell. If the plant is injured or
wounded, then the plant will secret or release the phenolic and these compounds would be
recognized by this bacterium and the bacterium will attack or enter into the plant. This bacterium
is
having
Tumor
inducing plasmid. That T-DNA will be transferred to the host cell that is plant. As the plant
divide, then the division of bacterium will also occur and a result it will cause disease.
Figure 3: Formation of gall tumor in plant affected by A. tumefaciens

Hairy root disease:
In this disease, A. Rhizogenes is a causative agent of the disease. It induces or effect the root
hairs of plant unlike the A. Tumiefacines, that is involved in gall formation into the plant.
Both of these diseases are showing that agrobacterium have the tendency to enter into the plant
cell and attach its DNA with plant DNA. As the plant will grow, the growth or division of
bacterium will also occur. So, agrobacterium can be used to introduce desired genes into the
plant.
METHOD:
For the transfer of the gene into the cell. First of all, the gen would be introduced into the Tumor
inducing plasmid. Then the plasmid containing the required gene would be transferred into the
plant. The selection of the plant is having two requirements; one is the plant have to produce
phenolic compounds, then it will be able to attract the bacterium and second is that, the tissue or
cell in which the plasmid has introduced should have the capability to generate a whole new
plant. Then this whole culture will be transferred into suitable medium and plant will be grown
containing the required gene or characteristic [9].

Figure 4: Indirect gene transfer
Direct gene transfer method:
In this method, there is not any need of the vector or plasmid to transfer the gene into plant.
Rather, the gene is directly transferred into plant without using second messenger. In this
method, direct transfer of naked DNA occurs. This method is effective and simple. Because of
this method, many transgenic plants have been produced.
This method is categorized into following:

 Chemical gene transfer method
 Physical gene transfer method
Physical gene transfer method:
It is divided into sub categories that are following:
Particle bombardment
Silicon carbide fibers
Microinjection
Electroporation
Liposome fusion
Electroporation:
It is a method in which electrical impulses are used in order to create hole to transfer the DNA
into plant. In this method, the plant material in buffer solution that is also containing the foreign
DNA is incubated and then subjected the electrical impulse to create holes in plasma membrane.
Through this plasma membrane, DNA will enter and become the part of host cell genome. In
past, protoplast was the only one that is used for gene transfer. Now, callus culture, premature
embryo has been using to produce transgenic plant.
Figure 5: Electroporation
Advantages:
 This method is simple, cheap, and less time consuming.
 By changing the electrical field, efficiency of this method can be changed or improved.
 The physiological state will remain same after this method.
Limitations:

 In case of protoplast, regeneration of plant is not facile.
 The amount of DNA transfer is very low.
Particle bombardment:
Another name of this method is biolistic.
In this method, DNA coated with material is shoot by the gun into
the selected plant tissue. The DNA coating material can either be
gold or tungsten. The gun is driven due to the high pressure. Due
to this process, DNA will be inserted into the genome.
Limitations:
 The target tissue can be damaged due to high velocity.
Microinjection:
In this method, a micropipette or capillary tube is used to transfer the solution of DNA into plant
tissue or part. Mostly, callus, embryo is used for this process. After microinjection, the receiver
or host cell is placed into agarose gel with the help of suction pipette. After that, this is placed
into medium containing required nutrients for the plant growth and as a result, it led to the
formation of transgenic plant.
Liposome mediated
transformation:
These are lipid vesicles.
They are also having
phospholipid membrane
and artificially prepared.
These are mostly use to transfer the drug at target site.
Figure 6: particle bombardment
gun
Figure 7: microinjection

In this method, plasmid containing liposomes will introduced and they will bind at attachment
site. After that, they will fuse and as a result, plasmid will enter.
Advantages:
 Can be applied on wide variety of plant.
 DNA can be stored in them for long time.
 It protects DNA from damage.
Limitation:
 The problem is only in case protoplast regeneration into whole plant.
Silicon carbide mediated transformation:
Silicon carbide fiber is used of 0.3-0.6pm width and of 10-100pm length. This fiber has ability to
enter cell wall and cell membrane and then inject the material. The DNA does not bind properly
to the fiber so, can easily transfer into call and bind with genetic material.
Advantages:
 This one is easy and cheap method.
Disadvantages:
 Carefully handling is required because silicon carbide is carcinogenic.
 In embryonic cell, SCF faces resistance.
Figure 8: Liposomes mediated transformation

Chemical gene transfer method:
It is sub categorized into following:
DEAE dextran mediated transfer
Calcium phosphate co-operation mediated transfer
Polyethylene glycol mediated transfer
Polyethylene Glycol mediated transfer:
In this method, PEG with the help of cations (e.g. Ca++) disturbs the plasma membrane and as a
result DNA enters to nucleus and then integrates with genome.
Advantages:
 Can be used for large variety of plant species.
Disadvantages:
 Degradation of DNA can be occurred in this process.
 Undesirable traits can be obtained due to random insertion of DNA in genome.
DEAE dextran mediated transfer:
DNA is combined with diethyl amino ethyl (DEAE) that is polymer containing high molecular
weight and then shift it. Stable transformation is not obtained through this method.
Calcium phosphate co-operation mediated transfer:
In this process, precipitates of DNA-calcium phosphate forms, when DNA is mixed with calcium
chloride solution in phosphate buffer. Continuously dividing cells then placed on these
precipitates, as a result, cell will be transferred after few hours. DMSO has ability to increase
efficiency [10] [11].
Conclusion:
It is a technique that is used for transfer of gene or genetic makeup from one organism to another
organism. There are different techniques for the editing of genome but the best one is CRISPR.
There are two methods for the transfer of gene; one is direct while another is indirect method. So
we can use both of these method in genetic engineering of plant and as a result variety of
transgenic plant can be produced.

References:
https://www.teachengineering.org/lessons/view/uoh_genetic_lesson01
https://www.genome.gov/genetics-glossary/Genetic-Engineering
https://en.wikipedia.org/wiki/Genetic_engineering
ynthego.com/learn/genome-engineering-history
https://en.wikipedia.org/wiki/History_of_genetic_engineering
https://www.iatp.org/sites/default/files/Brief_History_of_Genetic_Engineering_A.htm
https://www.slideshare.net/HikmetGekil/genetic-engineering-chapter-1-history-of-genetic-
engineering
https://en.wikipedia.org/wiki/Genetic_engineering_techniques
http://www.biologydiscussion.com/genetic-engineering/methods-of-gene-transfer-used-in-
plants/39218
http://www.biologydiscussion.com/genetics/engineering/methods-of-gene-transfer-in-plants-2-
methods/10824
https://www.slideshare.net/RIZWANABBAS3/gene-transformation-techniques