This document discusses various techniques for gene transformation, including biological, physical, and chemical methods. Biological methods use vectors like plasmids or viruses to transfer genes. The gene gun is described as a physical method that uses compressed gas to "shoot" gold or tungsten particles coated with DNA at high speeds into target cells. Calcium phosphate precipitation and liposomes are outlined as chemical methods that use calcium phosphate or lipid vesicles to transfer genes into cells. The document provides details on the procedures and components involved in gene gun transformation.

1. BAHUDDIN ZAKARIYA UNIVERSITY MULTAN
Institute of Molecular Biology & Biotechnology
Department of Biotechnology
Instructor: Dr Sumaira Rasul
Course: Recombinant DNA Technology
Course code: BSBT
Assignment Title: Physical and Chemical means of gene transformation
Submitted by: Group one
Class: BS Biotechnology
Semester: 7th
Members
All members of group had participated in collecting information and contributed their role in preparing this
assignment. The members include,
Muhammad Furqan: BSBT-13-F-005
Muhammad Junaid: BSBT-13-F-010
Salman Aslam: BSBT-13-F-029
Muhammad Ahmad: BSBT-13-F-030
Rizwan Abbas: BSBT-13-F-043

2. GENE TRANSFORMATION TECHNIQUES
Table of contents
s.no Title sub topics Page number
A Introduction
a. Gene transformation
b. Significance of gene transfer 01
B Gene transfer techniques a. Description 01
C Biological means of gene
transfer
a. Vectors & Plasmid
b. Advantages and limitations
02
03
D
Physical means of gene
transfer
a. Gene gun
b. Micro injection
c. Electroporation
d. Laser beam & ultrasound
04
05
06
07
E Chemical means of gene
transfer
a. Calcium phosphate mediated GT
b. Liposome mediated
c. Polyethylene Glycol
08
09
10
F Applications
a. Transgenic plants
b. Transgenic animals
c. Examples
d. limitations
11
G Future perspectives a. future perspectives
b. conclusion
12
H bibliography Bibliography 12

3. Physical and Chemical techniques of Gene transformation
Abstract
Gene transformation is descriptive word refers to genetic change, replacement of piece of DNA or transfer
of a new gene in the cell or genome. The phenomenon of genetic transformation was revealed by Fredrick
Griffith in 1928. Later the principle of transformation was elaborated by Ostwald Avery and coworkers. In
order to transfer the gene of our desire character we usually follow three different types of tools according to
availability, result and organism under research or manipulation and conditions. These techniques include,
 Biological means of gene transformation.
 Physical means of gene transformation.
 Chemical means of gene transformation
The main aim of all these techniques includes molecular cloning and production of
transgenic organisms. These transgenic organisms are applied for production of number of product and
function for benefit of mankind. All of given techniques have also some limitations. Generally as physical
methods are safer and effective method but they are also costly to afford.

4. Techniques of gene transformation
A. Introduction
a. What is gene transformation?
Gene transformation is descriptive word refers to genetic change, replacement of piece of DNA or transfer
of a new gene in the cell or genome. The phenomenon of genetic transformation was revealed by Fredrick
Griffith in 1928. Later the principle of transformation was elaborated by Ostwald Avery and coworkers. In
1953 the discovery of double helical model of DNA by James Watson and Francis crick gives huge
information about genetic material. This achievement is settled as base for new research and analysis. The
science of genetic transformation and recombinant DNA technology is rooted in restriction enzymes first
discovered by Hamilton Smith in 1970. In short words gene transformation technology is known as genetic
manipulation or modification which involves changing or transfer of DNA or gene segments within or in
different species. Genetic transformation has wide variety of significance and benefits. The term Transgenic
was coined by J.W. Gordon and F.H Ruddle in 1981 referring to organism having modification of gene.
The most important outcome of such technology is genetically modified organisms (GMOs).
b. Significance of Transformation
Nowadays genetic transformation has prime importance due to its number of outcomes and products. Why
we do genetic transformation? The answer of this question is quite simple and easy, that everyone can
answer this.
The manipulation and genetic transformation is well known techniques by which we can introduce, or
integrate our gene of interest in order to obtain the required product. This tool is used for molecular cloning.
Disease treatment, diagnostics, pharmaceutical products, qualitative and mass production of materials and
even organism is now easily processed through such mechanism. Insulin obtained from bacteria in 1972,
GMOs and genetic analysis like gene therapy are famous examples.
B. Gene Transfer Techniques
Genetic transformation techniques were developed after studying the natural transformation methods like
bacterial conjugation, transduction and Agrobacterium mediated gene transformation. The delivery of genes
categorized as follows.
 In-vivo
 Ex-vivo
Ex vivo gene delivery is (transformation outside the body of host) while In vivo delivery involves the
transfer of genes directly into the cells within host.
Gene transfer may be transient i.e. temporary or stable i.e. permanent and integrate for all time. Generally
we prefer stable transformation techniques.
In order to transfer the gene of our desire character we usually follow three different types of tools according
to availability, result and organism under research or manipulation and conditions. These techniques include,
 Biological means of gene transformation.
 Physical means of gene transformation.
 Chemical means of gene transformation.
Figure
A. Recombinant DNA formed by ligation of
desired gene-
B. Transfer of Insulin gene in plasmid

5. C. Biological means of gene delivery.
To deliver or transfer of our gene of interest we require a compatible carrier or delivery vehicle known as
vectors. Biological mean of gene delivery uses a biological system as gene carrier. These vehicles include
synthetic or natural parts of biological systems like Plasmid, viruses, artificial chromes etc. The Ti Plasmid
of Agrobacterium tumefaciens has natural ability to transfer DNA in plant cells by producing Crown Gall
disease. Biotechnologists use such plasmids for transformation of gene of interest in plant cells in growing
medium.
a. Vectors and plasmids
Some strains of bacteria contain circular double stranded plasmids which have capability of carry foreign
gene. They are used as delivery systems.
pBR322, pUC19, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are
common examples of non viral gene carriers.
Synthetic replication deficient viral genomes, phagemids, cosmids and SV40 etc are example of viral
mediated gene transfer carriers.
Major steps include in transformation.
The basic general steps include in gene delivery are as follows.
 Identification and Isolation of gene of interest.
 Cloning of desired gene.
 Generation of Vectors.
 Restriction of vectors and ligation of gene.
 Integration of gene.
Agrobacterium mediated gene transfer is shown as follows.
b. Advantages and limitations
 Biological mean of transformation have highest rate of success.
 HIV mediated gene transfer has up to 50% transformation rate, and are specific in their function.
 Plasmids are also specific and can perform target specific delivery and economical as well as
available.
 However they have some limitations including laborious, technical and sometimes pathogenic. HIV
mediated gene transfer may induce allergic reactions in host and activation of oncogene.
 The plasmids or vector need marker gene for screening making the task difficult.
Physical and chemical means of gene transfer are our main focus and are described in details as follows

6. D. Physical Means of Gene Transfer
This type of gene delivery technique uses the physical apparatuses and tool for transfer of desired gene in to
the target cell. This method involves the direct transformation of the target cell or tissue by transferring
DNA present on or in the apparatus. These techniques provide us sufficient as well as rapid transformation
of target cells within optimized conditions and handling. Another benefit is that unlike biological means
such methods need less number of molecular steps to perform and minimize the work burden and save time
also. The well known physical means of gene transformation techniques include,
 Gene Gun , Biolistic / Ballistic or Golden Gun method
 Microinjections
 Electropration.
 Laser beam technique
 Ultrasound / Sonication etc
 Stem cell mediated
The detail explanations are given below.
a. Gene gun/Golden gun/Biolistics
Gene gun is an instrument used to shoot the DNA on target sites. It is like a pistol and also known as
Biolistics, Ballistics and Golden gun as it uses gold particles for transformation process. This amazing and
successful invention was done by John C Sanford and coworkers of Cornell University in 1985. Gene gun
is carrier free i.e. direct transformation tool and usually used in both in-vitro as well as in-vivo gene transfer.
Gene gun mediated transfer is used for stable integration of gene.
Construction and Procedure of gene gun
Structurally gene gun is comprised of five main components.
 Firing pin with pressure source
 Macro projectile
 Micro projectile
 Safety vents
 Plates containing target cells.
Operating procedures
The gene transfer is followed by four basic operating steps.
 Preparation of DNA loaded beads
 Preparation of plastic tubes.
 Loading of Macro projectile
 Shooting (gene transfer)
The detail explanations are as follows.
Preparation of DNA loaded beads
About 250 mg Gold particle of specified size 1.6 µm is required, which is placed in a tube. Spermadine a
polyamine uses as cationic surfactant which creates positive charge on particle enhancing the affinity of
DNA binding. DNA solution is prepared and mixes both solutions; incubate subsequently for few minutes
which result in precipitation. Add precipitating agent and incubate for half an hour followed by
centrifugation at 10000 rpm .Discard the supernatant and the precipitated material is washed by absolute
ethanol in order to remove unbound genome.
Polyvinyl pyrolidine (PVP) an adhering agent is added to solution, which enhance adherence of particles.
Preparation of plastic tubes
About 2.6mm diameter and 2 to 6 feet long inert, cleaned plastic tubes are used for loading the DNA beads
of gold. The sample is loaded by help of sucking pumps. As the tube is loaded with DNA it is rotated in
rotators for uniform distribution of particles. After slight retention the tubes are cut in specific size e.g. 2cm
with help of cutters or choppers.
Loading of tubes in Macro projectile
The particles loaded in tube are loaded in the macro projectile and adjust properly .Macro projectile is just
like a cartridge or magazine of gun which holds tubes containing DNA beads.
Gene gun

7. Shooting
A firing pin associated with helium gas is used as a trigger which generates pressure on pressing. The
pressure is optimized according to the cell under transformation. Pressure (200 psi for nerve cells, while
195psi in case of the liver cells). The distance for transformation are also regulated and optimized according
to the cell and conditions. Plastic tube and pressure gas is exit from vents while DNA bead is inserted in
targets. Integration of targets cell is auto regulated and analyze transformation. This technique is amenable
for both plant and animal cell line transformation.
The block diagram of gene gun is shown below.
Advantages and disadvantages
 The success rate gene gun mediated transformation is less however it is safer method.
 Success rate is up to 30-50 percent.
 Safety regulation, proper handling is required for efficient transformation.
 Integration is auto regulated.
b. Micro injections
Microinjection technique is the physical mean of gene transfer. This technique is rooted in the work of Dr
Marshall Barber. It is the most efficient technique of transformation of animal cell as well as plant
protoplast. It is based on principle of direct transformation. The transformation is done by injection of gene
of interest through glass syringe i.e. pipette in target cells under observing lenses. The plates containing
target cells as well as injecting pipettes are hold by holders known as micromanipulators. Usually the tip of
pipettes ranges from 0.5µm-5µm in diameter.
The cells are placed in a medium with optimized parameters and gene is injected under observing lens.

8. Microinjection is also known as pronuclear transformation as it efficient in protoplast or nucleus of germ
line cells transformation. This technique has more potential to transform plant cells and requires proper
handling and expert. The transformation rate is 10 percent less then biolistics but more efficient then the
time consuming steps to follow in gene gun method. Stem cell mediated gene transfer is also carried by this
technique. Pollens, ovary etc are successfully transformed by this method.
c. Electroporation
Electroporation is another physical mean of gene transfer. This technique is also known as electro-
permeabilization which refers to induce permeability in cells. Electro-poration uses electric pulse to increase
the permeability of plant cells which allow or permit the entrance of foreign particle.
Operating mechanism
The target cells are placed in optimized medium already containing the gene to be inserted.
The temperatures and other parameters are optimized. Usually temperature is maintained up to 60-75
Celsius. Voltage of 150-160 volt is passed by power source which enables the gene transfer. Generally plant
cells, protoplasts are modified by this technique and are observed under microscope. After transformation
the electric pulse is released and cell membrane re maintains its structure.
Generally electroporation is applied in formation of double haploids and somatic embryogenesis and
protoplast culture.
d. Laser beam mediated gene transfer
In 1988 Weber and his team describe the application of laser beam for genetic transformation of plant cells.
An ultraviolet micro laser beam of specified wave length (200-400nm) is used to introduce DNA in cells
under monitored devices; like microscope. These beams create pores in cell membrane and enhance uptake
of DNA in medium. This technique was demonstrated in Brassica napus and have about 20 percent
success rate.
e. Ultrasound mediated gene transfer
Ultrasound mediated gene transfer is the principle generally use to transform the defective gene in human
cell. This technique is also known as sonication. It is also based on inducing permeability in cell for uptake
of gene placed in medium.
Beside these shock waves, macro injection, vacuum infiltration and agitation by glass rods mediated gene
transfer are other forms of physical tools which can also use for genetic modification and transfer.
Advantages and Disadvantages,
Physical methods of transformations are generally direct and has efficient success rate. It requires less
number of molecular steps to follow unlike biological means of gene transfer. The limitation is that
availability of such cost effective tools. These methods can generate efficient result under proper monitoring
and requires expertise. The success rate usually ranges from 30-40 percent.
Electroporation

9. E. Chemical means of Gene transfer
Genetic transformation can also be performed by means of chemical reagents. The chemical molecules
enhance and regulate targeted gene delivery in different systems .the first ever successful gene transfer was
carried out with the help of DEAE dex-tran, These chemicals facilitate transformation by targeted delivery
or by increasing the uptake affinity. The chemicals include positively charge particles. The principle of
chemical mean of transformation is the binding affinity between chemical particle and DNA. We use
positively charge polymers as DNA bears negative charge. DNA adheres to the molecule, which is transport
to target site.
Examples
The chemical mean of gene transformation include following examples.
a. DEAE dextran is used to transfer DNA in cell line in the presence of glycerol or chloroquinine or
DMSO (Dimethyl Sulfoxide) which provides osmotic shock as result enhances permeability.
b. Calcium phosphate mediated gene transfer.
c. Silicon carbide whiskers method.
d. Poly-l-ornithine mediated gene transfer.
e. Dendrimer mediated gene transfer.
f. Polyethyelenimine mediated gene transfer.
g. Peptide mediated gene transfer.
h. Polyethylene glycol method
i. Liposome’s mediated gene transfer.
Nowadays Poly ethylene Glycol and Liposome’s are use to transfer the gene of interest. These methods
yield more stable transformation in both plant and animal cells.
a. Calcium phosphate mediate gene transfer
This mechanism involves precipitation with DNA solution already prepared by isolating the interested gene.
The gene is precipitated in calcium chloride and potassium phosphate solutions. The transfer of the DNA is
held by endo-cytosis.
b. Polycation mediated gene transfer.
DEAE detx-ran method, DMSO method, Poly-l-ornithine, Dendrimer mediated gene transfer,
Polyethyelenimine mediated gene transfer.etc is also known as Polycation mediated gene transfer. These
methods are based on principle of inducing permeability in cells. Increased permeability of cells enhances
the uptake level of DNA from solution.
c. Liposomal mediated gene transfer
Liposomes are smallest spherical structure and lipid in nature. These smallest particles are usually
synthesized as nanosomes in laboratory according to the requirements. Conventional gene delivery system
may cause some effects so we use liposomes for targeted gene transfer to reduce these risks.
Synthetic liposomes of 5µm-10 µm sized are used in order to deliver the desired gene in th target cell or
tissue.
Structure of liposome
Liposome is composed of two phases or layers
 Lipid soluble layer
 Core i.e. water soluble layer
Core contents include
 Gene to be insert
 Nuclear localizing signals
 Integrases
Other components are
 Immunoprotective layer
 Homing peptides.
Preparation of gene Delivery Liposome
Liposomes mediated DNA transfer refers to encapsulation of gene in lipid bags or capsules. It refers to use
synthetic liposomes as delivery tool. It involves seven steps of formulation of such vehicles which are as
follows.

10. a. Preparation of core
b. Preparation of lipid bilayer
c. Sonication
d. preparation immune-protective specie
e. Addition of homing peptides
f. Confirmation
The explanation is given below
Preparation of core
First of all core component are encapsulated in the core. These components include DNA /plasmid, Nuclear
localizing signals, integrases and markers for identification.
 The core is prepared by preparing a solution of these particles in distilled water.
Preparation of Lipid Layer
 Lactosyl Ceramide is a polymer is added to construct the liposomes.
 Lactosy ceramide encapsulate the core component by forming lipid bi-layer.
The encapsulated core appears as spherical droplets.
Sonication
Sonication is physical technique usually use to sterilize and agitate the particles. The sound energy of known
frequency is passed to refurnished particles in desired size.
Formation of Immune protective layer
Proteoliposmes is a polymer which is used as protective agent. It is added to furnish encapsulated particles.
It covers the particles by forming a layer. These proteoliposomes prevent them from destruction from
immune system of host where these delivery tools are used.
Homing peptides
Oligo synthetic peptides are added over the layer of liposomes formed. The main function of homing
peptides is targeted deliver under signaling of NLS. Homing peptides works best according to the affinity of
recognition and binding. It helps is specific targeting. Colorants are also added for identification purpose
Confirmatory test
The liposome construct in observed or analyzed by different methods. These confirmatory tests are done to
confirm that desired gene is present within the construct. The methods include,
 RNA labeled probe detection
 X-ray and gel dock systems.
Advantages and limitations
 Liposomal gene transfer is best for both transient and stable integration of animal cell lines.
 It is cost effective and reliable technique.
 These species have not induced side effects and the transfer is target specific. The chance of success
is less but it is safer technique.
 The limitation include careful preparation is required.
 These may be destroyed by immune system in some cases.
d. Poly ethylene glycol mediated gene transfer
Polyethylene glycol is a chemical used as fusicogenic agent. PEG is used in gene transformation of
protoplast of plant cells. PEG along with some divalent cat ions like Ca2+ interfere the cell membrane of
plant cell. It fuses the protoplast by rendering and gets integrated with the genome.
The steps include,
 Preparation of protoplasts,
 addition of DNA,
 addition of PEG
Mechanism
Protoplast is prepared by removing or rendering cell membrane through mechanical or enzymatic action.
The desired DNA is isolated or collected from libraries.
40% PEG-4000 (w/v) dissolved in mannitol and calcium nitrate solutions are used as medium. These
mixtures are incubated, which enhance protoplasts and chloroplast transformation.

11. Advantages and Disadvantages
 This method allows transformation of number of protoplast at single process simultaneously.
 This technique is successfully used for stable transformation of plant cells.
 The manipulations are easy and require less time and are free of burden of steps to follow.
 However there are some chances of destruction of DNA may occur.
 This method is not specific strictly transformed cells. The production may difficult.
F. Applications of Gene transformations, with examples
The advantage and scope of gene transformation include number of productive outcomes.
a. Transgenic plants
 Transgenic plants are the outcome of gene transfer. First transgenic product or plant commercialized
was tomato. The transgenic crops and plants have number of significances.
 We can produce number of products, secondary metabolites.
 It is useful in production of disease resistant, herbicide and environmental competent crops.
 Disease free plants are produced. It defends the food scarcity.
 Mass production, quality enhancement, etc.
b. Transgenic Animals
The DNA transfer method have many significant importance, some of them are as follows.
 Different proteins can be produced. Insulin, growth hormone. Interleukin and interferon are common
example of transformational products.
 Monoclonal antibodies can also be produced.
 Stem cell mediated gene transfer can produce desired product in population of animal
simultaneously.
 Gene transfer and delivery is used as diagnostic purpose.
 Gene knock in and gene knock out can be done by different genetic manipulation and gene
transformation techniques has prime importance in health biotechnology.
c. Examples
 BT product like BT cotton and BT rice are produced by genetic transformation.
 Ripening of fruits is delayed, shelf life of fruits and vegetables are increased.
 Golden rice is produced to treat eye diseases.
 Herbicide resistant onions are produced by transformation through Agrobacterium mediated
transformation.
 Productivity of Barely (Hordeum vulgare) is enhancing by electroporation methods.
 Sorghum species productivity was enhanced by Biolistics or gene gun.
 Sunflower oilseed plants productivity is increased by PEG mediated gene transfer.
 Insulin was produced in 1972 for first time by plasmid /vector mediated transformation.
 Gene therapy is kind of treatment of diseases using gene transfer.
 Biopharmaceuticals can also be produce through gene transformation.
 Regeneration of organs by stem cells and gene manipulation etc.
d. Limitations and ethical issues
 Beside the vast applications of genetic transformations, there are some limitations and issues which
are as follows
 Generally all these techniques require skilled education persons,
 The tools are costly ant not available developing countries
 Some scientist thought genetic transformation result in super bugs and weeds which may harm the
environment.
 Pathogens also become more resistant.
 The negative approach may be used.
 Biological war and globally fear about safety may spread which interferes the life.
 Threat to life of some organism which is consider as immoral against natural environments.

12. G. Conclusion and future perspectives
Future perspectives
Gene transfer techniques generally result in genetic modification of organisms. The transgenic organisms
have enormous applications and benefits .In future physical and chemical means of gene transfer is widely
use for genetic transformation due to different reason. The main reason of this is pathogenicity which
reduced unlike biological means in such methods, these method are time effective , safer and have less
molecular steps which make them more efficient. Gene therapy for disease treatment shall more develop by
using the physical method of gene transfer.
Conclusion
Gene transformation is done in many ways. The techniques were designed by studying natural phenomenons
of gene transfer. Which are transduction, conjugation etc. gene transformation techniques are categorized
according to the path followed. These include Biological, Physical and Chemical means of gene
transformation. The main aim of all these techniques includes molecular cloning and formation of transgenic
organisms. These transgenic organisms are applied for production of number of product and function for
benefit of mankind. All of given techniques have also some limitations. Generally as physical methods are
safer and effective method but they are also costly to afford.
H. Bibliography
The given facts and figure are sort out from different sources which are given below accordingly
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Author: Gurbachan S.Migalani , published by Narosa
2. Genetic Transformation of Plants
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3. Gene Transfer techniques by Hongbao Ma, Guozhong Chen
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