Gene transfer techniques can be used to transfer genes between organisms. There are natural methods like conjugation, transformation, and transduction that transfer genes between bacteria. Artificial methods like microinjection, biolistics, calcium phosphate transfection, liposome transfection, and electroporation can be used to transfer genes into both bacteria and eukaryotic cells. Agrobacterium mediated transfer is used to transfer genes into plant cells and involves the T-DNA region of the Ti plasmid. The transferred gene is then integrated into the host genome.
This document discusses various techniques for gene transfer, including natural methods like conjugation, transformation, and transduction, as well artificial methods like microinjection, biolistics, calcium phosphate transfection, liposome-mediated transfection, and electroporation. It provides details on how each method works, such as how conjugation involves transfer of DNA between bacteria via sex pili, how transformation involves direct DNA uptake by competent bacteria, and how transduction involves transfer of DNA between bacteria via bacteriophages. The document also discusses Agrobacterium-mediated plant transformation and applications of gene transfer techniques.
This document discusses various techniques for gene transfer, including natural methods like conjugation, transformation, and transduction, as well artificial methods like microinjection, biolistics, calcium phosphate and liposome mediated transfer, and electroporation. It provides details on how each method works, such as how conjugation involves transfer of DNA between bacteria via sex pili, and how electroporation uses electrical pulses to create pores in cell membranes to allow DNA entry. The document also summarizes screening and applications of transgenic techniques.
This document discusses various techniques for transferring genes, including natural and artificial methods. Natural methods include conjugation, transformation, transduction, and Agrobacterium-mediated transfer. Artificial methods include microinjection, biolistics, calcium phosphate transfection, liposome-mediated transfer, and electroporation. The document provides detailed descriptions of conjugation, transformation, transduction, Agrobacterium-mediated transfer, microinjection, biolistics, calcium phosphate transfection, liposome-mediated transfer, and electroporation. It also discusses screening methods for transgenes and applications of gene transfer techniques.
This document discusses various gene transfer methods. It defines gene transfer as the insertion of genetic material into a cell. There are natural methods like conjugation, transformation, and transduction that involve the transfer of genes between bacteria. There are also artificial physical, chemical, and electrical methods to transfer genes into cells, including microinjection, gene guns, calcium phosphate, liposomes, and electroporation. The document provides examples of how these various gene transfer methods can be used to insert genes into bacteria, plants, and animals.
This document discusses various gene transfer methods. It defines gene transfer as the insertion of genetic material into a cell. There are natural methods like conjugation, transformation, and transduction that involve the transfer of genes between bacteria. There are also artificial physical, chemical, and electrical methods to transfer genes into bacteria, plants, and animals. These include microinjection, biolistics, calcium phosphate, liposomes, and electroporation. The document provides details on how each of these methods work and their advantages and limitations.
This document discusses methods for plant genetic engineering and transformation. It describes three main methods: electroporation, biolistics/particle bombardment, and Agrobacterium-mediated transformation. Agrobacterium transformation uses Agrobacterium tumefaciens bacteria to insert foreign DNA into plant cells. It involves removing crown gall genes from the Ti plasmid and replacing them with genes of interest. The process then involves inserting the modified Ti plasmid into Agrobacterium, mixing it with plant cells, and regenerating genetically modified plantlets.
1. There are two main methods of gene transfer - direct and indirect gene transfer. Indirect transfer uses Agrobacterium-mediated transformation while direct transfer uses physical or chemical methods.
2. Agrobacterium-mediated transformation uses Agrobacterium tumefaciens to transfer T-DNA containing the gene of interest into the plant genome. The process involves co-cultivation of plant explants with Agrobacterium followed by selection and regeneration of transgenic plants.
3. Direct physical methods include biolistic transformation, microinjection, electroporation, and macroinjection. Direct chemical methods include PEG-mediated, calcium phosphate co-precipitation, and liposome-mediated transformation
Gene transfer technology pharmacology biotechnology basic methods
Natural, physical, chemical methods of gene transfer.
Along with advantages and limitations, and applications.
This document discusses various techniques for gene transfer, including natural methods like conjugation, transformation, and transduction, as well artificial methods like microinjection, biolistics, calcium phosphate transfection, liposome-mediated transfection, and electroporation. It provides details on how each method works, such as how conjugation involves transfer of DNA between bacteria via sex pili, how transformation involves direct DNA uptake by competent bacteria, and how transduction involves transfer of DNA between bacteria via bacteriophages. The document also discusses Agrobacterium-mediated plant transformation and applications of gene transfer techniques.
This document discusses various techniques for gene transfer, including natural methods like conjugation, transformation, and transduction, as well artificial methods like microinjection, biolistics, calcium phosphate and liposome mediated transfer, and electroporation. It provides details on how each method works, such as how conjugation involves transfer of DNA between bacteria via sex pili, and how electroporation uses electrical pulses to create pores in cell membranes to allow DNA entry. The document also summarizes screening and applications of transgenic techniques.
This document discusses various techniques for transferring genes, including natural and artificial methods. Natural methods include conjugation, transformation, transduction, and Agrobacterium-mediated transfer. Artificial methods include microinjection, biolistics, calcium phosphate transfection, liposome-mediated transfer, and electroporation. The document provides detailed descriptions of conjugation, transformation, transduction, Agrobacterium-mediated transfer, microinjection, biolistics, calcium phosphate transfection, liposome-mediated transfer, and electroporation. It also discusses screening methods for transgenes and applications of gene transfer techniques.
This document discusses various gene transfer methods. It defines gene transfer as the insertion of genetic material into a cell. There are natural methods like conjugation, transformation, and transduction that involve the transfer of genes between bacteria. There are also artificial physical, chemical, and electrical methods to transfer genes into cells, including microinjection, gene guns, calcium phosphate, liposomes, and electroporation. The document provides examples of how these various gene transfer methods can be used to insert genes into bacteria, plants, and animals.
This document discusses various gene transfer methods. It defines gene transfer as the insertion of genetic material into a cell. There are natural methods like conjugation, transformation, and transduction that involve the transfer of genes between bacteria. There are also artificial physical, chemical, and electrical methods to transfer genes into bacteria, plants, and animals. These include microinjection, biolistics, calcium phosphate, liposomes, and electroporation. The document provides details on how each of these methods work and their advantages and limitations.
This document discusses methods for plant genetic engineering and transformation. It describes three main methods: electroporation, biolistics/particle bombardment, and Agrobacterium-mediated transformation. Agrobacterium transformation uses Agrobacterium tumefaciens bacteria to insert foreign DNA into plant cells. It involves removing crown gall genes from the Ti plasmid and replacing them with genes of interest. The process then involves inserting the modified Ti plasmid into Agrobacterium, mixing it with plant cells, and regenerating genetically modified plantlets.
1. There are two main methods of gene transfer - direct and indirect gene transfer. Indirect transfer uses Agrobacterium-mediated transformation while direct transfer uses physical or chemical methods.
2. Agrobacterium-mediated transformation uses Agrobacterium tumefaciens to transfer T-DNA containing the gene of interest into the plant genome. The process involves co-cultivation of plant explants with Agrobacterium followed by selection and regeneration of transgenic plants.
3. Direct physical methods include biolistic transformation, microinjection, electroporation, and macroinjection. Direct chemical methods include PEG-mediated, calcium phosphate co-precipitation, and liposome-mediated transformation
Gene transfer technology pharmacology biotechnology basic methods
Natural, physical, chemical methods of gene transfer.
Along with advantages and limitations, and applications.
Electroporation uses electric pulses to create temporary pores in the cell membrane, allowing DNA entry. DNA-coated microprojectiles are accelerated into cells using a gene gun. Microinjection precisely inserts DNA into cells through fine glass needles. Calcium phosphate precipitation forms DNA-calcium phosphate complexes taken up by cells. Cationic liposomes fuse with cell membranes, transferring DNA across. Adenoviruses and retroviruses can deliver DNA to dividing and non-dividing cells. Agrobacterium transfers tumor-inducing (T-DNA) from its Ti plasmid into plant cells at wound sites.
Gene transfer techniques can be direct or indirect. Direct techniques introduce foreign DNA into plant cells without a biological agent, using methods like microinjection, microprojectiles, protoplast fusion, electroporation, and polyethylene glycol treatment. Indirect gene transfer uses the bacterium Agrobacterium tumefaciens, which transfers DNA (T-DNA) from its tumor-inducing plasmid into the host plant genome, allowing genetic modification of plants. Techniques like bacterial transformation and transduction can also directly transfer genes between bacteria using viruses or naked DNA. Overall, a variety of methods have been developed to introduce foreign genes into organisms and achieve genetic modification.
Gene transfer methods in animals can be natural or artificial. Natural methods include conjugation, transformation, and transduction which transfer genes between bacteria. Artificial methods like microinjection, biolistics, liposome mediated transfer, calcium phosphate mediated transfer, and electroporation are used to directly insert genes into cells. These techniques transfer genes into organisms for genetic engineering applications such as producing transgenic animals, developing vaccines, and gene therapy to treat diseases.
Gene transfer techniques in plants by Kailash SontakkeKAILASHSONTAKKE
This document discusses various gene transfer techniques, including both direct and indirect methods. Indirect gene transfer uses Agrobacterium tumefaciens bacteria to transfer genes into plant cells. The Ti plasmid from Agrobacterium is used to introduce new genes between the plasmid's border sequences, which will then be integrated into the plant genome. Direct methods include calcium phosphate transfection, PEG-mediated transfection of protoplasts, electroporation, and biolistics/gene gun which coats DNA onto microparticles that are physically shot into plant cells. Both transient and stable transformation can occur, with stable integration allowing for inheritance of the transgene.
There are three main modes of gene transfer: transformation, transfection, and transduction. Transformation involves the natural uptake of foreign DNA by a cell. Transfection is the deliberate introduction of genetic material into animal cells. Transduction uses viruses to transfer genes between bacterial cells. It can occur through a lysogenic or lytic phase. Conjugation is also discussed, which involves the direct contact and temporary exchange of genetic material between two bacterial cells.
Genetic engineering and Transformation methodsManjunath R
Genetic engineering involves manipulating an organism's genome using modern DNA technology. This document discusses various genetic transformation methods, including both direct and indirect methods. Indirect methods involve using Agrobacterium tumefaciens to transfer DNA into plant cells. Direct methods discussed include particle bombardment, polyethylene glycol treatment, electroporation, and microinjection. The document provides details on the process, mechanisms, applications and history of genetic engineering and transformation techniques.
Transfection involves introducing foreign DNA into host cells to produce a new phenotype. There are two main methods of transfection - vector-mediated and non-vector mediated. Vector-mediated transfection uses bacteriophage, retroviral, cosmid, baculovirus, and plasmid vectors to introduce DNA. Non-vector mediated methods include direct techniques like microinjection, electroporation, and particle bombardment, and indirect techniques like calcium phosphate precipitation and DEAE-dextran. Retroviral vectors are modified retroviruses that can introduce foreign DNA into host chromosomal DNA. Microinjection involves injecting DNA directly into cells using a micropipette under a microscope. Electroporation uses electric pulses to create temporary
This document discusses the production of transgenic animals and plants. It describes three main methods for producing transgenic animals: DNA microinjection, retrovirus-mediated gene transfer, and embryonic stem cell-mediated gene transfer. It also discusses 11 methods for transforming plants, including Agrobacterium-mediated transformation, biolistic transformation, and floral dip transformation. Finally, it lists some beneficial traits that have been engineered in transgenic plants, such as stress tolerance, herbicide tolerance, and increased nutritional quality.
This document provides an overview of various gene transfer tools and techniques. It discusses vector-mediated methods like Agrobacterium and viral vectors as well as direct or vector-less methods such as electroporation, biolistics, microinjection, liposome mediated, and calcium phosphate mediated gene transfer. For each method, it describes the basic process and provides some key details and applications. It also notes some advantages and limitations of different techniques. The document aims to inform readers about the various options available for inserting genes into plant cells.
Hi, I am RAFi ,student of Genetic Engineering and Biotechnology , Jashore university of science & Technology. It is my first uploading slide in slideshare.I am so glad for doing this work.
This document discusses various methods of transfection, which is defined as the introduction of foreign DNA into eukaryotic cells. It describes transfection methods such as calcium phosphate transfection, liposome-mediated transfection, retroviral transfection, and electroporation. It provides details on how each method works and compares their strengths and weaknesses. Common transfection methods like calcium phosphate and liposomes are simple but have low efficiency, while retroviral transfection can generate stable cell lines but has limitations on DNA size. Electroporation is fast and applicable to many cell types.
This document discusses various gene transfer techniques used in genetic engineering. It describes direct techniques like chemically stimulated DNA uptake using polyethylene glycol (PEG), transduction using bacteriophages, electroporation using high voltage electricity, and microinjection of DNA into fertilized eggs. It also discusses indirect techniques like microprojectile bombardment which shoots DNA-coated particles into plant cells, and Agrobacterium-mediated transfer where the bacterium transfers tumor-inducing (T-DNA) from its Ti plasmid into the host plant genome.
Genetic transformation in prokaryotes has led to the discovery of the three major methods of transformationin bacteria i.e transformation, conjugation and transuction whichcommonly uses the bacterial- phages as vectors to transfer dna.
transformation in bacteria is a classical example of horizontal gene transfer which leads to enhanced survivability and also introduction of variations that may lead to evolution
Recombinant DNA technology involves manipulating DNA from different sources to produce novel DNA molecules. It has several key steps: isolating the desired DNA and vector, joining them using enzymes to create recombinant DNA, introducing this into a host cell, and selecting cells that express the gene. This technology has many applications including producing human insulin and growth hormones through bacteria, developing vaccines by cloning genes for antigens, and creating monoclonal antibodies. It allows mass production of important biological substances that were previously difficult to obtain.
This document discusses various gene transfer techniques including physical, chemical, and biological methods. It focuses on biological methods such as bactofection and transduction using viruses. Bactofection involves using bacteria to deliver genes directly into cells, while transduction uses viruses to package and deliver genes. The document also discusses chemical methods like calcium phosphate and lipofection, as well as physical methods such as electroporation, microinjection, and particle bombardment to introduce DNA into host cells.
Genetic transformation method in mammals cell by NIDHI MISHRA and tahura mari...Tahura Mariyam Ansari
this presentation includes method of gene transfer, factor that affect efficiency of gene transfer, fate of DNA in the recipient cells, autonomous replication vector and some other subtopics.
Genetically modified or transgenic plants are plants that have been modified using genetic engineering techniques to introduce new traits. This document discusses the history and process of creating transgenic plants. It describes how transgenic plants are generated by transferring genes from other species into the target plant using either indirect methods like Agrobacterium-mediated transformation or direct physical methods like biolistics. The document provides details on the various applications of transgenic plants including producing herbicide resistance, insect resistance, virus resistance, and improving nutritional quality.
This document provides an overview of various gene transformation techniques, including both vector-mediated and direct methods. It discusses natural transformation mechanisms like conjugation and transduction, as well as artificial vector-mediated techniques like Agrobacterium-mediated transformation. Direct methods like microinjection, electroporation, particle bombardment, and chemical methods using PEG or calcium phosphate are also covered. The applications, advantages, and limitations of different techniques are summarized. Overall, the document serves as an informative introduction to the key gene transfer methods used in plant biotechnology.
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This document discusses chemical methods of sterilization and disinfection. It defines disinfection as destroying pathogens but not all microorganisms, while sterilization kills all microorganisms including bacterial spores. Ideal disinfectants have wide antimicrobial spectrum, are effective in organic matter, have long shelf life and are non-toxic. Factors like temperature and concentration influence disinfection efficacy. Chemical agents act via different mechanisms including membrane damage. Disinfectants are classified based on antimicrobial spectrum into high, intermediate and low-level types with examples like aldehydes, alcohols and quaternary ammonium compounds discussed in detail.
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Electroporation uses electric pulses to create temporary pores in the cell membrane, allowing DNA entry. DNA-coated microprojectiles are accelerated into cells using a gene gun. Microinjection precisely inserts DNA into cells through fine glass needles. Calcium phosphate precipitation forms DNA-calcium phosphate complexes taken up by cells. Cationic liposomes fuse with cell membranes, transferring DNA across. Adenoviruses and retroviruses can deliver DNA to dividing and non-dividing cells. Agrobacterium transfers tumor-inducing (T-DNA) from its Ti plasmid into plant cells at wound sites.
Gene transfer techniques can be direct or indirect. Direct techniques introduce foreign DNA into plant cells without a biological agent, using methods like microinjection, microprojectiles, protoplast fusion, electroporation, and polyethylene glycol treatment. Indirect gene transfer uses the bacterium Agrobacterium tumefaciens, which transfers DNA (T-DNA) from its tumor-inducing plasmid into the host plant genome, allowing genetic modification of plants. Techniques like bacterial transformation and transduction can also directly transfer genes between bacteria using viruses or naked DNA. Overall, a variety of methods have been developed to introduce foreign genes into organisms and achieve genetic modification.
Gene transfer methods in animals can be natural or artificial. Natural methods include conjugation, transformation, and transduction which transfer genes between bacteria. Artificial methods like microinjection, biolistics, liposome mediated transfer, calcium phosphate mediated transfer, and electroporation are used to directly insert genes into cells. These techniques transfer genes into organisms for genetic engineering applications such as producing transgenic animals, developing vaccines, and gene therapy to treat diseases.
Gene transfer techniques in plants by Kailash SontakkeKAILASHSONTAKKE
This document discusses various gene transfer techniques, including both direct and indirect methods. Indirect gene transfer uses Agrobacterium tumefaciens bacteria to transfer genes into plant cells. The Ti plasmid from Agrobacterium is used to introduce new genes between the plasmid's border sequences, which will then be integrated into the plant genome. Direct methods include calcium phosphate transfection, PEG-mediated transfection of protoplasts, electroporation, and biolistics/gene gun which coats DNA onto microparticles that are physically shot into plant cells. Both transient and stable transformation can occur, with stable integration allowing for inheritance of the transgene.
There are three main modes of gene transfer: transformation, transfection, and transduction. Transformation involves the natural uptake of foreign DNA by a cell. Transfection is the deliberate introduction of genetic material into animal cells. Transduction uses viruses to transfer genes between bacterial cells. It can occur through a lysogenic or lytic phase. Conjugation is also discussed, which involves the direct contact and temporary exchange of genetic material between two bacterial cells.
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Genetic engineering involves manipulating an organism's genome using modern DNA technology. This document discusses various genetic transformation methods, including both direct and indirect methods. Indirect methods involve using Agrobacterium tumefaciens to transfer DNA into plant cells. Direct methods discussed include particle bombardment, polyethylene glycol treatment, electroporation, and microinjection. The document provides details on the process, mechanisms, applications and history of genetic engineering and transformation techniques.
Transfection involves introducing foreign DNA into host cells to produce a new phenotype. There are two main methods of transfection - vector-mediated and non-vector mediated. Vector-mediated transfection uses bacteriophage, retroviral, cosmid, baculovirus, and plasmid vectors to introduce DNA. Non-vector mediated methods include direct techniques like microinjection, electroporation, and particle bombardment, and indirect techniques like calcium phosphate precipitation and DEAE-dextran. Retroviral vectors are modified retroviruses that can introduce foreign DNA into host chromosomal DNA. Microinjection involves injecting DNA directly into cells using a micropipette under a microscope. Electroporation uses electric pulses to create temporary
This document discusses the production of transgenic animals and plants. It describes three main methods for producing transgenic animals: DNA microinjection, retrovirus-mediated gene transfer, and embryonic stem cell-mediated gene transfer. It also discusses 11 methods for transforming plants, including Agrobacterium-mediated transformation, biolistic transformation, and floral dip transformation. Finally, it lists some beneficial traits that have been engineered in transgenic plants, such as stress tolerance, herbicide tolerance, and increased nutritional quality.
This document provides an overview of various gene transfer tools and techniques. It discusses vector-mediated methods like Agrobacterium and viral vectors as well as direct or vector-less methods such as electroporation, biolistics, microinjection, liposome mediated, and calcium phosphate mediated gene transfer. For each method, it describes the basic process and provides some key details and applications. It also notes some advantages and limitations of different techniques. The document aims to inform readers about the various options available for inserting genes into plant cells.
Hi, I am RAFi ,student of Genetic Engineering and Biotechnology , Jashore university of science & Technology. It is my first uploading slide in slideshare.I am so glad for doing this work.
This document discusses various methods of transfection, which is defined as the introduction of foreign DNA into eukaryotic cells. It describes transfection methods such as calcium phosphate transfection, liposome-mediated transfection, retroviral transfection, and electroporation. It provides details on how each method works and compares their strengths and weaknesses. Common transfection methods like calcium phosphate and liposomes are simple but have low efficiency, while retroviral transfection can generate stable cell lines but has limitations on DNA size. Electroporation is fast and applicable to many cell types.
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Genetic transformation in prokaryotes has led to the discovery of the three major methods of transformationin bacteria i.e transformation, conjugation and transuction whichcommonly uses the bacterial- phages as vectors to transfer dna.
transformation in bacteria is a classical example of horizontal gene transfer which leads to enhanced survivability and also introduction of variations that may lead to evolution
Recombinant DNA technology involves manipulating DNA from different sources to produce novel DNA molecules. It has several key steps: isolating the desired DNA and vector, joining them using enzymes to create recombinant DNA, introducing this into a host cell, and selecting cells that express the gene. This technology has many applications including producing human insulin and growth hormones through bacteria, developing vaccines by cloning genes for antigens, and creating monoclonal antibodies. It allows mass production of important biological substances that were previously difficult to obtain.
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2. INTRODUCTION
• Gene transfer is to transfer a gene from one DNA molecule to
another DNA molecule.
• The directed desirable gene transfer from one organism to
another and the subsequent stable integration & expression of
foreign gene into the genome is referred as genetic
transformation.
• Transient transformation occur when DNA is not integreted
into host genome
3. • Stable transformation occur when DNA is integrated into host
genome and is inherited in subsequent generations.
• The transferred gene is known as transgene and the organism
that develop after a successful gene transfer is known as
transgenic.
4. METHODS OF GENE TRANSFER
DNA transfer by natural methods
• 1. Conjugation
• 2. Bacterial transformation
• 3. Retroviral transduction
• 4. Agrobacterium mediated transfer
5. DNA TRANSFER BY ARTIFICIAL METHODS
• Physical methods
• 1. Microinjection
• 2. Biolistics transformation
• Chemical methods
• 1. DNA transfer by calcium phosphate method
• 2. Liposome mediated transfer
• Electrical methods
• 1. Electroporation
6. CONJUGATION
• Requires the presence of a special plasmid called the F
plasmid.
• Bacteria that have a F plasmid are referred to as as F+ or
male. Those that do not have an F plasmid are F- of female.
• The F plasmid consists of 25 genes that mostly code for
production of sex pilli.
• A conjugation event occurs when the male cell extends his
sex pilli and one attaches to the female.
7. • This attached pilus is a temporary cytoplasmic bridge
through which a replicating F plasmid is transferred from
the male to the female.
• When transfer is complete, the result is two male cells.
• When the F+ plasmid is integrated within the bacterial
chromosome, the cell is called an Hfr cell (high frequency
of recombination cell).
8.
9. TRANSFORMATION
• transformation is the direct uptake of exogenous DNA from its
surroundings and taken up through the cell membrane .
• Transformation occurs naturally in some species of bacteria,
but it can also be effected by artificial treatment in other
species.
• Cells that have undergone this treatment are said to be
competent.
• Any DNA that is not integrated into he chromosome will be
degraded.
10.
11. TRANSDUCTION
• Gene transfer from a donor to a recipient by way of a
bacteriophag..
• If the lysogenic cycle is adopted, the phage chromosome is
integrated (by covalent bonds) into the bacterial chromosome,
where it can remain dormant for thousands of generation
• The lytic cycle leads to the production of new phage particles
which are released by lysis of the host.
12.
13. AGROBACTERIUM MEDIATED
TRANSFER
• Agrobacterium tumefaciens is a soil borne gram negative
bacterium.
• It invades many dicot plants when they are injured at the soil
level and causes crown gall disease.
• The ability to cause crown gall disease is associated with the
presence of the Ti (tumour inducing) plasmid within the
bacterial cell.
• Ti plasmid can be used to transport new genes into plant cells.
14. THE Ti-PLASMIDS
• A remarkable feature of the Ti plasmid is that, after infection, part of
the molecule is integrated into the plant chromosomal DNA .
• This segment, called the T-DNA, is between 15 and 30 kb in size,
depending on the strain.
• T-DNA contains eight or so genes that are expressed in the plant cell
and are responsible for the cancerous properties of the transformed
cells.
• These genes also direct synthesis of unusual compounds, called
opines, that the bacteria use as nutrient.
15. • The vir (virulence) region of the Ti- plasmid contains the
genes required for the T-DNA transfer process.
• The genes in this region encode the DNA processing
enzymes required for excision, transfer and integration of the
T-DNA segment.
16. • The T-DNA region of any Ti
plasmid is defined by the
presence of the right and the
left border sequences.
• These border sequences are 24
bp imperfect repeats.
• Any DNA between the borders
will be transferred in to the
genome of the plant.
17. Ti-Plasmid mediated
transfer of gene into a plant
• The Ti-Plasmid has an innate ability to transmit bacterial DNA
into plant cells.
• The gene of a donor organism can be introduced into the Ti
plasmid at the T-DNA region
• This plasmid now becomes a recombinant plasmid.
• By Agrobacterium infection, the donor genes can transferred
from the recombinant Ti- Plasmid and integrated into the
genotype of the host plant.
18. VECTORLESS or DIRECT GENE
TRANSFER
• Physical methods
• 1. Microinjection
• 2. Biolistics transformation
• Chemical methods
• 1. DNA transfer by calcium phosphate method
• 2. Liposome mediated transfer
• 3. Transfer of DNA by use of polyethene glycol
• Electrical methods
• 1. Electroporation
19. Electroporation
• Electroporation uses electrical pulse to produce transient
pores in the plasma membrane thereby allowing DNA into
the cells.
• These pores are known as electropores.
•
20. • The cells are placed in a solution containing DNA and
subjected to electrical pulse to cause holes in the
membrane.
• The foreign DNA fragments enter through holes into the
cytoplasm and then to nucleus.
21. Advantages of Electroporation
• 1. Method is fast.
• 2. Less costly.
• 3. Applied for a number of cell
types.
• 4. Simultaneously a large number
of cell can be treated.
• 5. High percentage of stable
transformants can be produced
22. Microinjection
The microinjection is the process of transferring the desirable
DNA into the living cell ,through the use of glass
micropipette .
Glass micropipette is usually of 0.5 to 5 micrometer,
easily penetrates into the cell membrane and nuclear
envelope.
The desired gene is then injected into the sub cellular
compartment and needle is removed
25. Biolistics or Microprojectiles
• Biolistics or particle bombardment is a physical method that
uses accelerated microprojectiles to deliver DNA or other
molecules into intact tissues and cells.
• The gene gun is a device that literally fires DNA into target
cells .
• The DNA to be transformed into the cells is coated onto
microscopic beads made of either gold or tungsten.
26. • The coated beads are then attached to the end of the plastic
bullet and loaded into the firing chamber of the gene gun.
• An explosive force fires the bullet with DNA coated beads
towards the target cells that lie just beyond the end of the
barrel.
• Some of the beads pass through the cell wall into the
cytoplasm of the target cells
27.
28. Liposome mediated gene transfer
• Liposomes are spheres of lipids which can be used to transport
molecules into the cells.
• These are artificial vesicles that can act as delivery agents for
exogenous materials including transgenes.
• Promote transport after fusing with the cell membrane.
• Cationic lipids are those having a positive charge are used for
the transfer of nucleic acid.
29. Advantages
• 1. Simplicity.
• 2. Long term stability.
• 3. Low toxicity.
• 4. Protection of
nucleic acid from
degradation
30. Calcium phosphate mediated DNA transfer
• The process of transfection involves the admixture of isolated
DNA (10-100ug) with solution of calcium chloride and
potassium phosphate so precipitate of calcium phosphate to be
formed.
• Cells are then incubated with precipitated DNA either in
solution or in tissue culture dish.
• A fraction of cells will take up the calcium phosphate DNA
precipitate by endocytosis.
32. Polyethylene glycol mediated transfection
• This method is utilized for protoplast only.
• Polyethylene glycol stimulates endocytosis and therefore DNA
uptake occurs.
• Protoplasts are kept in the solution containing polyethylene
glycol (PEG).
• After transfer of DNA to the protoplast in presence of PEG
and other chemicals, PEG is allowed to get removed
33. SCREENING OF TRANSGENE
• The presence of transgene or gene of interest is detected by
several methods:
• A selectable marker gene
• Southern blot techniques
• Northern bolt technique
• Western blot technique
34. APPLICATION
• Clinical gene transfer applications
• Vaccine Development
• Production of transgenic animals
• Treatment of Cancer, AIDS
• Gene Discovery
• Gene Therapy
• Enhancing the resistance of plants
• GMO
35. REFERENCES
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