Gene transfer technology pharmacology biotechnology basic methods
Natural, physical, chemical methods of gene transfer.
Along with advantages and limitations, and applications.
DNA ligation is the joining of two nucleic acid fragments through the action of an enzyme. Several factors can affect the ligation reaction, including the concentration of enzyme, DNA, and cofactors like ATP or NAD+. The DNA concentration is particularly important, as higher concentrations favor intermolecular ligation between separate DNA molecules, while lower concentrations favor intramolecular ligation where a DNA molecule joins its own ends. DNA ligase carries out ligation through a three-step catalytic mechanism involving adenylation of the enzyme and two phosphoryl transfers.
Selection & Screening of Recombinant cells & expression of recombinant (2) (1)SunandaArya
This document summarizes various methods for selecting and screening recombinant clones after introducing recombinant DNA into host cells. It discusses direct selection using antibiotic resistance genes, insertional inactivation by inserting DNA into antibiotic resistance genes, and blue-white screening using beta-galactosidase activity. It also covers colony hybridization using radioactive probes and immunological tests using antibodies to identify antigen-expressing colonies. Finally, it briefly discusses protein expression in different systems like bacteria, insect cells, and mammalian cells.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
The document discusses various methods of transfection in animals. Transfection is the process of introducing nucleic acids into eukaryotic cells. It describes viral transfection using bacteria like Agrobacterium tumefaciens and viruses. Non-viral methods include chemical transfection using calcium phosphate, liposomes, polyamines. Mechanical transfection employs microinjection or particle bombardment. Common chemical methods are calcium phosphate precipitation, polyplexes, and liposomes/lipoplexes. Viruses used are retroviruses, adenoviruses, adeno-associated viruses. Bacterial and viral vectors allow for integration into the host genome while chemical and mechanical are often transient.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
This document provides an introduction to gene transfer techniques. It discusses:
1. The process of gene transfer, which moves a specific piece of DNA into a cell, and genetic transformation, which is the stable integration and expression of a foreign gene into an organism's genome.
2. The two main methods of gene transfer - vector-based methods using organisms like Agrobacterium tumefaciens and direct gene transfer methods like particle bombardment.
3. The steps involved in transformation which include identifying a desirable gene, designing the gene for insertion, inserting the gene into a target plant, and identifying transformed cells.
This document summarizes the baculovirus expression system. Baculoviruses can be used as expression vectors by replacing a non-essential viral gene with a gene of interest. The recombinant baculovirus is produced through homologous recombination or using the Bac-to-Bac system. Insect cells are infected with the recombinant baculovirus, which drives high-level expression of the foreign gene. The baculovirus expression system allows safe, scalable production of recombinant proteins for research applications.
DNA ligation is the joining of two nucleic acid fragments through the action of an enzyme. Several factors can affect the ligation reaction, including the concentration of enzyme, DNA, and cofactors like ATP or NAD+. The DNA concentration is particularly important, as higher concentrations favor intermolecular ligation between separate DNA molecules, while lower concentrations favor intramolecular ligation where a DNA molecule joins its own ends. DNA ligase carries out ligation through a three-step catalytic mechanism involving adenylation of the enzyme and two phosphoryl transfers.
Selection & Screening of Recombinant cells & expression of recombinant (2) (1)SunandaArya
This document summarizes various methods for selecting and screening recombinant clones after introducing recombinant DNA into host cells. It discusses direct selection using antibiotic resistance genes, insertional inactivation by inserting DNA into antibiotic resistance genes, and blue-white screening using beta-galactosidase activity. It also covers colony hybridization using radioactive probes and immunological tests using antibodies to identify antigen-expressing colonies. Finally, it briefly discusses protein expression in different systems like bacteria, insect cells, and mammalian cells.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
The document discusses various methods of transfection in animals. Transfection is the process of introducing nucleic acids into eukaryotic cells. It describes viral transfection using bacteria like Agrobacterium tumefaciens and viruses. Non-viral methods include chemical transfection using calcium phosphate, liposomes, polyamines. Mechanical transfection employs microinjection or particle bombardment. Common chemical methods are calcium phosphate precipitation, polyplexes, and liposomes/lipoplexes. Viruses used are retroviruses, adenoviruses, adeno-associated viruses. Bacterial and viral vectors allow for integration into the host genome while chemical and mechanical are often transient.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
This document provides an introduction to gene transfer techniques. It discusses:
1. The process of gene transfer, which moves a specific piece of DNA into a cell, and genetic transformation, which is the stable integration and expression of a foreign gene into an organism's genome.
2. The two main methods of gene transfer - vector-based methods using organisms like Agrobacterium tumefaciens and direct gene transfer methods like particle bombardment.
3. The steps involved in transformation which include identifying a desirable gene, designing the gene for insertion, inserting the gene into a target plant, and identifying transformed cells.
This document summarizes the baculovirus expression system. Baculoviruses can be used as expression vectors by replacing a non-essential viral gene with a gene of interest. The recombinant baculovirus is produced through homologous recombination or using the Bac-to-Bac system. Insect cells are infected with the recombinant baculovirus, which drives high-level expression of the foreign gene. The baculovirus expression system allows safe, scalable production of recombinant proteins for research applications.
Site-directed mutagenesis is a molecular biology technique used to make specific changes to DNA sequences. It involves using a primer containing the desired mutation in a PCR reaction to introduce the mutation into the gene of interest. There are different approaches for site-directed mutagenesis using PCR, including using a mutated primer in normal PCR or a primer extension method. The technique is used for applications like protein engineering to study the impact of sequence changes or insert restriction sites. However, it can be difficult to replicate the mutated DNA and screening mutations requires sequencing.
Transformation is the process of altering an organism's genetic makeup by inserting new genes. Common transformation methods include Agrobacterium-mediated transformation, particle bombardment, protoplast transformation using polyethylene glycol or electroporation, and fibre-mediated DNA delivery. Agrobacterium transformation involves the bacteria transferring T-DNA from its Ti plasmid into the plant genome, while direct methods introduce naked DNA into plant cells using physical methods like particle bombardment or chemical treatments that make cell membranes permeable. Transformation allows improving crop traits like yield and stress resistance.
Selection and screening of recombinant clones neeru02
This document discusses several methods for selecting recombinant clones after introducing recombinant DNA into host cells:
- Direct selection involves using a gene from the inserted DNA that confers antibiotic resistance to select clones that grow on media containing that antibiotic.
- Insertional inactivation selection works by inactivating a host gene when foreign DNA inserts into it, allowing selection of recombinants.
- Blue-white screening uses a vector with a disrupted lacZ gene; foreign DNA insertion repairs the gene, allowing recombinants to be identified by colony color.
- Colony hybridization detects recombinants by transferring colonies to a membrane and probing for the inserted DNA sequence.
- Immunological tests identify clones expressing antigens encoded by the
This document discusses various enzymes used for genetic engineering and DNA manipulation. It describes restriction endonucleases and DNA ligase which cut and join DNA fragments. It also discusses other DNA modifying enzymes like nucleases which degrade DNA, and polymerases which synthesize DNA copies. Specific enzymes covered in detail include DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, terminal transferase, T4 DNA ligase, and T4 RNA ligase.
This document discusses key components of expression vectors that are important for efficiently expressing cloned genes. It explains that expression vectors contain regulatory sequences like promoters and terminators to control transcription, as well as elements like ribosome binding sites, fusion tags, and selection markers. Specifically, it provides details on tightly regulated promoters, commonly used viral and bacterial promoters, and considerations for promoters in prokaryotic and eukaryotic expression systems. The document also reviews other important vector elements and their functions.
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
Prokaryotic genomes are circular, double-stranded DNA contained within the nucleoid. They vary in length but are generally a few million base pairs. DNA supercoiling allows for tight packing of the genome.
Eukaryotic genomes are linear chromosomes associated with histone proteins within the nucleus. The DNA is wrapped around histone octamers to form nucleosomes, compacting the genome. Eukaryotic genomes are generally larger and contain more DNA than prokaryotic genomes.
Key differences between prokaryotic and eukaryotic genomes include genome size, number of chromosomes, ploidy level, association with histones, and method of compaction.
F plasmid is a conjugative plasmid found in Escherichia coli that was the first plasmid discovered. It plays an important role in bacterial reproduction by containing genes that code for the production of sex pili and enzymes required for conjugation. The F plasmid replicates through a rolling circle mechanism and transfers between bacteria via conjugation using an F pilus. During conjugation, the F plasmid unwinds and one strand is transferred to the recipient cell where it is replicated to form a double-stranded circular plasmid, converting the recipient into an F+ cell capable of plasmid transfer.
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
This document discusses various mechanisms for transforming and transfecting cells, including prokaryotic, eukaryotic, plant, and fungal cells. It describes the history of bacterial transformation and mechanisms such as natural competence, artificial competence using calcium chloride or electroporation, and lipofection. For eukaryotic transfection, it discusses lipofection, dendrimers, and nucleofection. It also outlines various mechanisms for transforming plants, including Agrobacterium, electroporation, viral transformation, and particle bombardment.
The document summarizes the mechanism of T-DNA transfer during Agrobacterium tumefaciens infection. It explains that T-DNA is a fragment of DNA transferred from the tumor-inducing (Ti) plasmid of A. tumefaciens into the host plant genome. The T-DNA is bordered by repeats and encodes genes that cause tumors in the plant. Virulence genes are expressed in response to plant signals and produce single-stranded T-DNA, which forms a complex with other proteins and is transported into the plant cell and integrated into the plant nuclear DNA, causing uncontrolled cell growth and tumor formation. The mechanism involves multiple virulence protein complexes and integration of T-DNA is directed by the
Restriction enzymes cut DNA molecules at specific recognition sites. Restriction mapping involves digesting an unknown DNA segment with restriction enzymes and analyzing the fragment sizes to determine the locations of restriction sites. One method involves single and double digestions with two enzymes followed by gel electrophoresis to separate the fragments by size. By comparing the fragment patterns between single and double digestions, the positions of each restriction site can be mapped, generating a restriction map of the DNA segment. Restriction mapping was previously important for characterizing cloned DNA but is now easier using DNA sequencing, though analysis of restriction sites remains useful for comparing chromosomal organization between strains.
A DNA library is a collection of DNA fragments that have been cloned into vectors. DNA libraries allow researchers to isolate and study specific DNA fragments of interest. To create a genomic library, DNA is extracted from an organism, cut into fragments, inserted into vectors, and introduced into host bacteria to generate clones containing all the organism's DNA sequences. This library can then be screened to identify and study particular genes. DNA libraries provide an efficient way to store, isolate, and analyze DNA sequences.
This presentation contains information about restriction enzymes, its nomenclature, restriction digestion, and its application. This also contains information about the chemicals used in restriction and also explains the general procedure of restriction digestion of DNA
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
Ti plasmids are found in Agrobacterium tumefaciens bacteria and contain genes that allow the bacteria to transform plant cells and cause crown gall tumors. The plasmid contains virulence genes that are activated by plant signals and mediate transfer of T-DNA into the plant genome. T-DNA integration results in tumor formation and production of opines that the bacteria can utilize. Ti plasmids have been engineered as vectors for plant transformation by removing oncogenes and adding gene of interest between the border sequences, allowing transformation via Agrobacterium infection of wounded plant tissues.
Introduction
Ti plasmid
Agrobacterium tumefaciens
Ti plasmid structure
Overview of infection process
Ti plasmid derived vector systems
Cointegrate vectors
Binary vectors
Agrobacterium mediated transformation of explants
Conclusions
References
Sequence tagged sites (STSs) are short DNA sequences that can be used as genetic markers. STSs were introduced in 1989 as a way to map genes along chromosomes using PCR. They serve as landmarks on physical maps of genomes. STSs are mapped by breaking genomes into fragments, replicating the fragments in bacterial cells to create libraries, and using PCR to determine which fragments contain STSs. Different types of STS markers include microsatellites, SCARs, CAPs, and ISSRs, each of which has distinct characteristics and applications in genetic mapping, population studies, and other areas.
The document provides information about bacterial transformation. It describes that transformation is the process by which bacteria take up extracellular DNA from their environment. Frederick Griffith first discovered transformation in 1928 while working with pneumococcus bacteria. His experiments showed that a non-virulent rough form could be transformed into a virulent smooth form by DNA from a heat-killed smooth strain. Later experiments by Avery, Macleod and McCarty demonstrated that DNA is the transforming principle and genetic material of bacteria. The document then discusses various methods of bacterial transformation including chemical and physical methods like electroporation and use of calcium chloride. It also explains the molecular mechanism of transformation involving DNA binding, penetration, synapsis formation and integration into the bacterial chromosome.
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.
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.
Site-directed mutagenesis is a molecular biology technique used to make specific changes to DNA sequences. It involves using a primer containing the desired mutation in a PCR reaction to introduce the mutation into the gene of interest. There are different approaches for site-directed mutagenesis using PCR, including using a mutated primer in normal PCR or a primer extension method. The technique is used for applications like protein engineering to study the impact of sequence changes or insert restriction sites. However, it can be difficult to replicate the mutated DNA and screening mutations requires sequencing.
Transformation is the process of altering an organism's genetic makeup by inserting new genes. Common transformation methods include Agrobacterium-mediated transformation, particle bombardment, protoplast transformation using polyethylene glycol or electroporation, and fibre-mediated DNA delivery. Agrobacterium transformation involves the bacteria transferring T-DNA from its Ti plasmid into the plant genome, while direct methods introduce naked DNA into plant cells using physical methods like particle bombardment or chemical treatments that make cell membranes permeable. Transformation allows improving crop traits like yield and stress resistance.
Selection and screening of recombinant clones neeru02
This document discusses several methods for selecting recombinant clones after introducing recombinant DNA into host cells:
- Direct selection involves using a gene from the inserted DNA that confers antibiotic resistance to select clones that grow on media containing that antibiotic.
- Insertional inactivation selection works by inactivating a host gene when foreign DNA inserts into it, allowing selection of recombinants.
- Blue-white screening uses a vector with a disrupted lacZ gene; foreign DNA insertion repairs the gene, allowing recombinants to be identified by colony color.
- Colony hybridization detects recombinants by transferring colonies to a membrane and probing for the inserted DNA sequence.
- Immunological tests identify clones expressing antigens encoded by the
This document discusses various enzymes used for genetic engineering and DNA manipulation. It describes restriction endonucleases and DNA ligase which cut and join DNA fragments. It also discusses other DNA modifying enzymes like nucleases which degrade DNA, and polymerases which synthesize DNA copies. Specific enzymes covered in detail include DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, terminal transferase, T4 DNA ligase, and T4 RNA ligase.
This document discusses key components of expression vectors that are important for efficiently expressing cloned genes. It explains that expression vectors contain regulatory sequences like promoters and terminators to control transcription, as well as elements like ribosome binding sites, fusion tags, and selection markers. Specifically, it provides details on tightly regulated promoters, commonly used viral and bacterial promoters, and considerations for promoters in prokaryotic and eukaryotic expression systems. The document also reviews other important vector elements and their functions.
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
Prokaryotic genomes are circular, double-stranded DNA contained within the nucleoid. They vary in length but are generally a few million base pairs. DNA supercoiling allows for tight packing of the genome.
Eukaryotic genomes are linear chromosomes associated with histone proteins within the nucleus. The DNA is wrapped around histone octamers to form nucleosomes, compacting the genome. Eukaryotic genomes are generally larger and contain more DNA than prokaryotic genomes.
Key differences between prokaryotic and eukaryotic genomes include genome size, number of chromosomes, ploidy level, association with histones, and method of compaction.
F plasmid is a conjugative plasmid found in Escherichia coli that was the first plasmid discovered. It plays an important role in bacterial reproduction by containing genes that code for the production of sex pili and enzymes required for conjugation. The F plasmid replicates through a rolling circle mechanism and transfers between bacteria via conjugation using an F pilus. During conjugation, the F plasmid unwinds and one strand is transferred to the recipient cell where it is replicated to form a double-stranded circular plasmid, converting the recipient into an F+ cell capable of plasmid transfer.
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
This document discusses various mechanisms for transforming and transfecting cells, including prokaryotic, eukaryotic, plant, and fungal cells. It describes the history of bacterial transformation and mechanisms such as natural competence, artificial competence using calcium chloride or electroporation, and lipofection. For eukaryotic transfection, it discusses lipofection, dendrimers, and nucleofection. It also outlines various mechanisms for transforming plants, including Agrobacterium, electroporation, viral transformation, and particle bombardment.
The document summarizes the mechanism of T-DNA transfer during Agrobacterium tumefaciens infection. It explains that T-DNA is a fragment of DNA transferred from the tumor-inducing (Ti) plasmid of A. tumefaciens into the host plant genome. The T-DNA is bordered by repeats and encodes genes that cause tumors in the plant. Virulence genes are expressed in response to plant signals and produce single-stranded T-DNA, which forms a complex with other proteins and is transported into the plant cell and integrated into the plant nuclear DNA, causing uncontrolled cell growth and tumor formation. The mechanism involves multiple virulence protein complexes and integration of T-DNA is directed by the
Restriction enzymes cut DNA molecules at specific recognition sites. Restriction mapping involves digesting an unknown DNA segment with restriction enzymes and analyzing the fragment sizes to determine the locations of restriction sites. One method involves single and double digestions with two enzymes followed by gel electrophoresis to separate the fragments by size. By comparing the fragment patterns between single and double digestions, the positions of each restriction site can be mapped, generating a restriction map of the DNA segment. Restriction mapping was previously important for characterizing cloned DNA but is now easier using DNA sequencing, though analysis of restriction sites remains useful for comparing chromosomal organization between strains.
A DNA library is a collection of DNA fragments that have been cloned into vectors. DNA libraries allow researchers to isolate and study specific DNA fragments of interest. To create a genomic library, DNA is extracted from an organism, cut into fragments, inserted into vectors, and introduced into host bacteria to generate clones containing all the organism's DNA sequences. This library can then be screened to identify and study particular genes. DNA libraries provide an efficient way to store, isolate, and analyze DNA sequences.
This presentation contains information about restriction enzymes, its nomenclature, restriction digestion, and its application. This also contains information about the chemicals used in restriction and also explains the general procedure of restriction digestion of DNA
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
Ti plasmids are found in Agrobacterium tumefaciens bacteria and contain genes that allow the bacteria to transform plant cells and cause crown gall tumors. The plasmid contains virulence genes that are activated by plant signals and mediate transfer of T-DNA into the plant genome. T-DNA integration results in tumor formation and production of opines that the bacteria can utilize. Ti plasmids have been engineered as vectors for plant transformation by removing oncogenes and adding gene of interest between the border sequences, allowing transformation via Agrobacterium infection of wounded plant tissues.
Introduction
Ti plasmid
Agrobacterium tumefaciens
Ti plasmid structure
Overview of infection process
Ti plasmid derived vector systems
Cointegrate vectors
Binary vectors
Agrobacterium mediated transformation of explants
Conclusions
References
Sequence tagged sites (STSs) are short DNA sequences that can be used as genetic markers. STSs were introduced in 1989 as a way to map genes along chromosomes using PCR. They serve as landmarks on physical maps of genomes. STSs are mapped by breaking genomes into fragments, replicating the fragments in bacterial cells to create libraries, and using PCR to determine which fragments contain STSs. Different types of STS markers include microsatellites, SCARs, CAPs, and ISSRs, each of which has distinct characteristics and applications in genetic mapping, population studies, and other areas.
The document provides information about bacterial transformation. It describes that transformation is the process by which bacteria take up extracellular DNA from their environment. Frederick Griffith first discovered transformation in 1928 while working with pneumococcus bacteria. His experiments showed that a non-virulent rough form could be transformed into a virulent smooth form by DNA from a heat-killed smooth strain. Later experiments by Avery, Macleod and McCarty demonstrated that DNA is the transforming principle and genetic material of bacteria. The document then discusses various methods of bacterial transformation including chemical and physical methods like electroporation and use of calcium chloride. It also explains the molecular mechanism of transformation involving DNA binding, penetration, synapsis formation and integration into the bacterial chromosome.
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.
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.
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.
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.
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
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 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 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.
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.
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.
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 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 presentation aims to provide an in-depth understanding of the science behind creating transgenic animals, explore their potential applications, and delve into the ethical considerations surrounding this emerging field of research.
Definition and Background:
We begin by defining transgenic animals as organisms that have had their genetic material intentionally altered through the introduction of foreign genes. This groundbreaking field of genetic engineering has its roots in the development of recombinant DNA technology in the 1970s, which enabled the transfer of genes across different species.
Genetic Engineering Techniques:
This section delves into the techniques employed to create transgenic animals, emphasizing the following key methodologies:
a. DNA Microinjection: The introduction of foreign DNA into the pronucleus of a fertilized embryo, allowing the foreign gene to be incorporated into the animal's genome and expressed in its cells.
b. Gene Targeting: The precise modification of an organism's genome by replacing or disrupting specific genes using technologies such as homologous recombination or CRISPR-Cas9.
c. Somatic Cell Nuclear Transfer (SCNT): The cloning technique involving the transfer of a nucleus from a somatic cell into an enucleated egg, resulting in the creation of an embryo with the same genetic makeup as the somatic cell donor.
Applications of Transgenic Animals:
This section explores the wide-ranging applications of transgenic animals across various fields, including:
a. Biomedical Research: Transgenic animals serve as invaluable models for studying human diseases and testing potential therapies, enabling significant advancements in medical research.
b. Agriculture: Transgenic animals can be engineered to possess desirable traits, such as increased resistance to diseases or improved meat quality, offering the potential to enhance agricultural productivity and sustainability.
c. Pharmaceutical Production: Transgenic animals can be designed to produce therapeutic proteins or antibodies in their milk or blood, providing a cost-effective means of manufacturing valuable pharmaceutical products.
d. Organ Transplantation: Research on transgenic animals has explored the possibility of generating organs that are genetically compatible with humans, addressing the shortage of donor organs for transplantation.
Transgenic Animals: The ability to manipulate the genome of the whole animal ...hilalahmad693671
Transgenic Animals
Since the early 1980s, fruit flies, fish, sea urchins, frogs, laboratory mice and farm animals, such as cows, pigs,
and sheep have been successfully produced.
The ability to manipulate the genome of the whole animal
and the production of transgenic animals has influenced the science dramatically in the last 15 years. The procedure for introducing exogenous donor DNA into
a recipient cell is called Transfection. Chromosomes are taken up inefficiently so that intact chromosomes rarely survived the procedure. Instead the recipient cell usually get a part of the DNA.
This document discusses various methods for creating transgenic animals. It describes how DNA can be injected into the pronuclei of fertilized eggs to produce transgenic mice. The DNA integrates randomly into the genome and may be passed down to offspring. Several methods are used to introduce foreign DNA, including retroviral vectors, microinjection of DNA into pronuclei, and using embryonic stem cells. The document provides details on pronuclei, the microinjection process, and challenges with different techniques.
This document discusses various methods for preserving pure microbial cultures, including short-term and long-term methods. Short-term methods include periodic transfer to fresh media, storage in paraffin or mineral oil, preservation using glycerol, and storage through drying or refrigeration. Long-term methods allow for extended preservation and involve oil storage, saline suspension, immersion in water, storage in soil, lyophilization, or cryopreservation through freezing in liquid nitrogen. Whichever preservation technique is used, it is important to routinely check the quality of preserved microbial stocks to ensure their viability and characteristics remain unchanged over long periods of storage.
Identification of bacteria by staining methodsNAGALAKSHMI R
The document discusses the importance of identifying bacteria, including determining clinical significance, guiding patient care, and identifying appropriate antibiotic therapy. It describes various identification methods, including traditional phenotypic methods examining morphology, staining characteristics, and biochemical tests, as well as newer genotypic and molecular methods. Specific staining techniques are explained in detail, including simple staining, differential staining, Gram staining, and acid-fast staining. The staining methods allow visualization of bacteria and differentiation of structures under a microscope.
Culture media are used to support the growth of microorganisms outside the body for laboratory experiments. They can be classified based on consistency (solid, semisolid, liquid), composition (synthetic, non-synthetic), purpose (general purpose, selective, differential), or oxygen requirement (aerobic, anaerobic). Solid media contain agar and allow study of colony characteristics. Selective media inhibit unwanted bacteria to isolate pathogens. Transport media maintain specimens during laboratory transport.
This document discusses various methods for cultivating anaerobic bacteria, which require an oxygen-free environment. Special pre-reduced culture media can be prepared by boiling and adding reducing agents to drive off oxygen. Anaerobic chambers maintain oxygen-free atmospheres for culturing. Anaerobic jars use hydrogen gas and catalysts to displace oxygen. Anaerobic bags and pouches also provide oxygen-free conditions using chemical oxygen removers. Additional techniques like shake cultures and pyrogallic acid methods pair anaerobes with aerobic bacteria to facilitate growth without oxygen. The rolling tube method developed by Hungate enabled culturing previously uncultivable anaerobes.
Bacteria can reproduce through vegetative, asexual and sexual methods. Vegetative reproduction includes budding, fragmentation, and binary fission. Binary fission is the most common, where the bacterial DNA replicates and the cell divides into two identical daughter cells. Asexual reproduction occurs through endospore formation, where spores form that can withstand harsh conditions until favorable conditions trigger germination. Sexual reproduction in bacteria involves three main methods - transformation, transduction, and conjugation - to facilitate genetic recombination between bacteria.
Bacterial cell structure and composition NAGALAKSHMI R
This document discusses the structure and composition of bacterial cells. It covers the key components of the bacterial cell envelope including the glycocalyx, cell wall, and cell membrane. The cell wall gives shape and structure, and its composition differs between gram-positive and gram-negative bacteria. The cytoplasm contains ribosomes for protein synthesis and may contain inclusion bodies like gas vacuoles or food reserves. The single, circular chromosome called the nucleoid contains the bacterial DNA. Some bacteria also contain extrachromosomal DNA in plasmids.
Bacteria and its classification. Microbiology NAGALAKSHMI R
Bacteria can be classified in several ways, including by their mode of nutrition, temperature and pH requirements, salt tolerance, gas needs, morphology, gram staining, presence of flagella and ability to form spores. Autotrophic bacteria can produce their own food while heterotrophic bacteria rely on organic compounds. Mesophilic bacteria generally grow best around human body temperature, while thermophilic and hyperthermophilic bacteria thrive at higher temperatures. Morphological classifications include cocci, bacilli, spirochetes and others. Gram staining distinguishes between gram positive and gram negative cell walls.
ABDOMINAL TRAUMA in pediatrics part one.drhasanrajab
Abdominal trauma in pediatrics refers to injuries or damage to the abdominal organs in children. It can occur due to various causes such as falls, motor vehicle accidents, sports-related injuries, and physical abuse. Children are more vulnerable to abdominal trauma due to their unique anatomical and physiological characteristics. Signs and symptoms include abdominal pain, tenderness, distension, vomiting, and signs of shock. Diagnosis involves physical examination, imaging studies, and laboratory tests. Management depends on the severity and may involve conservative treatment or surgical intervention. Prevention is crucial in reducing the incidence of abdominal trauma in children.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
- Video recording of this lecture in English language: https://youtu.be/kqbnxVAZs-0
- Video recording of this lecture in Arabic language: https://youtu.be/SINlygW1Mpc
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Rasamanikya is a excellent preparation in the field of Rasashastra, it is used in various Kushtha Roga, Shwasa, Vicharchika, Bhagandara, Vatarakta, and Phiranga Roga. In this article Preparation& Comparative analytical profile for both Formulationon i.e Rasamanikya prepared by Kushmanda swarasa & Churnodhaka Shodita Haratala. The study aims to provide insights into the comparative efficacy and analytical aspects of these formulations for enhanced therapeutic outcomes.
Integrating Ayurveda into Parkinson’s Management: A Holistic ApproachAyurveda ForAll
Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
Here is the updated list of Top Best Ayurvedic medicine for Gas and Indigestion and those are Gas-O-Go Syp for Dyspepsia | Lavizyme Syrup for Acidity | Yumzyme Hepatoprotective Capsules etc
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
2. •INTRODUCTION
• Gene transfer is defined as a technique to efficiently and stably introduce foreign
genes into the genome of target cells . It is the subsequent stable integration &
expression of a foreign DNA into the genome.
• The directed desirable gene transfer from one organism to another genome is referred
as genetic transformation.
The transferred gene is known as trans gene and the organism that develop after a
successful gene transfer is known as transgenic.
History: During the 1970’s Rogers made it became possible to introduce exogenous
DNA constructs into higher eukaryotic cells in vitro.
• In 1990’s, first approved gene therapy case in The United States took place on 14th
September 1990, at the national institute of health, under the direction of professor
William French Anderson.
• In 2012, Glybera (Alipogene tiparvovec) became the first gene therapy treatment
designed to reverse LIPOPROTEIN LIPASE DEFECIENCY(LPLD) a rare
inherited disease of pancreatitis. It was first approved for clinical use in either Europe or
The United States after its endorsement by the European commission.
3. METHODS OF GENE TRANSFER
DNA transfer by natural
methods
DNA Transfer by artificial methods
Physical methods Chemical method
•Conjugation
•Transformation
•Transduction
•Transposition
•Retroviral transduction
•Agrobacterium mediated
transfer
•Electro poration*
•Electro fusion
•Particle Bombardment*
or Biolistics transformation.
•Microinjection*
•Microinjection*
•Microlaser
•Ultrasound Mediated
Transfer
•Impalefection
•Magnetofection
•DNA transfer by calcium
phosphate co-precipitation method
•Liposome mediated transfer
DNA transfer by PEG mediated
method
•Silicon carbide fiber (scf) mediated
transfer
•DEAE dextran method
•DMSO polycation
•Rubidium Chloride Mediated DNA
Transfer.
•Viral delivery systems:
4. • CONJUGATION : It was discovered by Joshua Lederberg and Edward Tatum in
1946 in Escherichia coli.
• This process involved the transfer of DNA through a direct link between the
bacterial cells in the form of proteinaceous tube known as a pilus and the plasmid is
known as the F (for fertility) factor.
• The plasmid determine the F− to an F+ phenotype.
• In some cases, however, the F plasmid could integrate into the bacterial
chromosome, and conjugation could result in the transfer of chromosomal genes.
This process, which was used to construct the first genetic map of E. coli, was
termed sexduction.
• 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. A conjugation event occurs when the male cell
extends his sex pilli and one attaches to the female. 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).
5.
6. TRANSFORMATION: It 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.
7. • 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
8. 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. This disease is associated
with the presence of the Ti (tumour inducing) plasmid within the bacterial cell.
• Co-cultivate with the Agrobacterium:
• —Small pieces of leaf tissue placed into a culture of Agrobacterium for about 30 mins.
The explants then placed on MS medium without selective agent.
• Incubate explants with Agrobacterium for 2 days to allow transfer of the T-DNA.
• Kill the Agrobacterium with a suitable antibiotic:
• The explants are removed from the medium and washed in cefotaxime.
• Select for transformed plant cells: The explant are transferred to a selective
(kanamycin) medium with cefotaxime. Auxin, Cytokinin are used to encourage the
regeneration of by organogenesis.
• Regeneration of whole plant: —The shoot can be rooted by placing them on solid
medium containing a high auxin to cytokinin ratio.
10. ELECTROPORATION/ELECTRIC FIELD-MEDIATED MEMBRANE
PERMEABILIZATION: Microscopic pores are induced in biological membrane
by the application of high volt of electric pulse. These pores are known as
electropores which allow the molecules, ions and water to pass from one side of the
membrane to another and allow to accept exogenous DNA.
• Electroporation has been reported to enhance the level of gene expression and can
be used to increase efficiency of transformation or transfection of bacterial cells. It
significantly improve immune responses elicited to DNA vaccines in both large and
small animals.
• General applications of electroporation: Introduction of exogeneous DNA into
animal cell lines, plant protoplast, yeast protoplast and bacterial protoplast.
• Wheat, rice, maize, tobacco have been stably transformed with frequency upto 1%
by this method. Electroporation of early embryo may result in the production of
transgenic animals.
• Hepatocytes, epidermal cells, haematopoietic stem cells, fibroblast, mouse T and B
lymphocytes can be transformed by this technique.
• Naked DNA may be used for gene therapy by applying electroporation device on
animal cells.
11. • Procedure:
• During electroporation, protoplast or intact plant cells are taken in electroporation
chamber fitted with parallel steel electrodes.
• The chamber is initially filled with buffer containing DNA of interest and high
initial field strength of 1000-1500 volts with a short decay time in microseconds in
applied.
• Plant materials is incubated in a buffer solution containing DNA and subjected to
high-voltage electric pulse is applied by discharge of the capacitor across the cell.
• —The DNA then migrates through high-voltage-induced pores in the plasma
membrane and integrates into the genome.
• —It can be used to deliver DNA into plant cells and protoplasts.
• Even other tissues such as callus and immature embryos are suggested. Several
methods have been suggested to increase transformation efficiency.
13. Advantages: Efficient transformation.
• Large number of transformed cells can be obtained and least number of cells deaths.
• Method is fast and Low equipment cost.
• Does not require experties individual.
• Simultaneously a large number of cell can be treated.
• High percentage of stable transformants can be produced.
Disadvantages:
• Difficulties associated with regeneration of plants from protoplast.
• Rise of obtaining genetic variation in protoplast mediated regenerated plants.
• ~40 to 50% incubated cells receive DNA
• —~50% of the transformed cells can survive
14. •PARTICLE BOMBARDMENT /BIOLISTICS /MICROPROJECTILES /
GENE GUN METHOD : It is a physical method that uses accelerated micro
projectiles to deliver DNA or other molecules into intact tissues and cells.
Firstly used by Klein et al (1987) & Sanford et al (1987).
Gene gun is developed to enable penetration of the genetic material containing a
gene of interest in the cell.
1-2μm tungsten or gold particles (micro-projectiles)are used, coated with the DNA.
Acceleration is given to enter the micro-projectiles into the plant cells.
•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 down the barrel of
the gun towards the target cells that lie just beyond the end of the barrel.
•When the bullet reaches the end of the barrel it is caught and stopped, but the DNA
coated beads continue on toward the target cells. Some of the beads pass through the
cell wall into the cytoplasm of the target cells. Here the bead and the DNA dissociate
and the cells become transformed. Once inside the target cells, the DNA is solubilised
and may be expressed.
17. • General applications of biolistics.: It is used successfully to transfor soyabean,
cotton, spruce, sugarcane, papaya, sunflower, rice, maize, wheat, tobacco etc.
• Genomes of subcellular organelles have been accessible to genetic manipulation by
biolistic method.
• Method can be applied to filamentous fungi and yeast (mitochondria).
• The particle gun has also been used with pollen, early stage embryoids, meristems
and somatic embryos.
• Advantages: Requirement of protoplast can be avoided and this method can be use
to transform all plant species.
• Manipulation of genome of subcellular organelles can be achieved.
• Limitations: High cost of the equipment and microcarriers.
• Intracellular target is random (cytoplasm, nucleus, vacuole, plastid, etc.).
• Transfer DNA is not protected.
18. • MICROINJECTION : Microinjection where the DNA is directly injected into
plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine
tipped (0.5 - 1.0 micrometer diameter) glass needle or micropipette.
• This method of gene transfer is used to introduce DNA into large cells, normally
performed under a specialized optical microscope setup called a micromanipulator.
• The process is frequently used as a vector in genetic engineering and transgenetics
to insert genetic material into a single cell.
• Computerized control of holding pipette, needle, microscope stage and video
technology has improved the efficiency of this technique.
• Applications of microinjection: Process is applicable for plant cell as well as
animal cell but more common for animal cells and is ideally useful for producing
transgenic animal quickly.
• Procedure is important for gene transfer to embryonic cells. Applied to inject DNA
into plant nuclei such as cells and protoplast of tobacco, alfalfa etc.
• Microinjection is potentially a useful method for simultaneous introduction of
multiple bioactive compounds such as antibodies, peptides, RNAs, plasmids,
diffusion markers, elicitors, Ca2+ as well as nucleus and artificial micro or Nano
particles containing those chemicals into the same target single-cells.
19. • Procedure: During microinjection, plant protoplast or partially synthesized cells are
fixed to glass cover slips with the help of poly L. lysine.
• If any cell type is reluctant to attach to cover slips by binding agent, holding pipette
can be an essential factor in microinjection.
• These cell types are firmly retained on fixed place by blunt holding pipette.
• The exogenous DNA of 1 pm is taken in micro-injector and the cells or protoplasts
are firmly immobilized by holding pipette by exerting suction pressure.
• Microinjection containing approximate dosage of DNA is then directly delivered
inside the cells.
• In microinjection, it is possible to microinject 200-350 protoplasts intra nuclearly
and transformation frequency has been demonstrated with 20-60% success .
• By means of reference marking on the coverslip, it is possible to locate
microinjected cells/protoplast by recording with a video camera, which enables to
work more freely from one microinjected cell to next one without interception.
• Earlier microinjection studies were restricted to insect fluorescent dye and
introduction of virus. Microinjection of protoplast for transformation purpose is a
recent achievement. It was however, reasonably believed that injection of DNA
directly into the nucleus accelerates transformation frequency.
21. • Advantages of microinjection: Method is effective in transforming primary cells as
well as cells in established cultures.
• The DNA injected in this process is subjected to less extensive modifications.
• The amount of DNA delivered can be optimized.
• Precise and predictable delivery of DNA.
• Small cell structures like microspores, callus and proembyros can be precisely
targeted.
• Micro-culture is accomplished.
• Limitations of microinjection:
• Costly,
• Sophisticated equipment.
• Handling of protoplast for microinjection requires skilled personal required.
• Knowledge of mating timing and Only one cell receives DNA per injection.
• Method is useful for protoplasts and not for the walled cells.
22. • MACRO INJECTION
• Macroinjection is the method tried for artificial DNA transfer to cereals plants that
show inability to regenerate and develop into whole plants from cultured cells.
• Needles used for injecting DNA are with the diameter greater than cell diameter
(>10-100um).
• DNA injected with conventional syringe into region of plant which will develop into
floral tillers.
• Around 0.3 ml of DNA solution is injected at a point above tiller node until several
drops of solution came out from top of young inflorescence.
• Timing of injection is important and should be fourteen days before meiosis.
• This method was found to be successful with rye plants.
• It is also being attempted for other cereals plants.
23. • Advantages
• This technique does not require protoplast.
• Instrument will be simple and cheap.
• Methods may prove useful for gene transfer into cereals which do not regenerate
from cultured cell easily.
• Limitations
• Less specific,
• Less efficient,
• Frequency of transformation is very low (0.07%)
24. LIPOSOME MEDIATED GENE TRANSFER /LIPOFECTION
• Gene transfer mediated by liposomes was first described by Fengler in 1980
• Liposomes are microscopic vesicles developed in a laboratory environment. Each
liposome is a spherical ball like structure made up of phospholipid bilayers with a
hollow central space, allowing liposomes to interact directly with cells.
• Such vesicles when mixed with cells in culture, fuse with the cell membrane and
deliver DNA directly into the cytoplasm.
• It is a first non-viral technique devised specifically for in vivo DNA transfer.
• A liposome can fuse with the cell membrane of the taken host cell and can deliver its
content to it. The recombinant DNA enclosed in the liposome vesicles penetrates
into the protoplast of the host cell.
• These are artificial vesicles that can act as delivery agents for exogenous materials
including transgenes. Cationic lipids are those having a positive charge are used for
the transfer of nucleic acid.
• Liposomes are able to interact with the negatively charged cell membrane more
readily than uncharged liposomes. Due to fusion between cationic liposome and cell
surface results in the delivery of DNA directly across the plasma membrane.
25. • Procedure:
• In this technique the recombinant DNA, which is negatively charged at a near
neutral pH because of its phospho-diester backbone, is mixed with the lipid
molecules with positively charged (cationic) head groups. The lipid molecules form
a bilayer around the recombinant DNA molecules.
• This results in the formation of liposomes which are further mixed with the host
cells. Most eukaryotic cells are negatively charged at their surface, so the positively
charged liposomes interact with the cells.
• Cells take up the lipid-recombinant DNA complexes, and some of the transfected
DNA enters the nucleus. Fusion of liposomes will be resulted at the point of
attachment of DNA or plasmid DNA while entering the cell. This technique has no
obvious advantages over any other gene transfer methods.
• DNA containing liposomes can be directly microinjected into the vacuole, releasing
the content of liposome into the cytoplasm. However, micro-injected vacuole led to
fusion with tonoplast. This indicates that they could be used to transform even
vacuolated cells. Although this method is elegant on certain criteria, unfortunately,
regeneration plants are problematic with high vacuolated cells.
26. • Advantages
• High degree of reproducibility.
• Long term stability.
• Protection of nucleic acid from
degradation.
• Low toxicity.
• Disadvantage
• preparation of DNA-containing
liposomes is complicated and labor-
intensive.
27. • CALCIUM CHLORIDE (CACL2) MEDIATED DNA TRANSFER: This is used
for the transformation of prokaryotic host cells.
• Principle: In the process of transformation all bacterial cells cannot uptake the
exogenous DNA molecule. CaCl2 makes the cell wall of the bacteria more
permeable to the exogenous DNA and thus increases the competence of the host
cell.
• The process of transfection involves the admixture of isolated DNA (10-100ug) with
solution of calcium chloride and potassium phosphate under condition which allow
the 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.
• Transfection efficiencies using calcium phosphate can be quite low, in the range of
1-2 %. It can be increased if very high purity DNA is used and the precipitate
allowed to form slowly.
28. • Procedure: Growing E. Coli cells are isolated and suspended in 50 mM CaCl2 at a
concentration of 108-1010 cells/ml.
• The cells may be incubated for 12- 24 hr. to increase the frequency of
transformation. The recombinant DNA (10-100ug) is then added.
• Efficient transformation takes only a few minutes and the cells are plated on a suit-
able medium for the selection of transformed clones.
• The frequency of transformed cells is 106-107 per mg of plasmid DNA; this is about
one transformation per 10,000 plasmid molecules.
• The transformed cells are suitably diluted and spread thinly on a suitable medium so
that each cell is well separated and produces a separate colony.
• Generally, the medium is so designed that it permits only the transformed cells to
divide and produce colonies.
• This frequency can be further improved by using special E. Coli strains, e.g.,
SK1590, SK1592, X1766, etc.
29. Limitations
• Frequency is very low. Integrated genes
undergo substantial modification.
• Many cells do not like having the solid
precipitate adhering to them and the
surface of their culture vessel.
• Due to above limitations transfection
applied to somatic gene therapy is
limited.
RUBIDIUM CHLORIDE MEDIATED
DNA TRANSFER:
In this method a variant of the calcium
chloride method that offers somewhat
higher competency. The process
followed is same as before but just the
CaCl2 is replaced with RbCl2. This is
also used in the transformation of the
prokaryotic host cell.
30. • POLY ETHYLENE GLYCOL (PEG) MEDIATED TRANSFORMATION:
• Poly ethylene glycol (PEG) is inert, least toxic to cells and protoplast. Polyethylene
glycol (PEG), in the presence of divalent cations (using Ca2+), destabilizes the
plasma membrane of protoplasts and renders it permeable to naked DNA.
• PEG in complex with divalent cation can disturb molecular organization of the
plasma membrane of the protoplast.
• Positive charges of the calcium are attracted by the negative charge of the protoplast
membrane and alter its zeta potential and destabilize it. Finally DNA makes entry
inside the cell and integrates into the genome.
• The technique not only helps in assessment of transformation, but also involve in
regulating gene transfer into the plant cells. Once DNA gains entry inside the cell, it
is susceptible for degradation inside cytoplasm.
• Culture of protoplasts is taken into a tube and to this tube 40% PEG 4000 (w/v)
dissolved in mannitol and calcium nitrate is added slowly. Then incubated for few
min.
31. • Process
• Protoplast suspended in medium (Mg and Ca ions)
• Heat shock treatment (5min, 45 ˚C)
• PEG added (20-28% conc.)
• Incubation (calcium conc. enhenced)
• Cultured
• Advantages
• A large number of protoplasts can be simultaneously transformed.
• Can successfully use for a wide range of plant species.
• Limitations
• The DNA is susceptible for degradation.
• Random integration of foreign DNA into genome may result in undesirable traits.
• Regeneration of plants from transformed protoplasts is a difficult task.
32. ULTRASOUND MEDIATED TRANSFER/SONOPORATION:
• The uptake of foreign DNA by protoplast or cells can be facilitated by imposing
ultrasound. It involves the exposure of cells to a rapidly oscillating probe, such as
the tip of a sonicator. Test tube containing cells or protoplast in a buffer is made to
contact by inserting tip of ultrasonic device. The ultrasonic pulse generated by
ultrasonicator of 0.4 m/cm2 acoustic intensity is applied for 20-25 min.
• Vigorous vibration in the medium and violent collpase of bubbles generates high
hydrostatic pressure and shock wave may result in sporadic localized rupture in the
membrane and it can facilitates uptake of exogenous DNA.
• The transient appearance of such cavities allows DNA to cross the membrane into
the cytoplasm. It has been shown that the application of low-frequency ultrasound
allows the efficient delivery of nucleic acids into mammalian cells both in vitro and
in vivo, because the plasmid DNA is left structurally intact.
• Furthermore, the ultrasound waves appear to have no adverse effects when focused
on different anatomic locations in the human body.
• Hence, ultrasound-mediated gene delivery raises no safety concerns. Gene transfer
in vivo is generally achieved by injection followed by the application of a focused
ultrasound device.
33. • DNA TRANSFER BY DAE-DEXTRAN METHOD : DEAE-dextran was the first
transfection reagent to be developed and was very widely used until the advent of
lipofection reagents in the 1990s.
• It is a soluble polycationic carbohydrate that forms aggregates with DNA through
electrostatic interactions. It provides the entire complex with a net positive charge,
which allows it to interact with the negatively charged cell membrane and promotes
uptake by endocytosis.
• Like the calcium phosphate method, the reagents are inexpensive and the procedure
is simple and efficient.
• DEAE-dextran-mediated transfection is not particularly efficient for the production
of stably transformed cell lines.
• If DEAE-Dextran treatment is coupled with Dimethyl Sulphoxide (DMSO) shock,
then upto 80% transformed cell can express the transferred gene.
• It is known that serum inhibits this transfection so cells are washed nicely to make it
serum free. Stable expression is very difficult to obtain by this method. Treatment
with chloroquinine increases transient expression of DNA. The advantage of this
method is that, it is cheap, simple and can be used for transient cells which cannot
survive even short exposure of calcium phosphate.
34. • SILICON CARBIDE FIBER (SCF) MEDIATED TRANSFER: SCF does not
require any specialized equipment.
• In this approach, silicon carbide fibres in average of 0.4-0.6 µm in diameter and 10-
90 µm long are taken along with DNA in vortex tube.
• Plant cells or embryos are then introduced and vortexed gently.
• Entry of DNA into the cell is probably due to the penetration through the cell wall
and plasma membrane.
• Vortexing process results in the adhering DNA to silicon carbide fibres and gained
access to inside the nucleus and eventually stable integration into the nucleus
genome. Thus, passing of the DNA across the cell wall has advantage over other
methods.
• This approach does not involve regeneration of protoplast. Presently this technique
is applicable to a particular species, which produce friable nature of callus.
• Many cereals cannot be transformed by SCF as they produce non friable brittle
nature of callus.
35. • MICROLASER: Micro laser mediated gene transfer offers advantage only in
specific cases where other methods are not advantageous.
• This technique involves focusing micro laser beam into the light path or microscope
used to burn holes into the cell wall as membrane DNA uptake is possible through
penetrated cells during incubation.
• Several instances have shown that DNA gets adsorbed to the cell wall material even
before its entry inside the cell.
• IMPALEFECTION: Impalefection is a method of gene delivery using Nano
materials, such as carbon Nano fibres, carbon nanotubes, nanowires, etc. This
technique is used for the transfection of plant and animal cells. In this technique
needle-like nanostructures are synthesized perpendicularly to the surface of a sub-
strate. Recombinant DNA is attached to the nanostructure surface. A chip with
arrays of these needles is then pressed against cells or tissue.
• MAGNETOFECTION: Magnetofection, or Magnet assisted transfection is a
method, which uses magnetic force to deliver recombinant DNA into target host
cells. Nucleic acids are first associated with magnetic nanoparticles. Then,
application of magnetic force drives the nucleic acid particle complexes towards and
into the target host cells, where the cargo is released. This has been successfully
used to transfect the plant and animal cells.
36. • VIRAL DELIVERY SYSTEMS: Viruses are naturally evolved vehicles that
efficiently transfer their genes into host cells.
• Viral vectors that have been extensively studied and genetically manipulated for
safety concerns in laboratory research and for in vivo gene transfer protocols include
retroviruses, adenoviruses, herpes simplex viruses, lentiviruses, adeno associated
viruses and Sindbis viruses.
• Choice of viral vectors is dependent on gene transfer efficiency, capacity to carry
foreign genes, toxicity, stability, immune responses towards viral antigens and
potential viral recombination.
• There is a wide variety of vectors used to deliver DNA or oligo nucleotides into
mammalian cells, either in vitro or in vivo.
• Other viral vectors that are currently under development are based on lenti viruses,
human cytomegalovirus (CMV), Epstein-Barr virus (EBV), poxviruses, negative-
strand RNA viruses (influenza virus), alpha viruses etc.
• The three commonly used viral gene transfer systems are
• Retrovirus (RV), Adenovirus (AV), Adeno Associated Virus (AAV).
37.
38. References:
• Transposition - CoGepedia.mhtml
• https://www.takarabio.com/learning-centers/gene-function/viral-
transduction/retrovirus/retroviral-products
• Physical Methods of Gene Transfer _ Genetics.mhtml
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