Transgenesis techniques have evolved from early microinjection methods to more precise engineered nuclease approaches. Initial methods like pronuclear microinjection resulted in random integration with low efficiency. The development of embryonic stem cells and nuclear transfer enabled greater control over transgenic status but required extensive cloning. Newer tools like transposons, zinc finger nucleases, and CRISPR/Cas9 allow for stable, heritable integration at targeted genomic loci with higher efficiency and less mosaicism than early random integration methods. These advances facilitate the creation of transgenic and gene-edited animal models for agricultural and biomedical applications.
This document discusses genetic manipulation techniques for animals, including somatic cell nuclear transfer (SCNT) cloning. It provides details on the SCNT process, including the Roslin technique used to create Dolly the sheep. Applications of SCNT are described for agriculture, conservation, and medical therapeutics. The document also discusses the success of SCNT, limitations, and ethical concerns regarding genetic manipulation of animals.
Transgenic animals are organisms that have been genetically engineered to carry foreign DNA in their genome. This document discusses transgenic animals, including their definition, goals, benefits and risks, types, methods of production, and applications. Some key points covered are: transgenic animals are useful for studying gene function and producing human proteins; common types include mice, fish, cows, and pigs; methods to create them include pronuclear microinjection and using embryonic stem cells or retroviruses; they have applications in research, agriculture, and biotechnology.
This document discusses methods of embryo sexing. It begins with a brief history of embryo sexing and introduces invasive and non-invasive methods. For invasive methods, it describes cytological/karyotyping methods, identification of sex chromatin, use of Y-chromosome probes, and PCR. Advantages include low cost and accuracy, while disadvantages include potential harm to embryo viability. For non-invasive methods, it outlines detection of X-linked enzymes and H-Y antigens, noting advantages of maintaining embryo integrity but challenges around accuracy and availability of reagents. The document concludes by discussing applications and constraints of embryo sexing technologies.
This document discusses the production of transgenic farm animals. It begins by introducing transgenic technology which involves introducing foreign DNA into an animal using recombinant DNA methods. It then describes extracting the desired gene using PCR and discusses several methods for creating transgenic animals, including retroviral vectors, sperm-mediated gene transfer, microinjection, and embryonic stem cells. Potential applications are improved biomass, disease resistance, recombinant vaccines, and livestock pharming. Examples of transgenic animals produced include sheep, goats, pigs, cattle, fish, and chickens.
RETROVIRUS MEDIATED GENE TRANSFER AND EXPRESSION CLONINGSrishtiRoy10
- The retroviral virion is a spherical particle 80-100 nm in diameter composed of a lipid bilayer envelope containing glycoproteins and a capsid containing two copies of the viral RNA genome and enzymes.
- Retroviruses replicate by reverse transcribing their RNA genome into DNA which is then integrated into the host cell genome by an integrase enzyme to become a provirus, allowing transcription of viral genes.
- Retrovirus mediated gene transfer involves the virus producing a DNA copy of its genome using reverse transcriptase, with the DNA then integrating randomly into the host cell genome, allowing investigation of gene function.
Dr. B. Victor is a retired biology professor with over 32 years of experience teaching and researching reproductive technology in fishes. His presentation outlines various forms of reproduction including asexual, sexual, and parthenogenesis. It also discusses cloning technology such as embryo splitting, nuclear transfer, and the three main types of cloning - recombinant DNA cloning, reproductive cloning, and therapeutic cloning. The benefits and applications of cloning as well as techniques for transgenic animal production are also summarized.
Transgenic animals are animals that have had a foreign gene deliberately inserted into their genome using recombinant DNA methodology. The first transgenic mice were created in 1980, and the first cloned mammal, Dolly the sheep, was born in 1996. There are three main methods for creating transgenic animals: DNA microinjection, embryonic stem cell-mediated gene transfer, and retrovirus-mediated gene transfer. Transgenic animals produce useful products like monoclonal antibodies, blood clotting factors, and human milk proteins. They are used for medical research, toxicology studies, pharmaceutical production, and analyzing gene expression regulation. However, some ethical concerns exist regarding animal suffering during transgenic research experiments.
This document discusses genetic manipulation techniques for animals, including somatic cell nuclear transfer (SCNT) cloning. It provides details on the SCNT process, including the Roslin technique used to create Dolly the sheep. Applications of SCNT are described for agriculture, conservation, and medical therapeutics. The document also discusses the success of SCNT, limitations, and ethical concerns regarding genetic manipulation of animals.
Transgenic animals are organisms that have been genetically engineered to carry foreign DNA in their genome. This document discusses transgenic animals, including their definition, goals, benefits and risks, types, methods of production, and applications. Some key points covered are: transgenic animals are useful for studying gene function and producing human proteins; common types include mice, fish, cows, and pigs; methods to create them include pronuclear microinjection and using embryonic stem cells or retroviruses; they have applications in research, agriculture, and biotechnology.
This document discusses methods of embryo sexing. It begins with a brief history of embryo sexing and introduces invasive and non-invasive methods. For invasive methods, it describes cytological/karyotyping methods, identification of sex chromatin, use of Y-chromosome probes, and PCR. Advantages include low cost and accuracy, while disadvantages include potential harm to embryo viability. For non-invasive methods, it outlines detection of X-linked enzymes and H-Y antigens, noting advantages of maintaining embryo integrity but challenges around accuracy and availability of reagents. The document concludes by discussing applications and constraints of embryo sexing technologies.
This document discusses the production of transgenic farm animals. It begins by introducing transgenic technology which involves introducing foreign DNA into an animal using recombinant DNA methods. It then describes extracting the desired gene using PCR and discusses several methods for creating transgenic animals, including retroviral vectors, sperm-mediated gene transfer, microinjection, and embryonic stem cells. Potential applications are improved biomass, disease resistance, recombinant vaccines, and livestock pharming. Examples of transgenic animals produced include sheep, goats, pigs, cattle, fish, and chickens.
RETROVIRUS MEDIATED GENE TRANSFER AND EXPRESSION CLONINGSrishtiRoy10
- The retroviral virion is a spherical particle 80-100 nm in diameter composed of a lipid bilayer envelope containing glycoproteins and a capsid containing two copies of the viral RNA genome and enzymes.
- Retroviruses replicate by reverse transcribing their RNA genome into DNA which is then integrated into the host cell genome by an integrase enzyme to become a provirus, allowing transcription of viral genes.
- Retrovirus mediated gene transfer involves the virus producing a DNA copy of its genome using reverse transcriptase, with the DNA then integrating randomly into the host cell genome, allowing investigation of gene function.
Dr. B. Victor is a retired biology professor with over 32 years of experience teaching and researching reproductive technology in fishes. His presentation outlines various forms of reproduction including asexual, sexual, and parthenogenesis. It also discusses cloning technology such as embryo splitting, nuclear transfer, and the three main types of cloning - recombinant DNA cloning, reproductive cloning, and therapeutic cloning. The benefits and applications of cloning as well as techniques for transgenic animal production are also summarized.
Transgenic animals are animals that have had a foreign gene deliberately inserted into their genome using recombinant DNA methodology. The first transgenic mice were created in 1980, and the first cloned mammal, Dolly the sheep, was born in 1996. There are three main methods for creating transgenic animals: DNA microinjection, embryonic stem cell-mediated gene transfer, and retrovirus-mediated gene transfer. Transgenic animals produce useful products like monoclonal antibodies, blood clotting factors, and human milk proteins. They are used for medical research, toxicology studies, pharmaceutical production, and analyzing gene expression regulation. However, some ethical concerns exist regarding animal suffering during transgenic research experiments.
This document provides information about animal cloning, including its history, processes, examples of cloned animals, and ethical issues. It discusses the three main types of cloning - reproductive cloning, gene cloning, and therapeutic cloning. Reproductive cloning aims to produce genetically identical copies of animals and was used to create Dolly the sheep in 1996, the first mammal cloned from an adult somatic cell. While cloning may help protect endangered species and improve livestock, it also raises ethical concerns about technical safety, personal identity, and the commercialization of life.
Embryo transfer is a technique where embryos are collected from donor females and transferred to recipient females. The key steps are superovulation of the donor, artificial insemination, embryo collection 5-8 days later, grading the embryos, and transferring high quality embryos into a synchronized recipient. This allows one superior donor to produce many offspring, accelerating genetic improvement. Embryo transfer has advantages like faster breeding, disease control and conservation, but requires synchronization of donors and recipients.
Knockout mice are genetically engineered mice that have had specific genes inactivated through gene targeting. This document provides an overview of knockout mice, including their history, generation process, and uses. It describes how embryonic stem cells are isolated and genetically modified through homologous recombination before being injected into blastocysts to generate chimeric mice. Knockout mice are valuable research tools for studying gene function and modeling human diseases. They have contributed significantly to our understanding of immunology and the development of humanized antibody therapies.
This document discusses various types of vectors used for cloning, including bacteriophage vectors, plasmid vectors, cosmid vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and shuttle vectors. Key points include:
- Bacteriophage derivatives like lambda phage are suitable for cloning large eukaryotic DNA due to abilities like packaging millions of clones and size selection of DNA.
- Phage-based vectors can be insertional, containing a single cloning site, or replacement vectors with two cloning sites allowing DNA substitution.
- Cosmids are hybrid phage-plasmid vectors that can package DNA up to 48 kb into phage particles.
- Y
iPSCs are pluripotent; unlike ESC, iPSCs are not derived from the embryo, but instead created from differentiated cells in the lab through a process – cellular reprogramming.
This document discusses modern techniques for pre-selecting the sex of embryos, which can be done either through sperm sorting by looking for X or Y chromosomes, or by determining the sex of pre-implantation embryos. There are invasive methods like cytogenetic analysis of chromosomes and DNA probes, and non-invasive methods like detecting the H-Y antigen or differences in growth between male and female embryos. These techniques allow for altering the male to female ratio in farm animals to increase milk and meat production.
Animal Cloning Procedure, Problems and PerspectivesShafqat Khan
Cloning in farm animals has problems and perspectives. Key issues include developmental anomalies in cloned animals, the large offspring syndrome observed in cattle and sheep clones, and safety apprehensions regarding meat and milk from cloned animals. However, cloning also has potential applications for transgenic animal production, creating disease models, bioreactors, and research into xenotransplantation. It allows the propagation of elite livestock and conservation of endangered species. Further optimization is needed to improve cloning efficiency and resolve health issues.
Genetically engineered animals are created through genetic manipulation techniques like DNA microinjection or cloning to introduce new traits. They are being engineered for agriculture and medicine, such as producing human proteins and antibodies in their milk or blood for treating diseases. While this technology aims to increase productivity and quality, it raises ethical concerns about interfering with animal integrity and welfare, as well as potential environmental impacts. Regulations require ensuring modified animals and their products are safe for animal and human consumption.
Transgenic animals are produced by introducing foreign DNA into an animal's genome. The first transgenic animal was a mouse created in 1974. Since then, various methods have been used to generate transgenic fish, livestock, and other species. Transgenic animals have applications in biomedical research, agriculture, and industry. They can serve as models for human disease or help produce pharmaceuticals in their milk. However, transgenesis also carries risks if the inserted gene has unintended effects on the animal's development or physiology.
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.
Rishabh Maheshwari presents information on transgenic techniques. Transgenics involve introducing foreign DNA into a host organism's genome, typically using a mouse as the host. This allows for engineering organisms with DNA from another source as part of their genetic material. Common methods to create transgenic animals include DNA microinjection, retrovirus-mediated gene transfer, sperm-mediated gene transfer, and embryonic stem cell-mediated gene transfer. Transgenic technology has applications in disease models, pharmaceutical production, agriculture, and industry. While it has benefits, there are also concerns regarding animal welfare and environmental impacts.
This document discusses animal cloning, specifically somatic cell nuclear transfer (SCNT). It provides information on the history of cloning, animals that have been cloned, the SCNT process, challenges to successful cloning including reprogramming differentiated cells, and problems seen in cloned animals including embryonic and postnatal abnormalities. Applications of cloning such as restoring endangered species, generating transgenic animals, and gene knockout in farm animals are also covered.
This document discusses various applications of animal biotechnology. It outlines how transgenic animals can be used to improve biomass production, disease resistance, recombinant vaccine development, and production of pharmaceutical proteins. Specifically, it mentions that transgenic salmon can grow up to 6 times faster than wild salmon due to a growth hormone gene insertion. It also provides examples of using transgenic cattle and goats to produce therapeutic proteins in their milk for human disease treatments.
The document discusses embryo transfer, which is a process where an embryo is collected from a donor female and transferred to a recipient female to complete its development. Embryo transfer allows a genetically superior female to produce more offspring than through natural reproduction. Key aspects discussed include selecting donor females, inducing superovulation in donors to release multiple eggs, inseminating donors, non-surgical and surgical embryo recovery methods, evaluating and storing embryos, and transferring embryos into recipient females through non-surgical or surgical methods.
Microinjection is a gene transfer technique where DNA is directly injected into cells using a fine glass micropipette. It is highly efficient at the individual cell level and was originally used for transfecting hard-to-transfect cells. The procedure involves holding a cell using one pipette while another pipette is used to inject DNA into the cell's cytoplasm or nucleus. It allows for stable transfection efficiencies of around 20% and is used to generate transgenic animals by injecting DNA into oocytes, eggs or embryos. However, it is time-consuming and can only be done for a small number of cells.
Retroviral vectors are derived from wild type retroviruses like Moloney murine leukemia virus. They are engineered to carry foreign genes into target cells. The vectors contain cis-acting elements from the viral genome like LTRs and packaging signals but lack the trans-acting genes gag, pol, and env. This prevents replication but allows the vector and its gene of interest to integrate stably into the host cell genome. Retroviral vectors show promise for gene therapy applications but also have limitations like requiring actively dividing cells for transduction and potential risks of insertional mutagenesis leading to cancer.
a proper description about the process microinjection and also about gene transfer. and different types of DNA delivery methods.
with advantages, disadvantages, limitations and applications.
Introduction
History
Landmarks Events in Transgenic Livestock Research
Techniques/ Method for Gene Transfer
Examples of transgenesis
Importance
Application
Limitation
Issue related to Transgenic Technology
Ethical concerns and how to Overcome
Transgenic animals are produced by inserting foreign DNA into the animal's genome. There are several methods for producing transgenic animals. The first successful method involved microinjecting a rat growth hormone gene controlled by a promoter into mouse embryos, producing mice that grew larger. Other methods include using embryonic stem cells, viral vectors, cloning, and sperm-mediated gene transfer. Transgenic animals are useful for researching gene function and regulation, modeling human diseases, and potentially increasing agricultural production.
This document provides information about animal cloning, including its history, processes, examples of cloned animals, and ethical issues. It discusses the three main types of cloning - reproductive cloning, gene cloning, and therapeutic cloning. Reproductive cloning aims to produce genetically identical copies of animals and was used to create Dolly the sheep in 1996, the first mammal cloned from an adult somatic cell. While cloning may help protect endangered species and improve livestock, it also raises ethical concerns about technical safety, personal identity, and the commercialization of life.
Embryo transfer is a technique where embryos are collected from donor females and transferred to recipient females. The key steps are superovulation of the donor, artificial insemination, embryo collection 5-8 days later, grading the embryos, and transferring high quality embryos into a synchronized recipient. This allows one superior donor to produce many offspring, accelerating genetic improvement. Embryo transfer has advantages like faster breeding, disease control and conservation, but requires synchronization of donors and recipients.
Knockout mice are genetically engineered mice that have had specific genes inactivated through gene targeting. This document provides an overview of knockout mice, including their history, generation process, and uses. It describes how embryonic stem cells are isolated and genetically modified through homologous recombination before being injected into blastocysts to generate chimeric mice. Knockout mice are valuable research tools for studying gene function and modeling human diseases. They have contributed significantly to our understanding of immunology and the development of humanized antibody therapies.
This document discusses various types of vectors used for cloning, including bacteriophage vectors, plasmid vectors, cosmid vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and shuttle vectors. Key points include:
- Bacteriophage derivatives like lambda phage are suitable for cloning large eukaryotic DNA due to abilities like packaging millions of clones and size selection of DNA.
- Phage-based vectors can be insertional, containing a single cloning site, or replacement vectors with two cloning sites allowing DNA substitution.
- Cosmids are hybrid phage-plasmid vectors that can package DNA up to 48 kb into phage particles.
- Y
iPSCs are pluripotent; unlike ESC, iPSCs are not derived from the embryo, but instead created from differentiated cells in the lab through a process – cellular reprogramming.
This document discusses modern techniques for pre-selecting the sex of embryos, which can be done either through sperm sorting by looking for X or Y chromosomes, or by determining the sex of pre-implantation embryos. There are invasive methods like cytogenetic analysis of chromosomes and DNA probes, and non-invasive methods like detecting the H-Y antigen or differences in growth between male and female embryos. These techniques allow for altering the male to female ratio in farm animals to increase milk and meat production.
Animal Cloning Procedure, Problems and PerspectivesShafqat Khan
Cloning in farm animals has problems and perspectives. Key issues include developmental anomalies in cloned animals, the large offspring syndrome observed in cattle and sheep clones, and safety apprehensions regarding meat and milk from cloned animals. However, cloning also has potential applications for transgenic animal production, creating disease models, bioreactors, and research into xenotransplantation. It allows the propagation of elite livestock and conservation of endangered species. Further optimization is needed to improve cloning efficiency and resolve health issues.
Genetically engineered animals are created through genetic manipulation techniques like DNA microinjection or cloning to introduce new traits. They are being engineered for agriculture and medicine, such as producing human proteins and antibodies in their milk or blood for treating diseases. While this technology aims to increase productivity and quality, it raises ethical concerns about interfering with animal integrity and welfare, as well as potential environmental impacts. Regulations require ensuring modified animals and their products are safe for animal and human consumption.
Transgenic animals are produced by introducing foreign DNA into an animal's genome. The first transgenic animal was a mouse created in 1974. Since then, various methods have been used to generate transgenic fish, livestock, and other species. Transgenic animals have applications in biomedical research, agriculture, and industry. They can serve as models for human disease or help produce pharmaceuticals in their milk. However, transgenesis also carries risks if the inserted gene has unintended effects on the animal's development or physiology.
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.
Rishabh Maheshwari presents information on transgenic techniques. Transgenics involve introducing foreign DNA into a host organism's genome, typically using a mouse as the host. This allows for engineering organisms with DNA from another source as part of their genetic material. Common methods to create transgenic animals include DNA microinjection, retrovirus-mediated gene transfer, sperm-mediated gene transfer, and embryonic stem cell-mediated gene transfer. Transgenic technology has applications in disease models, pharmaceutical production, agriculture, and industry. While it has benefits, there are also concerns regarding animal welfare and environmental impacts.
This document discusses animal cloning, specifically somatic cell nuclear transfer (SCNT). It provides information on the history of cloning, animals that have been cloned, the SCNT process, challenges to successful cloning including reprogramming differentiated cells, and problems seen in cloned animals including embryonic and postnatal abnormalities. Applications of cloning such as restoring endangered species, generating transgenic animals, and gene knockout in farm animals are also covered.
This document discusses various applications of animal biotechnology. It outlines how transgenic animals can be used to improve biomass production, disease resistance, recombinant vaccine development, and production of pharmaceutical proteins. Specifically, it mentions that transgenic salmon can grow up to 6 times faster than wild salmon due to a growth hormone gene insertion. It also provides examples of using transgenic cattle and goats to produce therapeutic proteins in their milk for human disease treatments.
The document discusses embryo transfer, which is a process where an embryo is collected from a donor female and transferred to a recipient female to complete its development. Embryo transfer allows a genetically superior female to produce more offspring than through natural reproduction. Key aspects discussed include selecting donor females, inducing superovulation in donors to release multiple eggs, inseminating donors, non-surgical and surgical embryo recovery methods, evaluating and storing embryos, and transferring embryos into recipient females through non-surgical or surgical methods.
Microinjection is a gene transfer technique where DNA is directly injected into cells using a fine glass micropipette. It is highly efficient at the individual cell level and was originally used for transfecting hard-to-transfect cells. The procedure involves holding a cell using one pipette while another pipette is used to inject DNA into the cell's cytoplasm or nucleus. It allows for stable transfection efficiencies of around 20% and is used to generate transgenic animals by injecting DNA into oocytes, eggs or embryos. However, it is time-consuming and can only be done for a small number of cells.
Retroviral vectors are derived from wild type retroviruses like Moloney murine leukemia virus. They are engineered to carry foreign genes into target cells. The vectors contain cis-acting elements from the viral genome like LTRs and packaging signals but lack the trans-acting genes gag, pol, and env. This prevents replication but allows the vector and its gene of interest to integrate stably into the host cell genome. Retroviral vectors show promise for gene therapy applications but also have limitations like requiring actively dividing cells for transduction and potential risks of insertional mutagenesis leading to cancer.
a proper description about the process microinjection and also about gene transfer. and different types of DNA delivery methods.
with advantages, disadvantages, limitations and applications.
Introduction
History
Landmarks Events in Transgenic Livestock Research
Techniques/ Method for Gene Transfer
Examples of transgenesis
Importance
Application
Limitation
Issue related to Transgenic Technology
Ethical concerns and how to Overcome
Transgenic animals are produced by inserting foreign DNA into the animal's genome. There are several methods for producing transgenic animals. The first successful method involved microinjecting a rat growth hormone gene controlled by a promoter into mouse embryos, producing mice that grew larger. Other methods include using embryonic stem cells, viral vectors, cloning, and sperm-mediated gene transfer. Transgenic animals are useful for researching gene function and regulation, modeling human diseases, and potentially increasing agricultural production.
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.
Transgenic animals like goats and pigs have been generated using various techniques over the past few decades. Pronuclear injection was an early technique used to produce the first transgenic farm animals in 1985. Since then, other methods like somatic cell nuclear transfer (cloning) and viral vectors have been used. These techniques allow introduction of foreign genes encoding therapeutic proteins or genes modifying milk composition. Transgenic goats can express human proteins in their milk for pharmaceutical purposes. Pigs have been targeted as potential donors for xenotransplantation due to organ similarities to humans. New genome editing tools like CRISPR-Cas9 now enable more precise genetic modifications of goats and pigs.
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 provides an overview of transposable elements. It discusses how Barbara McClintock discovered transposable elements in corn in 1940 and was later awarded a Nobel Prize for this work. It defines key terms like transposition and transposase. It describes the mechanisms and types of transposable elements in prokaryotes and eukaryotes. Some applications of transposable elements like genetic mapping and gene therapy are mentioned. The functional impacts of transposable elements, like causing mutations that can lead to genetic disorders and antibiotic resistance, are briefly outlined.
Gene transfer technology pharmacology biotechnology basic methods
Natural, physical, chemical methods of gene transfer.
Along with advantages and limitations, and applications.
The document discusses several methods to generate transgenic animals, including:
1. Microinjecting DNA directly into embryos, which was the first successful method for mice but has low efficiency in other species.
2. Using transposons to insert DNA randomly throughout the genome, which is effective for insects, fish, and mammals.
3. Employing lentiviral vectors to integrate foreign genes into the host genome, which has proven highly efficient in several species.
4. Incubating sperm with DNA and using intracytoplasmic sperm injection for fertilization, which has generated transgenic mice and rabbits.
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.
Transgenic animal production and its applicationkishoreGupta17
A genetically modified animal with the heterologous gene of interest being inserted for the purpose of biopharming or make a diseased model to study the consequences of disease and its probable therapy
TRANSGENIC AND GENOME EDITED ANIMALS,.pptxPoojaJangir21
This document discusses transgenic and genome edited animals and some of their applications and ethical issues. Transgenic animals have foreign DNA randomly inserted into their genome, while genome edited animals have targeted changes made within their own DNA. Examples provided include glowing zebrafish, disease model mice, livestock that produce human therapeutic proteins, faster growing salmon, and efforts to create pigs and cattle with desirable traits. While these techniques aim to benefit research and agriculture, they also raise ethical concerns about animal welfare, environmental impacts, and unintended consequences that warrant consideration and oversight.
SYNTHETIC CHROMOSOME PLATFORMs IN PLANTS: CONCEPTS & APPLICATIONskundan Jadhao
This document outlines a seminar on synthetic/artificial chromosomes. It begins by discussing the need for synthetic chromosomes due to limitations of traditional genetic engineering approaches. It then describes various methods that have been used to develop artificial chromosomes, including the bottom-up and top-down methods. Several case studies are presented where telomere-mediated truncation was used to produce engineered minichromosomes in plants with endogenous centromeres. The document concludes by discussing ways that engineered minichromosomes can be amended in planta, such as through site-specific recombination systems and zinc finger nucleases.
Genetic Transformation of Maize: Conventional Methods and Precision Geno...Ananya Sinha
Genetic Transformation of Maize: Conventional Methods and Precision Genome Modification
This is a summary of the above chapter mentioned in "Biotechnology of Major Cereals".
The precision genome modification is defined majorly focused on comparing the conventional genome modification methods with the modern ones.
Introduction
Definition
History
Why are the transgenic animals being produced
Transgenic mice
Mice: as model organism
Methods of creation of transgenic mice
knock-out mice
Application of transgenic mice
Conclusion
References
This document provides information about a credit seminar on sperm sexing in farm animals. It discusses the importance of predetermining offspring sex in livestock for efficient food production. Various methods for sperm sexing are described, including albumin gradient, percoll density gradient, free-flow electrophoresis, identification of H-Y antigen, and flow cytometry. Flow cytometry works by staining sperm with a fluorescent dye that binds more to the DNA of X sperm, allowing separation of X and Y sperm based on their DNA content difference. The document discusses factors affecting sorting efficiency and the challenges and future possibilities of sperm sexing.
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.
KnockOut mouse technology By Bikash karkiBikash Karki
The document summarizes the process of creating a knockout mouse through genetic engineering techniques. Key points:
- Knockout mice are created by "knocking out" or inactivating specific genes in embryonic stem cells taken from early mouse embryos.
- There are two main methods - homologous recombination, which precisely replaces a gene with an inactive version, and gene trapping, which randomly inserts DNA to disrupt gene function.
- Genetically modified stem cells are injected into mouse blastocysts to generate chimeric mice, and breeding is used to produce mice that are homozygous for the knocked out gene. Studying these mice helps reveal the function of the targeted gene.
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
hematic appreciation test is a psychological assessment tool used to measure an individual's appreciation and understanding of specific themes or topics. This test helps to evaluate an individual's ability to connect different ideas and concepts within a given theme, as well as their overall comprehension and interpretation skills. The results of the test can provide valuable insights into an individual's cognitive abilities, creativity, and critical thinking skills
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
Brine shrimp (Artemia spp.) are used in marine aquaculture worldwide. Annually, more than 2,000 metric tons of dry cysts are used for cultivation of fish, crustacean, and shellfish larva. Brine shrimp are important to aquaculture because newly hatched brine shrimp nauplii (larvae) provide a food source for many fish fry (Mozanzadeh et al., 2021). Culture and harvesting of brine shrimp eggs represents another aspect of the aquaculture industry. Nauplii and metanauplii of Artemia, commonly known as brine shrimp, play a crucial role in aquaculture due to their nutritional value and suitability as live feed for many aquatic species, particularly in larval stages (Sorgeloos & Roubach, 2021).
3. What is a Transgenesis????
•Transgenesis is the process of introducing an exogenous gene into a living
organism
• which exhibit a new property
•transmit that property to its offspring
tomato
Growth hormone gene
Genetically modified tomato
4. 1985
1986
2000
First transgenic sheep and pigs
Hammer et al.
Embryonic cloning of
sheep Willadsen et al.
Somatic cloning of sheep
Wilmut et al.
Transgenic cattle Cibelli et al.
MMLV transgenic cattle Chan et
al. 1998
Gene targeting in Sheep
McCreath et al.
1997
5. 2006
2010
2007
2002Trans-chromosomal cattle
Kuroiwa et al.
Heterozygous knock-out in pigs
Dai et al., Lai et al.
Transposon transgenesis in
pigs
Jakobsen et al.
Gene knock-out in
cattle Richt et al.
Conditional
transgenesis in
pigs
Kues et al.
Homozygous gene
knockout in pigs
Phelps et al.
Lentiviral transgenesis in
pigs
Hofmann et al.
2003
6. • Classic method of gene transfer in farm animals
(Hammer et al., 1985)
Pronuclear DNA microinjection
Doyle, A., McGarry, M.P., Lee, N.A. and Lee, J.J., 2012. The construction of transgenic and gene
knockout/knockin mouse models of human disease. Transgenic research, 21(2), pp.327-349.
7. Species specific modifications are necessary
• Rabbits, pigs, sheep and goats- Embryo transfer (Brem, 1993)
• Cattle – IVM & IVF (Krimpenfort et al. 1991)
For visualisation of pronuclie
1. Cattle & pig: centrifugation prior to injection
2. Sheep : Nomarski phase contrast optics
Problem: discriminating between integrated and non integrated sequences,
(Seo et al. 1997)
• Neomycin phospho transferase (neo) (Bondioli & Wall, 1996)
• or green fluorescent protein (GFP) expression as selectable markers
Takada et al. 1997)
8. Pronuclear DNA microinjection in farm animals.
A, microinjection into a porcine zygote
B: efficiency of the DNA microinjection technique in various species
9. • Retroviral infection was the first method used to produce transgenic mice
(Jaenisch, 1976)
Viral Vectors
RNA
DNA
Integration in host genome
• Retroviruses can only integrate in dividing cells
Reverse transcriptase
Retro viral integrase
10. Chan
et.al.,
1998
• Moloney murine lukaemia virus with pseudo capsid glycoprotien
of stomatitis virus
Jaenisch,
1976
• Retroviral infection to produce transgenic mice .
Tsukui et
al., 1996
• Replication defective Adeno virus for transgenic mice
Whitelaw
et al.,
2004
• Lentiviruses for production of porcine zygotes
Ritchie et
al., 2009
• Lentivirus-mediated gene transfer in livestock
11. • mosaic foetuses were obtained (Haskell, 1995)
• limited size(< 10 kb) insertion (Brem, 1993)
• (LTRs) flanking interfere with mammalian promoters (Wells et al., 1999)
Other problems:
• gene silencing by DNA methylation due to the presence of viral sequences
• oncogene activation or insertional mutagenesis
(Hofmann et al., 2006)
Contd..
Disadvantages
12. • Sperm cells are incubated with DNA used for AI
• Plasmid DNA internalised in spermatozoa with the nuclear scaffold
recombination with genomic DNA (Spadafora, 1997)
• Generation of human decay accelerating factor (hDAF)transgenic pigs by
sperm mediated gene transfer (Lavitrano et al., 1999)
Sperm mediated gene transfer and
Intra-cytoplasmic sperm injection
Miao, X., 2013.
Recent advances in
the development of
new transgenic
animal
technology. Cellular
and Molecular Life
Sciences, 70(5),
pp.815-828.
13. • Intracytoplasmic sperm injection (ICSI)
• sperm cell membranes are damaged
• then immobile spermatozoa are used for ICSI
• Intratesticular transfection of germ cells (Kim et al. 1997)
• Male germ cells treated with busulfan
• LacZ gene introduced into each seminiferous tubule
• Transmission of the donor haplotype to the next generation after germ-cell
transplantation has been achieved in goats
(Honaramooz et al. 2003)
Contd..
14. • In mammals, the results of sperm mediated gene transfer has
low efficiency
repeatability (Gandolfi, 2000)
• Major obstacles of this strategy are the
lack of efficient in vitro culture methods for prospermatogonial cells
the lack of efficient gene transfer techniques into these cells.
(Keskintepe et al. 1997)
Disadvantages
16. • Pluripotent embryonic stem (ES) cells have the ability to participate in
organ and even germ cell development following injection into blastocysts
(Rossant, 2001)
• Targeted alterations of the genome can be induced in cultured cells by
homologous recombination
(Ledermann, 2000)
• General or tissue specific inducible gene knockouts site directed insertions
into defined loci
(Stacey et al., 1994)
• Possibility of engineering large chromosomal rearrangements
(Zheng et al., 1999)
Embryonic stem (ES) cells
17. Doyle, A., McGarry, M.P., Lee, N.A. and Lee, J.J., 2012. The construction of
transgenic and gene knock-out/knock-in mouse models of human
disease. Transgenic research, 21(2), pp.327-349.
18. • Nuclear transfer technology involves the transfer of donor nucleus into the
cytoplasm of a zygote or a metaphase II enucleated oocyte.
• Done by electrofusion of karyoplast and cytoplast
• The proof of this strategy was the generation of human factor IX transgenic
sheep by using nuclear transfer
Nuclear transfer technology
(Wilmut et al., 1997)
19. Nuclear transfer in cattle.
a—c: enucleation of a recipient oocyte to produce a cytoplast
d—f: transfer of a nuclear donor cell (karyoplast) under the zona
pellucida of the cytoplast
g: clone of four Simmental calves produced following electrofusion of
karyoplast—cytoplast complexes
20. Wilmut et al., 1997
Kato et al., 1998; Wells et al.,1999;
Zakhartchenko et al., 1999
Baguisi et al., 1999
Betthauser et al. 2000; Onishi et al.
2000; Polejaeva et al., 2000
21. Genetic modification of farm animals by DNA microinjection vs. nuclear
transfer using transfected donor cells
22. • Homologous recombination (HR) in the donor cells
(Kues, W.A. and Niemann, H. 2011)
• Gene knockout is feasible in large mammals. (Richt, J.A. et al. 2007)
Problem
• Uncontrolled, ‘passive’ nature of genomic insertion.
• Short life span of transgenic animal
Advantage and Disadvantage
24. • Small interfering RNA (siRNA) molecules 19–27 base pairs in length
• Transient gene knockdown, synthetic siRNAs can be transfected into cells
or early embryos
(Clark and Whitelaw, 2003; Iqbal et al., 2007)
• RNAi knockdown of porcine endogenous retrovirus (PERV) has been
demonstrated in porcine primary cells (Dieckhoff et al., 2007)
and in cloned piglets (Dieckhoff et al., 2008)
• siRNA mediated knockdown of the prion protein (PRNP) gene has been
accomplished in bovine embryos
(Golding et al., 2006).
RNA interference mediated gene
knockdown
25.
26. • Carry very large pieces of DNA that are maintained as episomal entities
(Niemann and Kues 2003)
• Expression of human immunoglobulin in bovine
Human Ig
heavy and
light chain
Bovine
fibroblast
Trans-
chromosomal
bovine
offspring
Artificial chromosomes as transgene
vectors
27. • “Jumping genes” – 1st discovered in maize
• Active transposons : Piggy Bac,Tol2 (Clark et al., 2007)
• Transposons reactivated from defective element :
Sleeping beauty (Ivics et al., 1997)
Frog prince, Hsmar1. (Clark et al., 2007)
• Use cut-and-paste mechanism
• first transposon transgenic pigs (Kues et al.,2010; Garrels et al., 2010)
Contains a transgene
flanked by inverted
terminal repeats
Transposons
29. • All animals are phenotypic positive
• No evidence of gene silencing
• No variegated transgenic expression
• F1 & F2 offspring shows no change in transposon mediated expression
Advantages
Contd..
30. Gene knock out mediated by
designer nuclease
• Target modification by homologus recombination : SCNT
• For non homologus recombination
Designer
nuclease
ZFN Meganuclease TALEN
32. +
Chemical or physical mutagen
UV-light, free radicals, irradiation, etc
3′-OH
5′-P OH-3′
P-5′
Transposase, viral integrase or ZFN
Depending on sequence specificity
one or more binding sites may exist
Ku70, Ku80 end binding factors,
DNA dependent protein
kinase,
XRCC4/DNA ligase IV
ZFNs create a DSB, in
combination with HR this may
result in a targeted integration
Passive DNA-Integration Active DNA-Integration
Exogenous protein
Double strand break (DSB)
Foreign DNA Cellular repair and
illegitimate recombination
Ectopic enzyme-catalyzed
Foreign DNA integration
Concatemeric arrays, Monomeric copy of transgene
antibiotic marker,
plasmid backbone
33. Comparison techniques
PNI SCNT ICSI Artificial
chromosom
e
Transposo
n systems
Retro- and
lentiviral
infection
ZNF
Integration Passive Passive Passiv
e
No
integration
the vector
is episomal
Active Active Active
Mechanism DSB
repair
DSB
repair
/HR
DSB
repair
Episomal
persistence
Transpo
sase
catalyse
d
Viral
integrase
catalysed
Binding
and
cleavage
of target
DNA + HR
34. PNI SCNT ICSI
mediat
ed
transg
enesis
Artificial
chromosom
e
Transposo
n systems
Retro- and
lentiviral
infection
ZNF
Transgenic
status
Stable,
often
concatem
eres
Stable,
often
concate
meres
Stable Stable in
founder
Stable,
majority
monom
eric
Stable,
monomeri
c
Stable
knock-in
Max.
construct
size (kb)
20–(500) 20–
(250)
20–
(250)
500 8–12
(60)
6–7 n.a.
Ratio of
transgenic
offspring
per born
offspring
3–10% 70–
100%
6% n.a 40–60% 50–90% n.a.
Contd..
35. PNI SCNT ICSI
mediat
ed
transg
enesis
Artificial
chromosom
e
Transposo
n systems
Retro- and
lentiviral
infection
ZNF
Preference
of
integration
sites
Random Random
or
targeted
for HR
Rando
m
n.a. Transpo
sasedep
endent,
random
or
semi-
random
Semi
random
preference
s
for
transcribed
genes, risk
of
insertional
mutagenes
is
Target
sequence/
off-target
effects
possible
Transgene
silencing
or
variegated
expression
Frequent Frequen
t
Frequ
ent
n.a. Rare Frequent Rare
39. • Research human disease
• Produce consumer products
• Human therapeutic use
• Enhance production
• Improve animal health
• Creation of neo organs
• Unpredictable outcome
1. Mutagenesis
2. Functional disorder
• Destruct natural breeding
• Disruption of natural genetic
information
• Low survival rate
CONSPROS
40. • Consequences of genetic modification
• Environment safety issue
• Crossing species boundries
• Xenotransplantation issue
Ethical issues
41. Since discovery of 1st transgenic mice all conventional techniques were
playing role.
But for precise integration new techniques in this field evolved.
Now transgenesis presenting difficult challenges for 21st century scientist
and ethicists
Two major consideration
How much a transgenic animal benefits human???
How much pain does it cause to the animal???
Summary
42. Reference
1. Garrels, W., Ivics, Z. and Kues, W.A., 2012. Precision genetic engineering in large
mammals. Trends in biotechnology, 30(7), pp.386-393.
2. Niemann, H. and Kues, W.A., 2007. Transgenic farm animals: an
update. Reproduction, Fertility and development, 19(6), pp.762-770.
3. Wolf, E., Schernthaner, W., Zakhartchenko, V., Prelle, K., Stojkovic, M. and Brem,
G., 2000. Transgenic technology in farm animals–progress and
perspectives. Experimental Physiology, 85(6), pp.615-625.
4. Kues, W.A. and Niemann, H., 2011. Advances in farm animal
transgenesis. Preventive veterinary medicine, 102(2), pp.146-156.
5. Miao,X.,2013. Recent advances in the development of new transgenic animal
technology. Cellular and Molecular Life Sciences,70(5), pp.815-828
6. Forsberg, C.W., Meidinger, R.G., Liu, M., Cottrill, M., Golovan, S. and Phillips, J.P.,
2013. Integration, stability and expression of the E. coli phytase transgene in the
Cassie line of Yorkshire Enviropig™. Transgenic research, 22(2), pp.379-389.