Transgenic Animal Technology (Animal biotechnology)

Here you will understand what actually Transgenic Animal Technology is and what is its important in the modern biological world
You'll also get to know different techniques through which we could create a Transgenic animal
Also there are some important examples mentioned in here,with there role in moder biotechnology and healthcare

1. What is Transgenic Animal Technology? Introduction and Strategies for the development of transgenic animals MICROINJECTION RETROVIRAL VECTORS INTRODUCTION OF ENGINEERED STEM CELLS INTO AN EARLY-STAGE EMBRYO Presented by: Umer majeed Bsc.Biotechnology (2023-2026);(5 th ) Roll no: 1013

2. Introduction: Transgenic animal technology refers to the process of creating animals that carry a foreign gene (transgene) deliberately inserted into their genome, allowing the expression of new traits or the study of specific biological functions. These genetically modified organisms are engineered using recombinant DNA techniques, where DNA from one species is introduced into another to alter its genetic makeup The term “transgenic” specifically applies to animals with integrated foreign DNA that can be passed on to offspring, distinguishing them from other forms of genetic modification. In this way, transgenic technology has led to the development of fishes, live stock and other animals with altered genetic profiles which are useful to mankind. 1.1 General overview

3. Introduction: This technology emerged in the early 1980s, building on advancements in molecular biology and recombinant DNA methods. The first transgenic animal was a SUPERMOUSE created by injecting DNA into fertilized eggs, marking a breakthrough in genetic engineering. This experiment was performed by Ralph Brinster (U Pennsylvania) and Richard Palmiter (University of Washington) in 1982. The offspring here was much larger comparatively Since then, the transgenic technology has evolved into a powerful tool for biomedical research, agriculture, and industry. 1.2 Historical background

4. Introduction: The first major step forward in the ability to chemically modify genes occurred when American biologist Martin Gellert and his colleagues from the National Institutes of Health purified and characterized an enzyme in Escherichia coli responsible for the actual joining, or recombining, of separate pieces of DNA (Zimmerman et al., 1967). They called their find “DNA-joining enzyme,” and this enzyme is now known as DNA ligase. A second major step forward in gene modification was the discovery of restriction enzymes, which cleave DNA at specific sequences. These enzymes were discovered at approximately the same time as the first DNA ligases by Swiss biologist Werner Arber and his colleagues while they were investigating a phenomenon called host-controlled restriction of bacteriophages. Although Griffith and Avery had had demonstrated the ability to transfer foreign genetic material into cells decades earlier, this “transformation” was very inefficient, and it involved “natural” rather than manipulated DNA. Only in the 1970s did scientists begin to use vectors to efficiently transfer genes into bacterial cells. The first such vectors were plasmids, or small DNA molecules that live naturally inside bacterial cells and replicate separately from a bacterium’s chromosomal DNA. The following year, Stanley Cohen and his colleagues were also the first to construct a novel plasmid DNA from two separate plasmid species which, when introduced into E. Coli, possessed all the nucleotide base sequences and functions of both parent plasmids. Cohen’s team used restriction endonuclease enzymes to cleave the double-stranded DNA molecules of the two parent plasmids. The team next used DNA ligase to rejoin , or recombine, the DNA fragments from the two different plasmids. Finally, they introduced the newly recombined plasmid DNA into E. Coli. A fourth major step forward in the field of recombinant DNA technology was the discovery of a vector for efficiently introducing genes into mammalian cells. Specifically, researchers learned that recombinant DNA could be introduced into the SV40 virus, a pathogen that infects both monkeys and humans. Indeed, in 1972, Stanford University researcher Paul Berg and his colleagues integrated segments of λ phage DNA, as well as a segment of E. coli DNA containing the galactose operon, into the SV40 genome. The significance of their achievement was its demonstration that recombinant DNA technologies could be applied to essentially any DNA sequences, no matter how distantly related their species of origin

5. 2. Key Methods for Creating Transgenic Animals Several techniques are used to introduce transgenes, each with varying efficiency and applications MICROINJECTION The most common method Involves introducing the injecting DNA directly into the pronucleus of a fertilized egg. This is then implanted into a surrogate mother. It’s widely used for mammals like mice, rats, and livestock Retroviral Vectors Genes are delivered using modified viruses that integrate into the host genome. This is effective for early embryos but can be less precise. Embryonic Stem Cell (ES Cell) Transfer Transgenes are introduced into ES cells in vitro, which are then injected into blastocysts to create chimeric animals. Offspring can inherit the modification. CRISPR-Cas9 and Gene Editing More recent advancements use CRISPR to precisely edit genomes, allowing targeted insertion or knockout of genes with higher accuracy and fewer off-target effect General overview

6. 2.1 MICROINJECTION Microinjection is a genetic engineering technique used to create transgenic animals by directly injecting foreign DNA (a transgene) into the pronucleus of a fertilized egg (zygote). The principle relies on recombinant DNA technology: the transgene integrates into the host genome during early embryonic development, allowing it to be expressed and passed to offspring. This method is precise because it targets the nucleus before cell division, increasing the chance of stable integration Key Concept: The pronucleus is the haploid nucleus from the sperm or egg before they fuse. Injection here ensures the DNA can incorporate into all cells of the developing embryo.

7. 2.1 MICROINJECTION The process is hands-on and requires specialized equipment like a micromanipulator, inverted microscope, and ultra-fine glass needles. Here’s a step-by-step breakdown : Procedure Steps Preparation of Transgene Construct: Design and isolate the DNA fragment (e.g., a gene of interest linked to a promoter for expression). Purify it to high concentration (typically 1-5 ng/µL) to avoid toxicity. Collection of Fertilized Eggs: Superovulate female animals (e.g., mice) with hormones to produce many eggs. Mate them, then harvest zygotes at the one-cell stage (usually 0.5 days post-fertilization). Microinjection: Pierce the pronucleus (usually the larger male one) and inject 1-2 picoliters of DNA. The egg swells slightly if successful. Inject 200-300 eggs per session for efficiency. Setup for Injection: Place zygotes in a culture medium under a microscope. Use a holding pipette to stabilize the egg and a injection needle (1-2 µm tip) loaded with DNA solution Culture and Implantation: Incubate injected eggs briefly to check viability, then transfer them into the uterus of a pseudopregnant surrogate mother (prepared via hormone treatment). Screening Offspring: After birth, use PCR or Southern blotting to confirm transgene integration in the pups. Breed positives to establish transgenic lines. Success Rate: Typically 10-30% of injected eggs develop into transgenic animals, varying by species.

8. 2.1 MICROINJECTION Advantages Microinjection is the gold standard for many species due to its reliability: High efficiency and specificity: Direct delivery ensures controlled dosage and integration. Versatile: Works for large DNA constructs and various species (e.g., mice, rats, pigs, cows). No vector limitations: Any DNA can be injected, unlike viral methods. Precise timing: Targets early embryos for germline transmission. Disadvantages Despite its strengths, it has some limitations too: Labor -intensive and time-consuming: Requires skilled technicians and can take hours per session
Low throughput: Only one egg injected at a time; success depends on embryo viability.
Potential mosaicism: Not all cells may integrate the transgene, leading to chimeric animals.
Animal welfare concerns: Involves surgery and hormone treatments; ethical oversight is crucial.
Costly: Needs advanced equipment and animal facilities. Applications Microinjection is widely used in research and in industrial area: Disease Modeling : Creating transgenic mice with human genes to study conditions like cystic fibrosis or cancer. Agriculture : Engineering livestock for disease resistance (e.g., mastitis-resistant cows) or enhanced products (e.g., goats producing antithrombin in milk). Basic Research: Investigating gene function, such as knockout mice for developmental biology. Notable Example: The first transgenic mouse (1981) was made via microinjection, inserting a growth hormone gene for larger “ supermice .”

9. 2.2 Retroviral vectors Retroviral vectors are modified viruses derived from retroviruses (e.g., lentiviruses or oncoretroviruses ) used to deliver foreign DNA (transgene) into the host genome of animals. The principle exploits the retrovirus life cycle: the viral RNA is reverse-transcribed into DNA, which integrates stably into the host chromosome via integrase enzyme, enabling heritable expression in transgenic offspring. Vectors are made replication-incompetent for safety, lacking genes for viral replication but retaining packaging signals. This method is particularly useful for species where direct DNA injection is challenging. Key Concept: Integration occurs in dividing cells ( oncoretroviruses ) or non-dividing cells (lentiviruses), leading to permanent genetic modification

10. ONCORETROVIRUSES & LENTI VIRUSES Oncoretroviruses , also referred to as onco -retroviruses or gamma-retroviruses, are a subgroup of retroviruses historically known for their oncogenic (cancer-causing) potential in animals due to mechanisms like insertional mutagenesis or carrying viral oncogenes. In the context of retroviruses used in transgenic animal biotechnology, oncoretroviruses serve as the basis for viral vectors designed to deliver and integrate transgenes into the host genome, enabling stable, heritable genetic modifications in animals like mice for research, disease modeling, or biopharmaceutical pr productio . T hese vectors, often derived from viruses like the murine leukemia virus, are introduced to fertilized eggs or embryonic stem cells via co-incubation, achieving transgenic rates of around 60-70% in founder animals, with integration at various genomic loci and germline transmission. However, a key limitation in this application is their requirement for host cell division during transduction, which reduces efficiency in non-dividing cells such as early embryos, and they frequently lead to transgene silencing (lack of expression) post-integration. This contrasts with lentiviruses, another retroviral subgroup commonly used in transgenic animal production, which can transduce non-dividing cells, support higher expression levels without routine silencing, and are thus often preferred for generating stable transgenic lines. Early developments in oncoretroviral vectors paved the way for retroviral applications in biotechnology, though their role has diminished in favor of safer, more efficient lentiviral systems to minimize risks like oncogenesis .

11. 2.2 Retroviral vectors The process involves vector construction, production, and delivery, typically in a biosafety lab. Steps for creating transgenic animals (e.g., via embryo infection): Vector Design: Clone the transgene into a retroviral plasmid with promoter, packaging signals (ψ), and long terminal repeats (LTRs) for integration. Remove viral genes to make it replication-defective. Virus Production : Transfect packaging cell lines (e.g., 293T cells) with the vector plasmid and helper plasmids providing gag, pol, and env proteins. Harvest pseudotyped viral particles ( titer : 10^6-10^8 particles/mL). Target Cell Infection: Expose early embryos, embryonic stem cells, or spermatogonia to the vector (e.g., via incubation or injection). Use polybrene to enhance uptake. For embryos, this is done at the blastocyst stage. Implantation and Development: Transfer infected embryos to surrogate mothers. Allow development to birth. Screening: Confirm integration in offspring via PCR, Southern blot, or fluorescence (if using reporter genes). Breed to establish stable lines. Success rate: 5-20% transgenic efficiency, depending on the type of vector used and the host Species Procedure Steps

12. Retroviral vectors offer unique benefits for transgenesis : High transduction efficiency: Infect a wide range of cell types with stable, single-copy integration.
Large cargo capacity: Up to 8-10 kb transgenes.
Natural integration mechanism: Minimizes DNA damage compared to physical methods.
Suitable for difficult species: Effective for birds, fish, or insects where microinjection fails.
Long-term expression: Integrated genes are heritable and expressed in progeny. Challenges include safety and precision issues: Random integration: Can disrupt host genes, causing insertional mutagenesis or oncogenesis .
Risk of contamination: Potential for replication-competent viruses or viral remnants in products.be25dd
Limited to certain cells: Oncoretroviruses require cell division; lentiviruses are broader but more complex.
Immunogenicity: Viral components may trigger immune responses in animals.
Biosafety concerns: Requires high-level containment; ethical issues with viral use.
Lower control: Multiple integrations possible, leading to variable expression Retroviral vectors are applied in research and biotech: Disease Modeling : Lentiviral vectors create transgenic models for HIV or neurological disorders in mice/rats. Agriculture: Engineering virus-resistant poultry (e.g., avian influenza-resistant chickens via retroviral delivery). Gene Therapy Research: Testing therapeutic genes in large animals like pigs for xenotransplantation. Basic Biology: Studying development in zebrafish or frogs with fluorescent reporters. Notable Example: Early transgenic chickens produced using retroviral vectors to express foreign proteins, overcoming eggshell barriers. 2.2 Retroviral vectors Advantages Disadvantages Applications

13. 2.3 Introduction of Engineered Stem Cells into an Early-Stage Embryo Definition and Principle : Introduction of engineered stem cells into an early-stage embryo refers to the process of modifying embryonic stem (ES) cells with foreign DNA in vitro and then injecting them into a host blastocyst to create chimeric transgenic animals. The principle is based on the pluripotency of ES cells, derived from the inner cell mass of blastocysts, which allows them to integrate into the host embryo and contribute to all tissues, including the germline, enabling heritable genetic modifications via homologous recombination or other editing techniques. This method ensures targeted gene insertion, knockout, or knock-in, with the engineered cells forming part of the developing organism. Key Concept: Chimerism results from the mix of host and donor cells; breeding chimeras passes the transgene to offspring for stable lines.

14. -This technique requires cell culture labs and micromanipulation, primarily for mice but adaptable to other species. Steps include: Procedure Steps 2.3 Introduction of Engineered Stem Cells into an Early-Stage Embryo ES Cell Isolation: Harvest blastocysts (3.5-4.5 days post-fertilization) from donor animals. Isolate the inner cell mass and culture on feeder layers with leukemia inhibitory factor (LIF) to maintain pluripotency . Selection and Cloning: Apply antibiotics to select modified cells (24-48 hours). Screen colonies via PCR, sequencing, or Southern blot; expand positive clones. Implantation: Transfer the chimeric blastocyst into the uterus or oviduct of a pseudopregnant surrogate mother prepared via hormone treatment. Genetic Engineering: Transfect ES cells with the DNA construct (e.g., via electroporation, liposomes, or CRISPR plasmids) for homologous recombination. Include selectable markers like neomycin or puromycin for enrichment. Blastocyst Injection: Microinject 10-15 engineered ES cells into the cavity of a host blastocyst (from a different strain for chimera tracking, e.g., by coat color ). Screening and Breeding: After birth, identify chimeras by markers. Breed with wild-type animals to confirm germline transmission and establish transgenic lines. Success Rate: Germline transmission in 10-50%; overall efficiency varies by species and edit type.

15. This method provides precision and ethical efficiencies:
Targeted editing via homologous recombination for specific modifications like knockouts.
In vitro manipulation allows easy screening, reducing animal numbers.
Versatile for complex traits, including conditional expressions.
High germline potential in rodents; supports disease modeling with human-like physiology.
Faster with CRISPR integration, lowering costs compared to direct embryo methods. Challenges limit widespread use: Species-limited: Reliable ES lines mainly for mice; difficult in livestock like pigs or sheep . Time-consuming and costly: Weeks for culture/selection; requires skilled technicians. Variable chimera contribution: May lead to mosaicism or low germline transmission. Ethical concerns: Animal welfare in surrogates and potential ecological risks if transgenics escape. Technical complexity: Risk of off-target effects or differentiation loss in culture. Widely used in research and biotech: Disease Modeling : Transgenic mice for Alzheimer’s, cancer, diabetes, or Parkinson’s to study mechanisms and test drugs. Biopharming: Goats producing anti-thrombin in milk for thrombosis treatment. Xenotransplantation: Gene-edited pigs with reduced rejection for human organ transplants (e.g., hearts/kidneys). Agriculture: Disease-resistant livestock, like cattle against foot-and-mouth disease. Notable Example: Knockout mice via ES cells to mimic human genetic disorders, accelerating drug discovery. Advantages Disadvantages Applications 3. Introduction of Engineered Stem Cells into an Early-Stage Embryo

16. 3. Important examples of transgenic animals TRANSGENIC RABBIT: A transgenic rabbit is a rabbit whose genome has been altered to include a foreign gene (transgene) from another organism, enabling it to express new proteins or traits. These genetically modified animals serve as valuable tools in biomedical research, acting as models for human diseases like cardiovascular disorders, cancer, and AIDS. Additionally, they function as bioreactors to produce biologically active proteins in their milk, which can be purified for therapeutic use. Alba , the EGFP (Enhanced Green Flurescent protein) bunny Created in 2000 as a transgenic artwork. TRANSGENIC MONKEY: Transgenic monkey is a genetically engineered primate that has received foreign DNA, typically to model human diseases for research and to test therapies. The first transgenic monkey, named ANDi , was created in 2001 at Oregon Health & Science University, carrying a marker gene from the jellyfish Aequorea victoria that confers green fluorescence. These models are crucial because monkeys, especially rhesus macaques, share significant genetic, physiological, and neurological similarities with humans, making them valuable for studying complex conditions like Huntington’s disease, which has been successfully modeled in transgenic monkeys. “ ANDi ” was the first transgenic monkey, born in 200 1. "ANDi" stands for "inserted DNA" spelled backwards. An engineered virus was used to insert the harmless gene for green fluorescence protein (GFP) into ANDi's rhesus genome. ANDi proves that transgenic primates can be created, and can express a foreign gene delivered into their genome.

17. TRANSGENIC LIVESTOCK: Transgenic livestock are  animals whose genetic makeup has been altered by the introduction of foreign DNA (a  foreign gene ), often using  DNA microinjection  into embryos, to introduce new or enhanced traits . These animals are engineered for various purposes, including producing pharmaceuticals in their milk or eggs, improving livestock traits like hornlessness , and serving as models for human diseases. Examples include goats producing a life-saving protein in their milk and cows with genes for human breast milk production.  Bioreactors whose cells have been engineered to synthesis marketable proteins.
More economical than producing desired protein in cell culture. Transgenic cattle: Transgenic cows are made to produce proteins lactoferrin and interferons in their milk.
Prion free cows resistant to mad cow disease. Transgenic sheep: For good quality wool production. Transgenic goat: Goats that could express tissue plasminogen activator, anti thrombin III, spider silk etc in milk.

18. T RANGENIC PIG : transgenic pig is  a pig whose genetic makeup has been altered by introducing foreign DNA, often to create models for human diseases, produce human proteins, or develop organs for xenotransplantation. such as studying diseases like diabetes and Alzheimer's, and to develop genetically modified organs, such as kidneys, that are more compatible with humans. Pig for organ transplant : Pigs with human genes, in order to decrease the chance of organ rejection by human body. TRANSGENIC MOUSE : A transgenic mouse is a genetically engineered mouse that has had foreign DNA introduced into its genome, enabling the study of gene function and the development of models for human genetic diseases. Scientists create these mice by microinjecting DNA into fertilized mouse eggs, which are then implanted into a foster mother; the foreign DNA becomes a permanent part of the mouse's cells and can be passed to offspring. These mice are crucial tools in biomedical research for understanding diseases like cancer, diabetes, and Alzheimer's, and for testing potential therapeutic approaches.   Alzheimer's mouse : In the brain of Alzheimer's patients, dead nerve cells are entangled in a protein called amyloid. Mouse made by introducing amyloid precursor gene into fertilized egg of mice .They are then used to study the essay of different drugs. Onco -mouse: Mouse model to study cancer m ade by inserting activated oncogenes. These are then used as a model in cancer biology. Sm art mouse: Biological model engineered to overexpress NR2B receptor in the synaptic pathway . This makes the mice learn faster like juveniles throughout their lives .

19. TRANSGENIC FISH: Transgenic fish are fish that have had an artificial foreign gene, or transgene, introduced and stably integrated into their genome through genetic engineering. This process, called transgenesis , allows for the creation of genetically modified fish with enhanced or novel traits, such as faster growth, increased disease resistance, or unique colors , for both aquaculture and research purposes. The technology is used to produce superior strains for commercial use and to develop useful model organisms for scientific study. Superfish : Increased growth and size
Growth hormone gene inserted into fertilized egg. Eg ; Transgenic salmon grows about 10-11 times faster than normal fish. Glo fish: Eg ; GM freshwater zebra fish ( Danio rerio )
Produce by integrating a fluorescent protein gene from jelly fish into embryo of fish.

20. 4. Concluding remarks ISSUES RELATED TO TRANSGENIC TECHNOLOGY: Blurring the lines between species by creating transgenic combinations.
There may be health risks associated with transgenics .
There may be long term effects on the environment when transgenic animals are released into the field.
Various bioethicist argue that it is wrong to create animals that would suffer as a result of genetic alteration. CONCLUSION : Transgenic technology is a field that is under constant evolution.
Many transgenic animals have been successfully created for a variety of purposes, and the prospects are enormous.
It holds great potential in many fields including agriculture, medicine and industry.
With proper research and careful use the transgenic animals can go a long way in solving several problems for which science doesn’t have a solution till now.