animal biotechnology ppt for biotechnology

animal biotechnology ppt for biotechnology . Includes monoclonal antibodies

1. Definition Animal biotechnology is a branch of biotechnology in which molecular biology techniques are used to genetically engineer animals to improve their suitability for agricultural, industrial, or pharmaceutical applications. Animal biotechnology has been used to produce genetically modified animals that synthesize therapeutic proteins, have improved growth rates or are resistant to disease. Gene manipulation technology/Recombinant DNA Technology/Gene cloning have revolutionized biotechnology.

2. Genetically modified organisms Scientific methods that include recombinant DNA technology are used to produce genetically modified organisms

3. Application of Gene Manipulation in Disease Management Nutritious food to overcome disease caused by nutrition deficiency. Prevention of pathogenic disease by vaccination/ immunization. Diagnosis of Disease. Treatment of Disease Understanding the new/ unknown disease.

4. How biotechnology helps in providing nutritious food? Golden Rice: a transgenic rice having high Vitamin A content. Carotene (Precursor of Vit A) biosynthetic gene has been introduced in paddy. High Iron containing transgenic Banana : The Ferritin gene has been introduced. Transgenic Soya with a high amount of Vitamin E.

5. How Biotechnology helps in prevention of Disease? Production of different categories of Vaccines: Vaccination is the administration of antigenic material to stimulate an individual’s immune system to develop immunity against pathogen. Types of Vaccines: Attenuated Vaccines, Inactivated Vaccines, Subunit Vaccines, DNA Vaccines etc.

6. Biotechnology in Diagnosis of Disease PCR based diagnosis of disease: PCR, RT-PCR, Real time PCR are used to diagnose disease. The presence of bacteria or viruses can be detected at an early stage of infection. Antibody based diagnosis of Disease: ELISA, Immunocytochemistry, Immunohistochemistry, Western blotting, radioimmuno assay, Immunofluorescence, etc. DNA Hybridization based diagnosis : Southern blotting, Northern blotting, DNA Microarray etc. DNA Sequence based diagnosis: Single nucleotide polymorphism, Gene Sequencing, Finding triplet codon repeats( Huntington’s Disease).

7. PCR Based Diagnosis Invitro amplification of a known(at least terminal sequence should be known) DNA segments. Increase quantity to million –billion times. Amplify only specific sequence from mixture of DNA RT-PCR and real time PCR are used to observe expression of genes so can be used to observe presence of pathogenic RNA viruses too.

8. Treatment of Diseases Therapeutic Recombinant Proteins: several human disorders are due to absence of malfunctioning of protein usually synthesized in the body. Human proteins are produced in bacteria or other organism using recombinant DNA technology. Eg : Insulin(Humulin), Growth hormone, blood clotting factor etc. Therapeutic nucleic acid used for Gene therapy: addition or deletion of gene which cause disease or mask the expression of disease causing gene by antisense technology. Therapeutic antibodies: antibodies conjugated with toxin kills cancer cell/tissue. Antibiotics: use to treat microbial disease. Antibiotics are secreted by Microorganism but to increase yield or get modified version of antibiotics scientist apply gene manipulation technology.

9. Immunofluorescences

10. Treatment of Diseases A. Therapeutic Recombinant Proteins: Several human disorders are caused by the absence of or malfunctioning of proteins that are usually synthesized in the body. Human proteins are produced in bacteria or other organism using recombinant DNA technology eg. Insulin ( humulin ), Growth hormone, blood clotting factor etc. B. Therapeutic nucleic acid used for Gene Therapy: Addition or deletion of gene which cause disease or mask the expression of disease causing gene by antisense technology. C. Therapeutic antibodies: Antibodies conjugated with toxin kills cancer cell/tissue. D . Antibiotics: Use to treat microbial disease. Antibiotics are secreted by Microorganism but to increase yield or get modified version of antibiotics scientists apply gene manipulation technology. E. Biosynthesis and production of natural drugs are being increase applying metabolic engineering. Eg. Vinblastine, vincristine etc.

11. Gene Therapy This is a technique whereby a working gene replaces the absent or faulty gene, so that the body can make the correct enzyme or protein and consequently eliminate the root cause of the disease. Gene therapy may also used for curing cancer.

12. Monoclonal Antibodies Monoclonal antibody ( MAb ) is a single type of antibody that is directed against a specific antigenic determinant (epitope). George Kohler and Cesar Milstein (Nobel Prize, 1984) achieved large scale production of MAbs . They could successfully hybridize antibody—producing B-lymphocytes with myeloma cells in vitro and create a hybridoma Monoclonal antibodies are used in various medical applications, including treating cancer, autoimmune diseases, and infectious diseases.

13. Steps involved in MAb production (Hybridoma Technology)

14. Immunization: An animal, typically a mouse, is injected with an antigen (the molecule the antibody will target). This stimulates the animal's immune system, leading to the production of B cells that produce antibodies against the antigen.

15. Cell Fusion Spleen cells (containing the antibody-producing B cells) are harvested from the immunized animal. Myeloma cells (immortal cancerous cells) are also obtained. These cells are chosen for their ability to grow indefinitely in culture but lack the enzyme HGPRT, which is necessary for nucleotide synthesis. The B cells and myeloma cells are fused together using agents like polyethylene glycol (PEG) or electrofusion. This fusion creates hybridoma cells, each potentially producing a specific antibody.

16. Selection and Screening The fused cells are cultured in a special medium called HAT medium (hypoxanthine-aminopterin-thymidine). HAT medium inhibits the growth of non-fused myeloma cells (since they lack HGPRT) and non-fused B cells (which are short-lived). Only the hybridoma cells, which can utilize the hypoxanthine and thymidine pathways due to the presence of HGPRT from the B cell, will survive and proliferate. The surviving hybridomas are then screened to identify those that produce the desired antibody.

17. Principle for creation of hybridoma cells The myeloma cells used in hybridoma technology must not be capable of synthesizing their own antibodies. The selection of hybridoma cells is based on inhibiting the nucleotide (consequently the DNA) synthesizing machinery. The mammalian cells can synthesize nucleotides by two pathways—de novo synthesis and salvage pathway

18. The de novo synthesis of nucleotides requires tetrahydrofolate which is formed from dihydrofolate. The formation of tetrahydrofolate (and therefore nucleotides) can be blocked by the inhibitor aminopterin . The salvage pathway involves the direct conversion of purines and pyrimidine’s into the corresponding nucleotides. Hypoxanthine guanine phosphoribosyl transferase (HGPRT) is a key enzyme in the salvage pathway of purines.

19. When cells deficient (mutated cells) in HGPRT are grown in a medium containing hypoxanthine aminopterin and Thymidine (HAT medium), they cannot survive due to inhibition of de novo synthesis of purine nucleotides (Note : Salvage pathway is not operative due to lack of HGPRT). Thus, cells lacking HGPRT, grown in HAT medium die. The hybridoma cells possess the ability of myeloma cells to grow in vitro with a functional HGPRT gene obtained from lymphocytes (with which myeloma cells are fused). Thus, only the hybridoma cells can proliferate in HAT medium, and this procedure is successfully used for their selection.

20. Antibody Production and Purification Once a hybridoma producing the specific antibody is identified, it is cloned and grown in large quantities. The monoclonal antibodies ( mAbs ) secreted by these cells are then purified from the culture medium.

21. Therapeutic Monoclonal Antibodies

22. Genetic Modification GM is a technology that involves inserting DNA into the genome of an organism. To produce a GM plant, new DNA is transferred into plant cells. Usually, the cells are then grown in tissue culture where they develop into plants. The seeds produced by these plants will inherit the new DNA.

23. First Genetically Modified Bacteria The first genetically modified bacteria were developed in 1973 by biochemists Herbert Boyer and Stanley Cohen . They successfully transferred a kanamycin-resistant gene from one bacterium to another, creating the first example of recombinant DNA technology and a genetically modified organism. They achieved this by inserting a gene for antibiotic resistance into a plasmid, which was then introduced into E. coli bacteria, enabling the bacteria to express the new trait.

24. The first genetically modified animal, a mouse, was created in 1974 by Rudolf Jaenisch. The first genetically modified plant was an antibiotic-resistant tobacco plant, produced in 1982. This plant was created by inserting a gene that confers resistance to antibiotics into tobacco cells. China was the first country to commercialize a transgenic plant, introducing a virus-resistant tobacco plant in 1992.

25. A genetically modified organism (GMO) is an animal, plant, or microbe whose DNA has been altered using genetic engineering techniques. For thousands of years, humans have used breeding methods to modify organisms. Corn, cattle, and even dogs have been selectively bred over generations to have certain desired traits. Within the last few decades, however, modern advances in biotechnology have allowed scientists to directly modify the DNA of microorganisms, crops, and animals.

26. Conventional methods of modifying plants and animals—selective breeding and crossbreeding—can take a long time. Moreover, selective breeding and crossbreeding often produce mixed results, with unwanted traits appearing alongside desired characteristics. The specific targeted modification of DNA using biotechnology has allowed scientists to avoid this problem and improve the genetic makeup of an organism without unwanted characteristics tagging along.

27. Transgenic Animals A transgenic animal is one whose genome has been changed to carry genes from another species or to use techniques for animal genome editing for specific traits. Animal features can be changed by purposefully altering the gene (or genes). A mouse was the first successful transgenic animal. Then pigs, sheep, cattle, and rabbits came a after few years.

28. The foreign-interested genes prepared using a variety of methods like vectors, including yeast artificial chromosomes, bacterial plasmids, and cosmids. Several techniques, including heat shock, electroporation, viruses, the gene gun, microinjection, and liposomes, are used to deliver the created vector, which includes the interesting gene, into the host cell. Transgenesis can be carried out in the gonads, sperm, fertilized eggs, and embryos through DNA microinjection, retroviruses, stem cells, and cloning.

29. Method A cloned gene is injected into the nucleus of a fertilized egg. Fertilized eggs are implanted into a receptive female. Some of the offspring carry the cloned gene in all of their cells. Animals with the cloned gene cells are bred to establish new genetic lines.

30. Most animals that are GMOs are produced for use in laboratory research. These animals are used as “models” to study the function of specific genes and, typically, how the genes relate to health and disease. Some GMO animals, however, are produced for human consumption. Salmon, for example, has been genetically engineered to mature faster, and the U.S. Food and Drug Administration has stated that these fish are safe to eat.

31. Genetically engineered mice have induced mutations, including transgenes, targeted mutations (knockouts or knockins ), and retroviral, proviral or chemically induced mutations. Genetically engineered mice are useful for elucidating basic biological processes, studying relationships between gene mutations and disease phenotypes, and modeling human disease.

32. Enviropig™ is genetically engineered to produce the enzyme phytase in its salivary glands to enable more effective digestion of phytate, the from of phosphorus found in pig feed ingredients like corn and soybeans.

33. In vitro fertilization In vitro fertilization (IVF) is a type of assisted reproductive technology (ART) where sperm and an egg are fertilized outside of the human body. IVF is a complex process that involves retrieving eggs from ovaries and manually combining them with sperm in a lab for fertilization. Several days after fertilization, the fertilized egg (now called an embryo) is placed inside a uterus. Pregnancy occurs when this embryo implants itself into the uterine wall.

34. Ovarian Stimulation: The woman takes medication to stimulate her ovaries to produce multiple eggs. Egg Retrieval: Once the eggs are mature, they are retrieved from the ovaries, usually through a minor surgical procedure. Sperm Collection: A sperm sample is collected from the male partner or a donor. Fertilization: The eggs are fertilized with sperm in a laboratory dish. Embryo Culture: The fertilized eggs (embryos) are grown in the lab for several days to allow them to develop. Embryo Transfer: One or more embryos are transferred to the woman's uterus, where they may implant and result in pregnancy.

35. Cloning Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. The copied material, which has the same genetic makeup as the original, is referred to as a clone. The most famous clone was a Scottish sheep named Dolly. There are three different types of cloning: Gene cloning, which creates copies of genes or segments of DNA Reproductive cloning, which creates copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body.

36. Cloning of Dolly These experiments were carried out at The Roslin Institute by a team led by Professor Sir Ian Wilmut . Because of the nature of the research, the team was made up of many different people, including scientists, embryologists, surgeons, vets and farm staff. Dolly was cloned from a cell taken from the mammary gland of a six-year-old Finn Dorset sheep and an egg cell taken from a Scottish Blackface sheep. She was born to her Scottish Blackface surrogate mother on 5th July 1996. Dolly’s white face was one of the first signs that she was a clone because if she was genetically related to her surrogate mother, she would have had a black face.

37. Stem cells Stem Cells are defined as precursor cells that can self-renew and to generate multiple mature cell types . Self-renewal refers to the ability of a stem cell to divide and make identical copies of itself to assure that the stem cell population is not depleted. Stem cells may be slow to divide and are mostly thought to be quiescent in the body, preserving their potential until needed. Differentiation is the process whereby stem cells transform into more specialized cell types and can perform new functions through the expression of new genes, mRNA, and proteins.

38. Types of Stem Cells Embryonic stem cells Adult stem cells Induced Pluripotent stem cells

39. Embryonic Stem cells Around 3–5 days after a sperm fertilizes an egg, the embryo takes the form of a blastocyst or ball of cells. The blastocyst contains stem cells and will later implant in the womb. Embryonic stem cells come from a blastocyst that is 4–5 days old. The embryo (blastocyst), contains an outer cell mass that become part of placenta and an inner cell mass that is capable of generating all the specialized tissues that develop into the human body. ESCs are derived from the inner cell mass of an embryo that has been fertilized in vitro .

40. Adult Stem cell A body contains stem cells throughout their life. The body can use these stem cells whenever it needs them. ASCs are undifferentiated, multipotent cells found in living differentiated tissues in our bodies that can renew themselves or generate new cells that can replace dead or damaged tissue. It is also called tissue specific or somatic stem cells, adult stem cells exist throughout the body from the time an embryo develops. ASCs are present in different tissue such as the brain, bone marrow, blood and blood vessels, umbilical cord, placenta, skeletal muscles, skin, the liver, fat tissue etc. ASCs generate new cells to replace those that are lost through normal repair, disease, or injury

41. Types of ASC Hematopoietic Stem Cells (Blood Stem Cells) Mesenchymal Stem Cells. Neural Stem Cells. Epithelial Stem Cells. Skin Stem Cells.

42. Induced pluripotent stem cells (iPSCs) Induced pluripotent stem cells (iPSCs) are adult cells that have been reprogrammed to an embryonic stem cell-like state, capable of differentiating into various cell types. iPSCs are created by taking adult cells (like skin or blood cells) and introducing a specific set of genes (like Oct4, Sox2, Klf4, and c- Myc ) that are normally active in embryonic stem cells. A key advantage of iPSCs is that their creation doesn't require the use of human embryos, addressing ethical concerns associated with embryonic stem cells.

43. Techniques to produce Stem cells

44. Stem Cells from Cord Blood and Amniotic Fluid Cord blood and amniotic fluid contain several different types of stem cells, with the predominant cell type being HSC. The various cell types have different potencies, ranging from ESC-like cells to multipotent stem cells. Cord blood (and placental blood) has several benefits for clinical therapy. The tissue source is freely and widely available, with no extra risk to the donor and no ethical issues. In contrast, amniotic fluid is captured by amniocentesis, so there is a small chance of harm to the fetus.

45. Cord blood contains a large number of stem cells; therefore, substantial numbers of cells can be easily obtained without many passages. This has three advantages: first, the decreased time in culture and number of cell divisions reduces the potential for chromosomal changes; second, cord blood stem cells, although they can give rise to all three germ layers, do not seem to be tumorigenic; third, the ability to rapidly generate large numbers of cells affords the use of high numbers of cells in therapies, which may increase efficacy and reduce the waiting time for treatment.

46. Cancer stem cells (CSCs) In 1997, it was hypothesized that any cancer tissue may contain a small fraction of cancer stem cells (Bonnet and Dick 1997). Cancer stem cells (CSCs) are a rare population of cancer cells within a tumor that possess characteristics similar to normal stem cells, specifically the ability to self-renew and differentiate into various cell types found in that tumor. This allows them to drive tumor growth, metastasis, and recurrence, and also contributes to resistance to conventional therapies.

47. Stem Cells in Plants In plants, stem cells are found primarily at the meristems, the areas where new growth takes place. The apical meristems are at the tips of the shoots and roots, where the majority of growth takes place. Stem cells are also present in lateral (procambium) and intercalary meristems. A plant callus is a mass of undifferentiated cells, derived from previously differentiated plant cells, that have the characteristics of stem cells and are thus able to give rise to all plant tissue types.

48. Tissue Engineering Despite recent technological advances, thousands die each year while waiting for organ transplants due to : lack of organ donors or efficient organ substitutes So, this lack of donor organs has caused many to consider “tissue engineering” methods or “regenerative medicine” as means to replace diseased or damaged organs

49. Tissue Engineering Is an emerging technology where artificial organs and tissues are constructed in vitro and transplanted in vivo for the recovery of lost or malfunctioned organs or tissues. It is the use of a combination of cells, engineering methods and materials, and suitable biochemical and physico -chemical factors to improve or replace biological functions.

50. Tissue engineering refers to the practice of combining scaffolds, cells, and biologically active molecules into functional tissues. The goal of tissue engineering is to assemble functional constructs that restore, maintain, or improve damaged tissues or whole organs. Artificial skin and cartilage are examples of engineered tissues that the FDA has approved, however, currently they have limited use in human patients.

51. The triad of tissue engineering. The combination of cells, scaffolds, and signals is used to engineer functional tissues. Basic Principle of Tissue Engineering

52. Different tissue-engineered organs. Scaffold prepared from synthetic biodegradable polyglycolic acid (PLA) in the shape of a 3-year-old auricle. Scaffolds implanted subcutaneously on the back of an immunodeficient mouse. First trachea organ transplant using human's bone marrow stem cells. Constructed artificial bladder seeded with human bladder cells and dipped in a growth solution. Bioengineered kidney that mimics the function of a normal kidney concerning the control of the urinary system and blood filtration. Tissue-engineered heart valve using human marrow stromal cells.

53. Sources of Cells Autologous cells are obtained from the same individual to which they will be reimplanted. Autologous cells have the fewest problems with rejection and pathogen transmission, however in some cases might not be available. Allogeneic cells come from the body of a donor of the same species. While there are some ethical constraints to the use of human cells for in vitro studies, the employment of dermal fibroblasts from human foreskin has been demonstrated to be immunologically safe and thus a viable choice for tissue engineering of skin. Xenogenic cells are these isolated from individuals of another species. In particular animal cells have been used quite extensively in experiments aimed at the construction of cardiovascular implants.

54. Isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models. Stem cells are undifferentiated cells with the ability to divide in culture and give rise to different forms of specialized cells. According to their source stem cells are divided multipotent, pluripotent& totipotent

55. SCAFFOLDS Cells are often implanted or 'seeded' into an artificial structure capable of supporting three-dimensional tissue formation. These structures, typically called scaffolds

56. Scaffolds usually serve at least one of the following purposes: Allow cell attachment and migration Deliver and retain cells and biochemical factors Enable diffusion of vital cell nutrients and expressed products Exert certain mechanical and biological influences to modify the behaviour of the cell phase

57. To achieve the goal of tissue reconstruction, scaffolds must meet some specific requirements. A high porosity and an adequate pore size are necessary to facilitate cell seeding and diffusion throughout the whole structure of both cells and nutrients. Biodegradability is often an essential factor since scaffolds should preferably be absorbed by the surrounding tissues without the necessity of a surgical removal

58. Scaffolds may also be constructed from natural materials: in particular different derivatives of the extracellular matrix have been studied to evaluate their ability to support cell growth. Protein materials, such as collagen or fibrin, and polysaccharidic materials, like chitosan or glycosaminoglycans (GAGs), have all proved suitable in terms of cell compatibility, but some issues with potential immunogenicity remains. Functionalized groups of scaffolds may be useful in the delivery of small molecules (drugs) to specific tissues

59. Examples of Scaffolds in Tissue Engineering: Collagen Chitosan Hyaluronic Acid (HA) Fibrin Alginate Silk Fibroin

60. Synthetic Polymer Poly(lactic acid) (PLA) Polyglycolic Acid (PGA) Poly(lactic-co-glycolic acid) (PLGA) Polycaprolactone (PCL) Poly(vinyl alcohol) (PVA)

61. Other Forms Hydrogels Nanofibers Microspheres Composite Scaffolds

62. Methods used for synthesis of tissue engineered scaffolds Nanofiber Self-Assembly Textile technologies Solvent Casting & Particulate Leaching (SCPL) Gas Foaming Emulsification/Freeze-drying Thermally Induced Phase Separation (TIPS) ElectroSpinning CAD/CAM Technologies Laser-assisted BioPrinting ( LaBP )

63. Growth Factors In tissue engineering, growth factors are crucial for promoting cell growth, differentiation, and tissue regeneration. Commonly used growth factors include platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-β), bone morphogenetic proteins (BMPs), and insulin-like growth factors (IGFs). These factors are often incorporated into scaffolds or delivered via specialized systems to enhance their therapeutic effects.

64. Why is it necessary? Congenital abnormalities require tissue reconstruction. Most tissues cannot regenerate following a disease or injury. Even tissues that regenerate spontaneously (e.g. skin, bone …) may not completely do so. The scarcity of donor tissue limits transplantation. Permanent implants have a lot of success, but also many problems

65. Tissue culture It includes the creation of functional tissues and biological structures in vitro It requires extensive culturing to promote survival, growth and inducement of functionality The major problem is maintaining culture conditions

66. Thank You