ABR Animal Biotechnology TRANSGENICS o o o aka Recombinant DNA transfer of a specific gene from one organism to another changing

the organisms protein makeup

Creates a possible source of replacement organs for humans

XENOTRANSPLANTATION Process of transplanting cells, tissues, or organs from one species to another Pigs major animal

p21 a gene that inhibits regeneration ability Main Goals of Animal Biotech More nutritious livestock Increasing growth and development of agriculturally important animals Antibody and vaccine production Can engineer animals for many purposes Gene Therapy o o Treatment or cure for disorders caused by mutated genes Involves adding a normally functioning copy of the gene(s) to enough affected cells to restore normal function

Gene Splicing used to introduce one or more genes of an organism into a second organism ***vector an organism that can carry the donor DNA into the host Creating a Transgenic Animal Gene of choice is manipulated and prepared in the laboratory Transgene is injected into the animal Egg is implanted into a surrogate

CLONING o o to produce multiple copies of mammals cattle, sheep, pigs, goats, horses, mules, cats, rats, and mice

Types: Germline Gene Therapy

Dolly sheep; first cloned animal; 1996 o Reproductive cloning begins with somatic cell nuclear transfer Scientists remove the nucleus from an egg cell and replace it with a nucleus from a donor adult somatic cell The embryo is implanted into a uterus of a surrogate female, where it can develop into a live being. Permanent transfer of a gene into sperm or egg cells

Somatic Cell Gene Therapy Transfer of genes into affected cells

Genetic Disorders o o Medical problems caused by a mutation in 1 or more genes in the chromosomes Inborn or developed

GENE KNOCK OUT TECHNOLOGY

Categories of Genetic Disorders

Single Gene Changes A mutation in one gene can result in a change in the protein product or possible the elimination of the entire protein Example: Sickle Cell Anemia

The gene must be turned on at the right time Gene Delivery Methods Ex vivo cells are modified outside the body and then transplanted back in again In vivo genes are transferred to cells inside the body September 4, 1990 first gene therapy was performed (Ashanti DeSilva was treated for SCID Severe Combined Immunodeficiency) Other Examples of Gene Therapy Cystic fibrosis caused by defective ion transport molecule (CFTR cystic fibrosis transmembrane conductance regulator) in the plasma membrane of the cell ***Virotherapy viruses are engineered to infect and kill tumor cells, leaving normal cells intact Stem Cell Therapy undifferentiated biological cells that can differentiate into specialized cells

Multigene Disorders Aka multifactor disorders Result from mutations in more than one gene, and sometimes along with environmental influences Example: heart disease, diabetes, cancer, obesity

Mitochondrial Disorders Caused by mutations in the mitochondrial DNA Example: Leber hereditary optic neuropathy, Leigh syndrome

Chromosomal Disorders Complete chromosomes or large regions of a chromosome are missing, duplicated, or modified Example: Down syndrome, Klinefelters syndrome

Factors for Gene Therapy Only a single gene causing the disorder can be a candidate The normal and mutated genes must be well-identified and studied The disease must be well-understood An approved method for gene delivery must be available The gene must be delivered to the correct cell

Stem Cell Types Embryonic o Most valuable; any type of cell Fetal

Natural source of dopamine neurons; can treat Parkinsons disease Umbilical cord blood o Multipotent stem cells; naturally become blood cells and immune cell types Adult o Multipotent cells that develop into cells of a specific type of tissue; less flexible Gene Transfer Embryo A multicellular diploid eukaryote in its earliest stage of development; 8th wk of fertilization Fetus Intra uterine baby; 9th wk of fertilization Germ Layers: Ectoderm give rise to neural tissue and epidermis Mesoderm develops into somites which differentiate into skeletal and muscle tissues, the notochord, blood vessels, dermis, and connective tissues Endoderm gives rise to the epithelium of digestive and respiratory systems GENE TRANSFER METHODS Microinjection

Steps: 1. Identification of foreign interest 2. Insertion of the foreign gene into an appropriate vector 3. Microinjection of DNA directly into the male pronucleus of a single fertilized egg 4. Implantation of the microinjected egg into surrogate mother 5. Allowing the embryo to develop to birth 6. Demonstrating that the foreign gene has been stably incorporated into the host organism 7. Demonstrating that the gene is expressed and regulated correctly in the host organism Embryonic Stem Cell Gene Transfer Prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem cells

Steps: 1. ES cells are removed from the embryo 2. The gene of interest is inserted into the embryo (transfection). 3. The gene is transferred by homologous recombination and selective markers. 4. The ES cells with the correct gene are injected into partially developed embryos called blastocysts which are implanted into the mother animal. 5. The offspring are screened for the gene of interest. Gene Targeting in Mice

Injection of the gene of interest into the fertilized egg of a donor animal

Insertion of DNA into a specific chromosomal location

Knockout Mouse Had both alleles of a particular gene replaced with an inactive allele; normal gene is missing or engineered

Knockin Mouse Transgenic mouse

Genomics Genetic Linkage Maps Depicts the relative positions of known genes or genetic markers Show the order and distance between pairs of linked genes Determine the arrangement of genes

Generation of high-resolution genetic and physical maps Establishment of DNA sequence landmarks Identification of all genes in the genome using bioinformatics and comparative genomics Establishment of polymorphism databases that will aid in disease diagnostics 1994 first high-resolution map for the human genome was established 2001 public human genome draft was published 2003 99.9% of the gene-containing portion of the human genome was sequenced to 99.9% accuracy Organ Transplantation Autografts organs/tissues that are transplanted within the same persons body Allografts transplants that are recently performed between 2 subjects of the same species Isografts subset of allografts in which organs/tissues are transplanted from a donor to a genetically identical recipient Xenograft transplant of organs/tissues from one species to another

Cytological Maps Help to align the genetic linkage map with the more specific physical map Banding pattern of a chromosome through karyotyping Used to diagnose human genetic diseases

Physical Maps Assembly of contiguous stretches of DNA called contigs where the distances between landmark DNA sequences are shown in kilobases

Human Genome Contains about 30000-40000 genes Largest chromosome Chrom 1 (2968 genes) Smalles chromosome Y Chrom (231 genes) Began in Oct 1990 Goals

Antigen substance that binds specifically to a respective body; ANTIbody GENerator Antibody aka immunoglobin; used by the immune system to identify and neutralize foreign objects

Types of Rejection Hyperacute rejection after few minutes after the transplant Acute rejection 1 week to 3 months after the transplant Chronic rejection many years Plant Biotechnology Plant Tissue Culture o Sterile in vitro cultivation of plant parts

through differentiation and dedifferentiation Types of Culture: Callus culture differentiated tissue from an explant that differentiates Cell culture cells or cell aggregates in liquid medium Protoplast culture plant cells with their cell walls removed Embryo culture isolated embryos Seed culture - seeds to generate plants Organ culture isolated plant organs Micropropagation In vitro clonal propagation Provides the ability to produce thousands of genetically identical plants by asexual reproduction

1902 initiated first experiments 1939 first true plant tissue culture; carrot BENEFITS: Promote the mass propagation of desirable plants through in vitro propagation Specific characteristics can be selected Production of virus-free plants and plant products

Stages: 1. 2. 3. 4. Initiation of sterile explant culture Shoot initiation Root initiation Transfer of plants to sterile soil/other substrates under controlled conditions A. Somatic Embryos Embryoids from cultured somatic plant tissues; embryo-like structures Somatic embryogenesis induces cultured somatic or nonsex cells to form embryoids from vegetative tissues Plantlets mini plants Pectinase digest cell walls Cellulase yield protoplasts ***liquid propagation more preferred method

STEPS: 1. A piece of tissue/organ is removed from the plant. (explant) 2. It is grown on a specific nutrient medium in the presence of specific ratios of growth hormones. 3. The nondividing cell revert to an undifferentiated, meristematic state whereby they form callus tissue. (dedifferentiation) a. Dedifferentiation ability of callus tissue to differentiate into a whole plant/plant organ b. Totipotency ability of a plant cell to give rise to a whole plant

B. Chemicals from Plants More complex arrays of secondary metabolites at least 50000 Plant products flavouring, coloring, fragrances, pharmaceuticals Shikonin red pigment found in the purple roots of a perennial herb, Lithospermum erythrorhizon; used as creams in cosmetics

First application Transplantation of human insulinproducing porcine islet cells from knockout pigs for diabetes treatment ***HEART AND KIDNEY - first transplants Birds

Other Uses of Tissue Culture Transgenic Animals Benefits: Mice o CFTR cystic fibrosis transmembrane regulator; chloride channel; allows chloride ions to move in and out of cells Increase production Provide healthier, more rebust animals Provide healthier foods Produce disease-resistant animals Increase wool quality and quantity 1. Protoplast Fusion - protoplasts can be transformed with foreign genes using microinjection and electroporation

2. Somoclonal Variation - the genetic variability produced by plant tissue culture - variability can be exploited to improve characteristics obtained in different crop species

Cow Pigs Examples: Sheep *corn- herbicide resistance Goats *wheat- gain color, height *barley- grain yield Xenotransplantation *soybean- height, maturity Use of animal organs in human patients Baby Fae survived 20 days with a baboon heart in 1984 Pigs - animal of choice *carrot- higher carotene content TRANSGENE - a gene that has been introduced into the genome of a eukaryotic cell that originated from another source

PRONUCLEAR MICROINJECTION method by which one of the nuclei in a fertilized egg is injected with DNA prior to fusion of gamete nuclei CALLUS mass of undifferentiated cells CHIMERA recombinant DNA molecule/organism that contains sequences from more than one organism EMBRYO SPLITTING twinning/cloning ARTIFICIAL INSEMINATION allows a diluted sperm from one bull to inseminate 500 to 1000 females VACCINE promote an antibody response that protects the organism against infection PG gene instructions for making proteins called proteoglycans PECTIN major component of primary cell walls