16-Transgenic Animals.ppt

Transgenic Animals 1 Learning objective • Define transgenesis. • Learn about the elements/requirements for creating a transgenic animal. • Importance/applications of transgenesis • Transgenic animal vs cloned animal • Learn about transgenic plants 2 * History • The term ‘transgenic animal’ was...

Transgenic Animals 1 Learning objective • Define transgenesis. • Learn about the elements/requirements for creating a transgenic animal. • Importance/applications of transgenesis • Transgenic animal vs cloned animal • Learn about transgenic plants 2 * History • The term ‘transgenic animal’ was coined in 1981 by Gordon and Ruddle to describe an animal in which an exogenous gene was introduced into its genome. • In the late 1980s, the term transgenic was extended to gene-targeting experimentation and the production of ‘knockout’ mice in which a gene (or genes) has been selectively removed from the host genome. • Today, a transgenic animal can be defined as one having any specific, targeted genetic modification. • Genetic material of an animal can be manipulated so that extra genes may be inserted (transgenes), replaced (i.e. human gene homologs coding for related human proteins), or deleted (knock-out). 3 * Elements of Transgenic animal Technology: 1) Recombinant DNA: Using recombinant DNA technology, the exogenous DNA is built to have: • The desired gene (restriction enzymes, Polymerase Chain Reaction (PCR), DNA cloning). • DNA vector to enable the molecule to be inserted into the host DNA. • Promoter and stop codons to enable the gene to be expressed in the host cells. 4 2) Microinjection: - The approach requires the following: 1) ‘egg holding’ pipette with a ~60–70 μm tip diameter 2) negative pressure is used to hold the embryo (fertilized egg). 3) a fine glass microinjection pipette (1 mm tip diameter) is used to pierce the embryo and subsequently deliver a picoliter volume of a gene. * A viral vector can be used for gene transformation. 5 3) Cell type and animal cell Culture: • Fertilized eggs: In vitro manipulation of fertilized eggs requires a culture system that supports physiological and metabolic processes and ensures a high level of viability for embryos ex utero. The establishment of proper ova culture media is currently available due to the considerable improvement in culture conditions and media formulations. 6 • Embryonic stem cells (Blastocysts) are derived from early preimplantation embryos and are maintained in culture for a sufficient period to perform various in vitro manipulations by introducing foreign DNA using techniques such as, microinjection, retroviral insertion, precipitation reactions, transfection or electroporation. • Hematopoietic bone marrow stem cells (Adults). 7 * Creation of Transgenic Animals Implementation of the Elements of Transgenic animal Technologies: 1) DNA microinjection: - DNA microinjection generally involves the use of micromanipulators and a microinjection apparatus to inject a solution containing recombinant DNA into embryos. - Microinjected gene constructs generally integrate randomly into the genome of the embryo, but typically only in a single chromosomal location. - Mosaic animal ? 9 • Advantages of DNA microinjection method: 1) a high frequency of generating transgenic animals. 2) a lack of constraint on the size or type of DNA constructs used. 3) the stability of the transgene as it is transmitted from generation to generation. 10 • Disadvantages of DNA microinjection method: 1) Positional effect: the levels of expression of the same transgene can vary dramatically between individual founder animals and the resulting transgenic lines. 2) The potential for undesired insertional mutagenesis. 3) the time and expense required developing the necessary micromanipulation and microinjection skill sets. 11 * Notice: • The animals produced using DNA microinjection must be thoroughly tested to determine: - Whether all the animal cells contain the transgene. - Whether the desired cell types are producing the gene’s protein product. 13 2) Retrovirus-mediated gene transfer: * High rates of gene transfer, approaching 100% efficiency, are achieved with the use of retroviruses. 14 • Limitation of Retrovirus-mediated gene transfer: 1) a general limitation on the size of the foreign DNA insert. 2) a potential for undesired genetic recombination with other viruses. 3) the additional steps required to produce retroviruses (preparation difficulties). 15 Clinical Application of Transgenic animals: 1- Lab Models for human disease Transgenic models of human disease are used extensively to assess the validity of therapeutic applications before clinical trials. Many of the transgenic mouse models created to study the pathogenesis of Alzheimer’s disease have been used to investigate therapeutic strategies. 16 2- Production of pharmaceuticals in transgenic animals: The production of therapeutic proteins from transgenic animals usually involves their expression from mammary-gland specific promoters to drive secretion of the transgene into milk and also in eggs of the transgenic chicken using egg-whitespecific gene promoters. 17 • U.S. Food and Drug Administration (FDA) approved ATryn for the prevention of perioperative and peri-partum thromboembolic events in hereditary antithrombin deficient patients. ATryn is the first ever transgenically produced therapeutic protein and the first recombinant antithrombin approved in the U.S. 18 19 3- Xenotransplantation Xenotransplantation (xenos- from the Greek meaning "foreign"), is the transplantation of living cells, tissues or organs from one species to another. Such cells, tissues or organs are called xenografts or xenotransplants. 20 * Immunological barriers: • To date no xenotransplantation trials have been entirely successful due to the many obstacles arising from the response of the recipient’s immune system. Ultimately results in rejection of the xenograft, and can in some cases result in the immediate death of the recipient. 21 • How to overcome the immunological barriers: 1) Knock out engineered pigs (Slide 23). 2) Transgenic pigs which have been developed with the gene that codes for human cell-surface protein(s). As a result , these pigs have organ with “human cell label” proteins, which prevents the component of the human immune system from attacking and destroying the transplanted organ. 22 - The transplantation of pig organs to humans (xenotransplantation) is now receiving serious consideration because of the shortage of human donors for organ transplants of kidney, liver and heart, and of islet cell transplantation for diabetes. The problem with such xenografts would be hyperacute rejection-mediated by natural antibodies in humans to pig antigens. 23 - Pig alpha 1,3-galactosyl transferase knock-out pig (lacking the alpha 1,3-galactosyl transferase) could serve as donor for human transplantation. Cloned Vs Transgenic animals 24 • The technique of nuclear transfer, the replacement of the nuclear genomic DNA of an oocyte (immature egg) or a single-cell fertilized embryo with that from a donor cell is such an alternative breeding methodology. Animal “cloning” can result from this nuclear transfer technology. The cloned sheep Dolly is NOT a transgenic animal because she does not possess a transgene. 25 Animal Cloning 26 Transgenic Plants (Genetically Modified Crops) 27 • A genetically engineered plants is generated in a laboratory by altering its genetic makeup. This is usually done by adding one or more genes to a plant's genome. • Most genetically modified plants are generated by: 1) the biolistic method (particle gun) or 2) Agrobacterium tumefaciens bacteria. In research tobacco and Arabidopsis thaliana are the most genetically modified plants and used as test models, due to well developed transformation methods, easy propagation and well studied genomes 28 • In the biolistic method, DNA is bound to tiny particles of gold or tungsten which are subsequently shot into plant tissue or single plant cells under high pressure. The accelerated particles penetrate both the cell wall and membranes. The DNA separates from the metal and is integrated into plant genome inside the nucleus. 29 • Agrobacteria are natural plant parasites and it is used as a vector, enabling transportation of foreign genes into plants. 30 Genetically modify plants can posses one or more of the following traits : – produce vaccines in their fruit (e.g. polio vaccine) – be resistant to disease and pests – require less fertilizer, pesticides and herbicides – have a higher nutritional value (NEXT slide) 31 * Transgenic (GM) Rice Genetically modified rice to produce betacarotene 32 Learning outcome • Transgenesis is an application in biotechnology where recombinant DNA technology is utilized to generate transgenic animals for medical and pharmaceutical use. • Cloned animal is a replica of the parent animal but transgenic animal is an animal bearing a foreign gene. • Transgenic plants are generated to improve plant properties and characteristics to solve problems related to agriculture and food production. 33