Chapter 21: Transgenic Organisms and Organismal Cloning PDF

Because learning changes everything. ® Chapter 21 Lecture 24 Transgenic organisms and organismal cloning © McGraw Hill ©Michael Goldberg, Cornell University, Ithaca, NY 1  What is a transgenic organism? Transgenic organis...

Because learning changes everything. ® Chapter 21 Lecture 24 Transgenic organisms and organismal cloning © McGraw Hill ©Michael Goldberg, Cornell University, Ithaca, NY 1  What is a transgenic organism? Transgenic organism— Contains a transgene from another individual of the same species or a different species For transgene to be propagated between generations in a multicellular organism transgene must be in cells that become gametes Transgene— Can be made in vitro using recombinant DNA technology Introduced into eukaryotic cells by chemical treatment (single cell organisms) or injection (multicellular organisms) © McGraw Hill 2  Injecting transgenes © McGraw Hill a: ©Martin Oeggerli/Science Source; b: ©Solvin Zankl 3  Procedure involved in pronuclear injection Fertilized eggs harvested from female mouse. Transgene is injected into one of the pronuclei of the fertilized egg. 25-50% of the time the injected transgene integrates into the mouse genome in a random location. Access the text alternative for slide images. © McGraw Hill 4  Injected embryos are implanted into a pseudo-pregnant female mouse Mice that were injected as embryos are born If the transgene integrated after cell division, the transgene will be in some cells and not others (mosaic). Mice are bred to produce stable transgenic lines. © McGraw Hill 5  Uses of transgenic organisms Transgenes can clarify which gene causes a mutant phenotype Transgene reporter constructs can tell us where and when a gene is actively transcribed Transgenic organisms serve to produce proteins needed for human health. Use of transgenic animals to model gain-of-function genetic diseases in humans. © McGraw Hill 6  Uses of transgenic organisms Transgenes can clarify which gene causes a mutant phenotype Transgene reporter constructs can tell us where and when a gene is actively transcribed Transgenic organisms serve to produce proteins needed for human health. Use of transgenic animals to model gain-of-function genetic diseases in humans. © McGraw Hill 7  Connecting genes with phenotypes can be difficult For example, a mutation that deletes two genes results in malformed Drosophila eyes. Which of the two genes is involved in eye development? Access the text alternative for slide images. © McGraw Hill a: ©Janice Fischer, The University of Texas at Austin 8  Transgenes can connect genes with phenotypes To determine which gene is involved in eye development: Construct two transgenes, one with gene A and one with gene B Determine which gene restores the wild type phenotype Access the text alternative for slide images. © McGraw Hill 9  Uses of transgenic organisms Transgenes can clarify which gene causes a mutant phenotype Transgene reporter constructs can tell us where and when a gene is actively transcribed Transgenic organisms serve to produce proteins needed for human health. Use of transgenic animals to model gain-of-function genetic diseases in humans. © McGraw Hill 10  Fluorescent tags can be fused to genes to determine where proteins act in the cell Chalfie M., GFP: lighting up life, PNAS (2009) © McGraw Hill  Multiple fluorescent tags can be fused to different genes to visualize proteins simultaneously Your favorite gene (YFG) fused to GFP gene Protein produced with GFP tagged to YFG YFG GFP Glofish: transgenic fish expressing various fluorescent proteins (commercially GFP and RFP variants available as pets) © McGraw Hill  Fluorescent tags can be fused to genes to determine where proteins act in the cell Single pair of motor neurons in the fly nerve cord Johnson et al, Nat Comm (2021) Fly brainbow Hampel et al. Nat Methods (2011) © McGraw Hill  Uses of transgenic organisms Transgenes can clarify which gene causes a mutant phenotype Transgene reporter constructs can tell us where and when a gene is actively transcribed Transgenic organisms serve to produce proteins needed for human health. Use of transgenic animals to model gain-of-function genetic diseases in humans. © McGraw Hill 14  Pharming: Use of transgenic animals & plants to produce human protein drugs Transgenic mammals produced by pronuclear injection. Expression directed to mammary glands and gene product is secreted in milk. Identical clones of high producing animals can be made using reproductive cloning. Access the text alternative for slide images. © McGraw Hill 15  Drugs produced by pharming Atryn®️ - Blood factor antithrombin II Produced in goats, used to treat clotting Ruconest®️ - C1 esterase inhibitor protein Produced in rabbits, used to treat hereditary angioedema (hereditary disorder that causes severe swelling, usually in the limbs, face, airway, and intestines) © McGraw Hill 16 Organismal cloning Transgenic animals used for pharming have variable production rates. Thus, high producing animals are maintained by organismal cloning. Dolly the sheep was the first animal to be cloned from an adult somatic cell in 1996 Organismal cloning is also being used to save endangered species Also, used for recreational uses- cloning pets © McGraw Hill  Tools of Genetics: Somatic cell nuclear transfer Somatic cell nuclear transfer used to create reproductive clones Diploid nucleus of a somatic cell from one individual inserted into an egg cell whose nucleus has been removed Access the text alternative for slide images. © McGraw Hill 18  Genetically modified crops are widely used More than 100 different transgenic plant species have been created Roundup®️ Ready soybeans are herbicide-resistant, >90% of US soybeans are Roundup ®️ Ready. Corn expressing Bt protein protects the plant from corn- borer moth caterpillars. 10 billion acres are used to grow Bt crops. In 2015, the first GM animal - GM salmon that express growth hormone - was approved for human consumption GM crops are important to limit environmental problems caused by large- scale agriculture and to meet food requirements of world population © McGraw Hill 19 Golden Rice Severe Vitamin A deficiencies can cause blindness and death and are prevalent in many developing countries Rice is the primary food source in many of these countries, but do not normally contain Vitamin A Genetically modified rice to produce Vitamin A © McGraw Hill  Concerns about GM organisms A few large agricultural companies have all the power. Farming communities may be disrupted. Potential environmental consequences, such as transfer of transgenes to wild organisms. © McGraw Hill 21  Uses of transgenic organisms Transgenes can clarify which gene causes a mutant phenotype Transgene reporter constructs can tell us where and when a gene is actively transcribed Transgenic organisms serve to produce proteins needed for human health. Use of transgenic animals to model gain-of-function genetic diseases in humans. © McGraw Hill 22  Transgenic models of human gain-of-function genetic disease Generate an animal with a mutation that corresponds to a human disease allele and a similar disease phenotype. Transgene added to normal organisms, so only dominant mutations can be modeled. Mice are common model organism. Primates may be better models for complex neurological disease. First primate model was for Huntington disease (Rhesus macaque) Future of primate models is unclear because of ethical issues. © McGraw Hill 23  More advanced technology allows targeted mutagenesis Targeted mutagenesis enables scientists to change specific genes in virtually any way desired. The process is called gene targeting. Specific gene mutagenized in vitro Mutant DNA put into cells Rare homologous recombination replaces normal gene with mutant gene © McGraw Hill 24  Knockout mice Knock out mouse–functional allele of a specific gene replaced with an amorphic (nonfunctional) allele Null allele constructed by inserting drug resistance gene Mutant DNA construct is taken up by embryonic stem (ES) cells made from agouti mouse (yellow fur) Access the text alternative for slide images. © McGraw Hill 25  ES cells are used to target genes in mice Embryonic stem (ES) cells are undifferentiated cells from the blastocysts of embryos They can grow and divide in culture They are totipotent— can become any type of cell, including germ cells Access the text alternative for slide images. © McGraw Hill 26  Homologous recombination replaces wild-type allele with mutant allele in ES cells Access the text alternative for slide images. © McGraw Hill 27  Drug resistant ES cells are injected into blastocyst Blastocysts come from mating of black mice 10 cells from same colony injected Access the text alternative for slide images. © McGraw Hill 28  Knock out mice are raised Blastocyst implanted into uterus of foster mother Resulting mice are chimeras Access the text alternative for slide images. © McGraw Hill 29  Knockins introduce specific mutations in specific genes A knockin is a variation on a knockout The gene is altered, not deleted. Access the text alternative for slide images. © McGraw Hill 30  Knockins can be used to make models of human disease Mouse model for achondroplasia was constructed to contain the same mutation in the FGFR3 gene as seen in the human gene Wild-type Mutant Access the text alternative for slide images. © McGraw Hill ©1999 National Academy of Sciences, U.S.A. 31  Summary and key concepts from today Understand what transgenes and transgenic organisms are Know the different methods used to introduce transgenes into organisms Know the different uses for transgenic organisms we talked about today Understand basic process of organismal cloning Know the difference between knock-out and knock-in and when you would want to use each. © McGraw Hill