Genetics: From Genes to Genomes Sixth Edition Leland H. Hartwell, Michael L. Goldberg, Janice A. Fisher, and Leroy Hood ©McGraw-Hill Education 2018. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or further distribution permitted without the prior written consent of McGraw-Hill Education. PART IV - Using Genetics Ch13 Manipulating the Genomes of Eukaryotes 1. Creating Transgenic Organisms 1) Transgenic mice made by pronuclear injection 2) Plant transgenesis by using Agrobacterium Ti plasmid vectors 2. Targeted Mutagenesis 3. Human Gene Therapy ©McGraw-Hill Education 2018 3 13.1 Creating Transgenic Organisms Transgenic organism— • Contains a gene from another individual of the same species or a different species. • Progeny inherit the transgene because cells containing the transgene are able to form gametes. Transgene— • The transferred gene. ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 13.1.1 Transgenic mice can be made by pronuclear injection ©McGraw-Hill Education 2018 a: © Martin Oeggerli/Science Source; 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. Jump to long description ©McGraw-Hill Education 2018 Injected embryos are implanted into a pseudopregnant 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. Jump to long description ©McGraw-Hill Education 2018 7 Pharming: Use of transgenic animals 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. ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 8 13.1.2 Plant transgenesis using Agrobacterium Ti plasmid vectors Agrobacterium-mediated T-DNA transfer—DNA transfer to the host cell during infection is mediated by the Ti (tumor inducing plasmid. The Ti plasmid contains left and right border sequences (LB and RB) surrounding vir genes. The transferred DNA is integrated into the plant genome through the action of trans-acting Vir enzymes. ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 9 Plant transgenesis using Agrobacterium Ti plasmid vectors Recombinant T-DNA plasmid is constructed. T-DNA plasmid and helper plasmid are sprayed on plants. DNA is transferred and integrated into plant cell genome. Individual transgenic plant cells are selected by resistance to herbicide and grown into plants. ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 Bt corn , an example of plant gene engineering Bacillus thuringiensis (Bt) is a soil bacterium that produces a protein called Bt toxins that has insecticidal qualities. After Bt toxins binding to the appropriate receptor on the surface of midgut epithelial cells, thereafter insect pest stops feeding and dies soon. Crop plants have now been engineered to contain and express the genes of Bt toxin. Any organism that lacks the appropriate receptors in its gut cannot be affected by Bt toxins. Bt toxins is nontoxic to vertebrates ©McGraw-Hill Education 2018 Bt plant reduced dependence on pesticides 11 Genetically modified crops are widely used • Corn expressing Bt protein protects the plant from corn-borer moth caterpillars. 10 billion acres are used to grow Bt crops. • Roundup Ready soybeans are herbicide-resistant, >90% of US soybeans are Roundup Ready. • They belong to Genetically Modified Organism (GMO) ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 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 Education 2018 13.2 Targeted Mutagenesis Targeted mutagenesis enables scientists to change specific genes in virtually any way desired. • Knockout (making deletion to) a gene at a defined locus. • Knockin ( introducing ) a gene at a defined locus. ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 13.2.1 Gene Targeting The gene targeting process: 1. Establish embryonic stem (ES) cells culture. 2. Specific gene mutagenized in vitro. 3. Mutant DNA put into cells. 4. Rare homologous recombination replaces normal gene with mutant gene (knockout) ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 Embryonic Stem (ES) Cells Purebred agouti mice Embryonic stem (ES) cells are undifferentiated cells from the inner cell mass of embryos. • They can grow and divide in culture. • They are totipotent—can become any type of cell, including germ cells. ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 Specific Gene Mutagenized in vitro Gene Knockout: • A functional allele of a specific gene is replaced with an amorphic (nonfunctional) allele. • Amorphic allele constructed by inserting drug resistance gene. • The insertion of neor inactivate the gene. ©McGraw-Hill Education 2018 neor : resistant to neomycin. Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 Homologous recombination replaces wild-type allele with mutant allele in ES Cells neor : resistant to neomycin. Add the mutant DNA construct to ES cells. Mitotic recombination occurs between mutant DNA and wt cellular DNA ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 Drug resistant ES cells are injected into blastocyst Add Neomycin ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17 • Blastocyst implanted into uterus of foster mother. • Chimera mouse. --- (Genetic mosaicism) • Some targeted ES cells may develop into germ line cells. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16 ©McGraw-Hill Education 2018 Knock out mice are raised • Chimera mates with black mouse. • Agouti progeny should be heterozygous for knock out allele. (Agouti is dominant to black) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16 ©McGraw-Hill Education 2018 Knockins introduce specific mutations in specific genes A knock in is a variation on a knockout. Mouse model for achondroplasia was constructed to contain the same mutation in the FGFR3 gene as seen in the human gene. ©McGraw-Hill Education 2018 Copyright © McGraw-Hill Education. Permission required for reproduction or display Hartwell et al., 5th ed., Chapter 17
