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Modern Genetics

by: Isobel Spencer

Modern Genetics BIOL 450

Isobel Spencer
GPA 3.66

Mark Ungerer

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Mark Ungerer
Class Notes
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This 4 page Class Notes was uploaded by Isobel Spencer on Monday September 28, 2015. The Class Notes belongs to BIOL 450 at Kansas State University taught by Mark Ungerer in Fall. Since its upload, it has received 17 views. For similar materials see /class/214964/biol-450-kansas-state-university in Biology at Kansas State University.

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Date Created: 09/28/15
Lecture 22 Genetic Engineering 1 History a Conventional traditional plant and animal breeding is genetic engineering i The willful manipulation oforganisms genetics to advance human interests ii Has been occurring since the dawn of animal domestication and agriculture 1 0K years ago iii This has involved interspecific hybridization 1 Many modern grains are hybrid species 2 Donkey X horse 9 mule iv Incurred risks but highly acceptable program of biological manipulation b Modern genetic engineering is driven by the same desire in advancing human interests i New tools and methods for manipulation ii The universe of possible modi cations has expanded iii lnterspecific hybridization common iv Not a highly acceptable program of biological manipulation v Differences between conventional breeding and genetic engineering at the heart of debates 0 Genetic engineering raises many valid and interesting concerns that are worthy of discussion 2 Why genetically modify organisms a Basic Research i Powerful tools for studying gene structure and function ii Allows the development of models ofhuman disease in model organisms b Medicine i Used for production oftherapeutics ii Gene therapy using genes as therapeutics 0 Agriculture i Improving crop yields ii Improving or changing nutritional qualities of plants iii Improving or changing agronomic qualities of plants temperature tolerances drought tolerances salt tolerances etc iv Biopharming using plants or animals to produce medicinal products d Environment i Using genetically modi ed organisms to clean the environment 1 Eg Plantsthat take up heavy metals ii Biosensors 1 Create organisms that respond to threats pollutants toxins e Many other applications 3 General Considerations creating transgenic organisms involves some common problems a Transgene design and construction i Details of gene expression must be carefully considered including promoter sequences splice signals codon usage ii Need to make transgenes using target hostappropriate components and design considerations 1 eg no introns in genes to be expressed in bacteria 2 no operons to be expressed in animals 3 promoters are often species limited and will not work everywhere b Transgene Delivery i The objective is to get transgene to the nucleus oftarget cells ii Must traverse cell membrane ii Must get transported to nucleus and traverse nuclear membrane iv Must be able to get to cells within the context of a multicellular organisms usually v Must get to germ cells ifa permanent heritable change is desired c Transgene Integration i Once DNA is in the nucleus integration is required if you want transgene to persist through mitosis othenNise it will be degraded ii Integrations can be random based on random breaks and repairs of chromosomal DNA and introduced DNA 1 Recall translocations deletions and inversions Double strand breaks are happening all of the time iii Integrations may be driven by linking transgene to sequences that have the ability to integrate into chromosomes 1 Transposable elements 2 Viruses d Transgene Expression i Once integrated everything must work but position effects may be important closed chromatin heterochromatin methylated DNA e Transgene Detection i How do you recognize a transgenic cell or organism 1 If frequency of integration is high then you can screen gDNA for presence of transgene 2 If frequency is low then it might require using a genetic marker a second transgene linked to your transgene that confers a visible phenotype a Eg Green fluorescent protein screen for transgenics b Eg antibiotic resistance select fortransgenics 4 Transgenic Mice a Pronuclear injections i Highly technical but pretty ef cient 1 1020 of pups arising from injection of plasmid DNA will have integrated plasmid DNA integrations are random many copies of the plasmid are integrated as concatamers this is good for adding genes to genome for expression studies b Blastocystinjections i Make transgenic embryonic stem cells cell culture techniques 1 Can find rare recombinants using powerful cell selection methods antibiotic resistance a Homologous recombination knockout mice knockin mice b Transposon vectors c Retrovirus vectors 2 Select expand and grow up transgenic embryonic stem cells ii Transplant transgenic embryonic stem cells to developing blastocyst iii Introduce blastocyst into surrogate mother iv Pups will be chimeras transgenic and nontransgenic cells 1 lftransgenic cells include gonads then subsequent progeny will be 100 transgenic start lines from these ACHN 5 Transgenic Plants a Agrobacterium tumefaciens a pathogenic bacteria crown gall disease plant cancer i Bacteria mates with plant cell transfers tumor inducing plasmid Ti plasmid ii Ti plasmid hastransposonlike sequences that enable it to integrate into plant genome Sort of like a transposition event but different biochemistry iii A t is a natural genetic engineer Use Ti plasmid to carry transgenes use At to delivertransgenes to plant cells iv Or Ti plasmid can be delivered directly to cells without A t b Transgenic Cotton Corn and Soybean i Herbicide tolerance is populartransgenictrait ii Glyphosate Round Up by Monsanto a popular and effective herbicide iii Targets plant speci c biochemical pathway aromatic amino acids iv lnhibits key enzyme in synthesis pathway EPSPS v Resistance by different strategies 1 Overexpress EPSPS 2 Mutant EPSPS that can t see glyphosate most popular 3 Detoxify glyphosate break it down 4 Note need to express in right place at the right time c Transgenic Rice i Golden rice make beta carotene in the endosperm ii Beta carotene used to make vitamin A ii Nutritional supplement iv Couldn t do this without genetic engineering 6 Human gene therapy somatic cell engineering no germline modifications QOC39N 39D Delivery oftransgenes by retroviruses or transposons integrate Delivery by adenoviruses don t integrate episomal Deliver DNA or RNA directly no integration transient expression Delivery problems are severe lots of cells in body how to get DNA to target cells Example i SClD severe combined immunediffiency 1 Many causes 2 Supply gene to produce wild type proteins and complement inherent mutations 3 Blood stem cells are accessible and transplantable 4 Delivery by retrovruses originally but this caused cancer in some patients integrated near oncogene 5 Has been successful ii Making stem cells 1 Can introduce and express 4 transgenes involved in regulating cell growth into differentiated fibroblast cells in culture 2 Converts broblasts to pluripotent stem cells 3 Eliminates some problems associated with using pluripotent stem cells usually come from human embryos 7 Beyond Transgenics Direct Genome Modi cation using Enzymes a Zince nger nucleases 39 Custom designed DNA binding proteins with an endonuclease attached Permits site specific double strand DNA breaks Can influence DSB repairto result in mutations gene corrections new DNA insertions 8 Synthetic Biology a b Start with a genome of a bacteria and start eliminating genes O Q Build now biological systems to do things not necessarily biological things Build a minimal genome what is minimal essential genes for life Mycoplasma laboratorium lab constructed life form Can start adding functions 9 Concerns O 39D a Rational and irrational fears and concerns b c Concerns come from all directions not just biology Most of the noisy arguments and debates tend to frame issues in black and white talk radio approach to discussion There are many interesting serious nuanced ideas that are worth serious consideration and from which we can learn much Attitudes vary widely among cultures an interesting phenomenon


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