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Lecture 22

Biology 2581B Lecture 22: Lec 22 – Transgenics

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Western University
Biology 2581B
David R Smith

Lecture 22 – Transgenics Model organisms - Transform organisms to make mutants - Can’t work with all organisms – must pick and choose - Conditions that organisms must correspond to to be usefull: o Size (mice vs. elephant) ▪ Want small space to raise the organisms o Numbers of organisms that can be raised ▪ When have genetic problems, we are dealing with populations ▪ Want to raise a large number of offsprings to genetically analyze them o Generating mutants ▪ Cannot do this with all organisms o Number of off springs ▪ How long it takes to get the next generation • Don’t want an organism that takes many years to get the first generation o Genome sequence – this is becoming more available for a larger number of organisms as technologies have improved o Community support ▪ Don’t want to be the only person working on an organism ▪ Want to share information with other people working with the same organism – use the same resources and work on community projects ▪ Make resources available for everyone working with a particular organism o Funding ▪ Must justify the organisms we are working with and how data obtained will be helpful to understand large concepts - Chose organisms that let us work around these things o Easy to be maintained under laboratory conditions ▪ To raise large numbers ▪ Controlled environmental conditions o Easy induction/recognitions/tracking of mutations o Produces large number of offspring ▪ In a short period of time o Cloning/transformation procedures are in place o Genome sequence o Community support o Can address interesting questions to obtain funding What they should model - Different model organisms can address different questions o Organisms are good for different things - There are different scales - There are different model organisms for different biological/genetic questions Different scales - Universal properties - Different groups of organisms o Bacteria, protists, worms, insects, mammals, fungi, plants….. - Many of the basic mechanisms are similar o Can generally apply mechanisms to other organisms - All organism use basic processes o Transcription o Translation o Replication o Mutation etc - Choose an organism based on a question you want to ask o Comparing different insects = choose different insects ▪ Address evolutionary questions ▪ See how mechanisms have evolved in different species ▪ Drosophila and match it with butterflies – see what the changes are in different groups of insects ▪ This could be too broad o Taxon specific properties o Species specific properties ▪ For example, comparing different Drosophila insects • There are many different species of this fly ▪ Different species within the genus ▪ Compare how reproductive mechanisms have evolved in closely related species and what differences exist - Choosing model is and allows us to do different types of comparisons Saccharomyces cerevisiae - Alias: budding yeast o Single cell eukaryote o Grown similarly to bacteria – raise large number of cells in mL (attractive organism) o Has a haploid and diploid cycle ▪ Switch between the two and follow a genetic trace - Genome: 16 chr, ~6600 genes, 12 Mb - Attractiveness: beer brewing, single cell eukaryote o Haploid or diploid, large quantities - Transgenics: transformation with plasmids followed by recombination o Easily transformed with plasmids and it can stay extrachromosomal o Make constructs to integrate DNA into the genome of the yeast using recombination - Markers: nutritional auxotrophs, drug resistance - Advantages: properties of eukaryotes, cell cycle, cell division, meiosis, secretory mechanisms, protein trafficking, chromosome structure, signal transduction, replication, transcriptions etc - Limitations: extracellular signaling, centromers are small, microRNAs?, not multi cellular - Use shuttle vectors when working with yeast - Must use shuttle vectors because we go back and forth between e. coli and yeast o E. coli is the organism used to clone plasmids or any gene of interest o Cloning = assembly of the plasmid (it is done in e.coli) - Shuttle vectors o Allows for more then one host system o Clone in E. coli but express the gene in yeast o Minimal components: ▪ Origin of replication for each host ▪ Selectable marker for each host - Use shuttle vectors when working with different types of organisms o This one allows you to go between e. coli and yeast Minimal components for shuttle vector - Have an area where you clone the gene of interest o What you want to get into the yeast organism later on - Promoter and termination signal that are apart of the host system to express a gene o YEAST promoter and YEAST termination signal o Otherwise the gene will not be expressed in the yeast host - Track the plasmid: need a selectable marker that works in yeast o Yeast use nutrient markers o LEU2 marker that allows the yeast go grow in absence of leucine o Track the plasmid by plating the yeast strain on a plate that lacks leucine ▪ If it contains the plasmid, it will supply it with leucine and the yeast can grow (LEU2 encodes leucine) - Origin of replication o Without this, the plasmid will not be maintained in yeast o CEN/ARS ORI: ▪ Part of a centromere that is integrated into plasmids ▪ Plasmids are kept at a copy number of one ▪ Replicating like a normal chromosome in yeast at the same time during the cell cycle as the other chromosomes in yeast and get divided to the daughter cells afterwards o 2 µm ORI: ▪ Allows the plasmid to exist a multi-copy plasmid in yeast ▪ Advantage for some experiments - Chose the origin the replication we want depending on the goal of the experiment - Chose multi copy plasmid if want to express a protein in high yield of the protein o If have several plasmids coexisting in the cell, all make the transcript and all make the protein o High yield of the protein in a particular cell - Shuttle vector requires components to maintain it and select for in e. coli o Selectable marker: ampicillin resistance (antibiotic resistance) o ORI E that works in e. coli (bacterium) - Need all of the components to clone the plasmid in e. coli and then transform it into yeast - These types of cassettes were used to disrupt EVERY known ORF in the yeast genome – knockouts simply can be purchased - Use shuttle vectors for knock out scenarios o Yeast is VERY good in knockout scenarios ▪ Needs short region of homologous sequences in order to recombine a sequence that is on the vector - Knockout construct: o There will be homologous sequences in the construct that can align with identical sequences in the genome o Have a selectable marker to track the recombination event - Want 2 recombination events o Outcome: replace sequences in between the recombination event with whatever is in the vector - Replace ORF with a selectable marker that creates a knockout scenario o Remove that particular gene from the genome of the yeast strain - Yeast are very good at this o Community has made the effort to knock out every ORF in yeast (in different strains) o Can go online, pick a gene and order a knockout line in the yeast database - Use yeast strain with a knockout in an experiment to check what happens if a particular gene is missing Caenorhabditis elegans - Alias: “the worm” - Genome: 6 chr, ~18500 genes, 105 Mb - Attractiveness: defined cell lineages o It is tiny (1-2mm) - Attractive feature of this model organism: o Know what happens to very cell after every cell division - Have a map of the cell divisions: o Start with zygote that continuously divides o What what happens to every single cell ▪ Know if the cells form neurons, or muscles or other tissues - There is 302 neurons in the organism o Can do very particular experiments o Destroy a particular cell at a particular stage and follow what the consequence of the lack of that cell for the development of that worm - Sensitive system for this approach - Don’t have detailed information for every cell and every cell line for the mature individual in any other organism - Transgenics: injection with dsDNA o Maintained as extrachromosomal copy - Inject 2 constucts with dye to check if the solution gets into the worm o 2 different sequences: DNA of interest and a selectable marker (rol-6) - Inside the worm, the 2 pieces of DNA form a concatemer o Alternating DNA sequences in the construct o Kept as an extrachromosomal piece that is not integrated into the genome - Put the selectable marker to know if the gene of interest gets into the worm - Rol-6: makes a protein that changes the behavioral patterns of the worm o Usually a worm waves over a plate o When rol-6 is present, the worm moves differently o From the movement pattern, can see if the worm received extra DNA o Can follow the morphological DNA o It is easy to distinguish between the two different phenotypes - Identify transgenics very easily though morphological changes - Markers: morphological change in shape or affecting movement - Advantages: o Eorms are used to study embryology o Programmed cell death (can be implemented, or followed naturally in the cells) o Neurobiology (have neurons that can be studied) o Behaviour o Signal transduction pathways o microRNA and RNA interference ▪ miRNA were detected for the fist time in this organism ▪ miRNA exist in many organisms but the worm guided us to the existence and allowed us to study them initially - Limitations: o No established tissue culture ▪ Cannot extract cells and grow – always deal with the whole organism o Targeted integration difficult o Many genes are transcribed as polycistronic RNAs ▪ When look at the native system, have many coding units on a single RNA making the manipulations different o Must deal with the whole organisms - Hermaphrodites have female and male characteristics o No male-female system o Have a male-hermaphrodite system o Hermaphrodite can produce sperm AND eggs (XX) ▪ Get reproduction from using sperm and egg from an individual ▪ Don’t necessarily have crosses Modes of reproduction - Set up crosses: o Can mate a hermaphrodite with a male and track it - Hermaphrodite cross with hermaphrodite o Hermaphrodite self fertilization generate mostly hermaphrodites o Small percentage of males - Classical cross fertilization: o 50% will be male and 50% will be hermaphrodite - Morphological difference: o Hermaphrodite is larger than the male organism = can be identified easily Drosophila melanogaster - Alias: fruit fly - Genome: 4 chr, ~13750 genes, 170 Mb - Used extensively as a model organism - Attractiveness: easily grown, huge number of mutant lines, long history of research o Many tools available to us to grow it - They are very small – raise 100s of individuals in small vials o Kept and propogated - Transgenics: traditional by injection with P elements, random integration o Done with P elements (transposons) in fruit flies o P elements: integrating transpsoson randomly into the genome o P elements INSERT (ADDITION) into the genome ▪ Do NOT replace – cannot replace what is already there - Newer systems: CRISPR/Cas9, targeted integration and changes o Allow us to do integration and replacements very specifically - P elements are still used (classical approach) - Markers: morphological, eye color - Advantages: o Long history: know a lot about the insect already o All topics of animal genetics o Polytene chromosomes: easy to track - Limitations: o No recombination in males ▪ Mix up of genetic material is limited o Target disruptions of genes difficult ▪ New technologies are arising to introduce targeted disruptions and replacements P elements from Drosophila - Activity of transposase is what happens in P elements - Transposition event using inverted repeats is what happens with P elements o P elements move from one location in the genome to another using inverted repeats - Modify natural systems so transposition event is in our control and we can control where things end up
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