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44 Cards in this Set
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Animal models |
Any species that has been widely studied Cheap/easy to maintain and breed Known genome sequence Experimental advantages 3 types – genetic models, embryological modelsand genomic models |
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Genetic models |
Lookingat the expression of genes in the animal and their effect on the organism Cancarry out gene knock-outs or investigate the genotype of mutants Large number of mutants available Large number of offspring Short generation/gestation time |
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Embryological models |
Want to find out about embryology Robust embryos that can be easily manipulated Large number of embryos External development |
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Genomic models |
Comparingwhole genome and epigenetic effects between species, e.g. mice and humans Relevance to human genome (gene conservation) Disease models Drug testing |
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Nematode (worm) |
Caenorhabditis elegans Invertebrate developmental model Only has around 400 cells which are visible as the worm is transparent so individual cell development can be tracked Genetics is very simple Very easy to maintain - can be kept in Petri dishes |
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Fruit fly |
Drosophila melanogaster Invertebrate developmental model Easy to breed and maintain Large numbers of mutants |
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Zebrafish |
Danio rerio Vertebrate developmental model External development – eggs can be monitored ina dish Can watch movement of cells Clear embryo for imaging Can generate mutants |
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African clawed frog |
Xenopus laevis Vertebrate developmental model External development Good for experimental embryology |
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Chicken |
Gallus gallus domesticus Vertebrate developmental model Good embryological model - if you remove the top of the egg, the embryo is visible |
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Mouse |
Mus musculus Genomics Genetic modification |
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Early animal development |
Cleavage begins forming blastomeres After 3 divisions, synchronous radial cleavage occurs until it reached the blastula stage Gastrulation Neurulation |
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Gastrulation |
Cells begin moving into the blastula from the edges of the embryo staring at the dorsal lip During gastrulation, cells move inside theblastula through the blastopore and are dragged along the roof of theblastocoele Gastrulation forms three different layers of cells: the ectoderm, the mesoderm and the endoderm |
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Neurulation |
Forms the primitive brain and spinal cord Notochord gives off signalling molecules whichcause neurulation to occur Ectoderm cells begin dividing and spreadingand fatten up along the midline forming the neural plate Either side of them, the ectoderm starts tomove around to form a tube, which then becomes dissociated |
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Animal cloning |
Also called somatic cell nuclear transplantation Wanted to know whether DNA from one organism isable to direct the development of another organism Took an unfertilised Xenopus egg and irradiatedit using UV radiation to remove its DNA, this gave an enucleated egg Separated a nucleus from a cell from the animalpole of a blastula Injected the nucleus into the enucleated egg Eggs developed into a blastula stage embryo andeventually developed into an adult frog |
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Studying transcription factors |
Homeotic mutants are studied One cell will secrete an inducing signal(protein), which can bind to a transmembrane receptor on the cells surroundingit This gives a signal down the signal transductionpathway from the cell membrane to the nucleus of the cell Cells closer to the one producing the signalwill get the highest dose of the inducing signal, therefore more of thetranscription factor will be produced This results in different cells being formed in different regions |
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Finding the signalling centre Hans Spemann (1920s) |
Ligature tightened around fertilised egg When ligature bisected grey crescent region, it resulted in 2 normal embryos When the grey crescent region was on one side, the other side developed into a "belly" piece with only ventralised tissues An embryo with 2 grey crescent regions formed a two-headed conjoined embryo with two anterior to posterior axis |
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Finding the genes responsible theory |
This failed until the advent of moleculartechnologies Links with generating and rescuing mutants To generate mutants, you can over-express geneproducts in embryos This is done by injecting mRNA into the embryoand this causes lots of the protein coded for to be produced You then check whether the protein altersdevelopment. If it does, then it is likely that the gene that coded for thatmRNA is responsible for differentiation |
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Finding the genes responsible procedure |
Make a plasmid library containing thousands ofrandom genes Using these plasmid libraries, mRNA can beproduced in vitro Many eggs were injected with many differentstrands of mRNA Once the mRNA is in the egg, processing such aspolyadenylation can occur so the strands of mRNA are in a form that can betranslated This was repeated to form a long list of genesthat may be involved in axis generation |
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Identifying the specific gene for axis generation |
Experiments with rescue mutants carried out Eggs were UV radiated, which should cause veryventralised embryos to develop Lots of different mRNAs were injected intodifferent eggs to find one which rescued head development mRNA from a gene called Noggin rescued thephenotype when given in low doses but when given in high doses formed a mutantof the opposite type, which was basically just a headFound that Noggin is a secreted signallingmolecule that can promote dorsal structures |
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Finding where the gene is expressed |
In situ hybridisation mRNA probe complementary to the mRNA sequenceis produced and labelled with a DIG (Digoxigenin) label Anti-DIG antibodies with alkaline phosphataseenzymes attached recognise DIG Alkaline phosphatase will give a colouredprecipitate when a substrate is added Embryos are washed so only specifically boundprobes remain and produce a colour |
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Expression of Noggin |
Noggin is expressed in the organiser Cells close to the organiser follow dorsal rates and cells far away follow ventral rates |
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Drosophila lifecycle |
Larval stage insect Takes around 9 days from birth to maturity |
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Superficial cleavage in drosophila |
Genetic material divides 9 times, forming manydifferent nuclei. After 9 divisions, the nuclei move to the outside of the blastodermleading to a syncytial blastoderm Nuclei that will later form gametes (pole cells)are sequestered to the pole of the blastoderm After 11 divisions, membranes form around thesenuclei, leading to a cellular blastoderm |
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Gastrulation in drosophila |
Starts at the ventral furrow –cells start rolling around and go into the blastoderm Cells start invaginating and moving into theblastoderm forming the mesoderm External cells are the ectoderm First cells that are invaginated form themesoderm, forming a tube initially, but then the mesoderm cells begin spreadingaround the edges Invagination occurs again forming the endoderm The two groups of endoderm cells are dragged around the inside of theblastoderm and form the anterior and posterior gut |
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Germ band extension |
Occurs at the same time as gastrulation Movement of ventral tissue around the posteriorend and onto the dorsal side. Cells of the future anterior and posterior midgutinvaginate into the embryo and eventually fuse to form the gut |
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Neurulation in drosophila |
No neurulation as flies are not vertebrates.Instead, the ventral furrow closes, then ectodermal cells either sidedissociate and start moving as individual cells into a position between theexternal ectoderm and the internal mesoderm. These cells become the neuroblastswhich form the nerve cord on the ventral side |
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Segmentation |
Segmentation occurs leading to different bodyparts forming (mouthparts, thorax and abdomen) These segments are formed in early stages in theembryo but will also specify later on for adult segmentation to occur |
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Genetic screens |
Generate the mutants Identify genes that are mutated Find where the genes are expressed |
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Generating mutants |
Mutate the male using chemical mutagens andthen cross it with a female Offspring are the F1 individualsand can be mutated or non-mutated, either +/- or +/+ Theseare bred with a wild type mate as the mutations are often recessive so aren’texpressed. These are called F2 families It is still not possible to know whichfish are mutated as they will have the same possible phenotypes as the F1individuals. This is to increase the population of the fish. After this, the F2families are interbred to get the F3 generation. In this generation, we can nowget -/- fish so 1/16 of them will be phenotypically mutant |
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Homeotic genes |
Foundnear each other in the genome. The order of expression in the embryo matchesthe order on the chromosome (colinearity) Homeotic genes are Homeobox (HOX) transcriptionfactors. They activate genes required to specify organs or cell types (e.g.wing). Homeobox transcription factors affect protein folding once they’ve beenproduced |
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Mutations in homeotic genes |
If Antp and no Ubx gives a pair of legs and anextra pair of wings but no halteres in thoracic segment 3 Antp promotoes leg identities. In the head,lab/Dfd and Antp causes the fly to lose the antennae and gain an extra pair oflegs There are also Hox genes in mammals (38) |
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Early mouse development |
Morula formation Blastocyst formation Hatching Implantation Gastrulation Neurulation |
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Morula formation |
A fertilised mouse egg is called an oocyte It divides to form 2 blastomeres, then continuesdividing to form a blastocyst At the 16 cell stage it is called a blastula andat the 32 cell stage it is called a morula In the morula cells begin to clump together in aprocess called compaction The zona pellucida forms. It is a thicktransparent membrane that surrounds the clump of cells in a morula Atthis stage, the embryo is not increasing in size, the cells are just dividing |
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Blastocyst formation |
After the morula stage, the cells begin to movemore and form a more structured blastocyst, which is made up of three mainparts, the inner cell mass, the trophoblast (outer lining) and the blastocoele(fluid filled cavity) Cells divide rotationally – once equatoriallyand then once meridionally Isn’t synchronous, so could end up with an oddnumber of blastomeres at any time as one cell may divide before the others do |
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Hatching |
Eventually the blastocyst hatches out of thezona pellucida by squeezing out and releasing enzymes that will digest themembrane It needs to move out of the zona pellucida to beable to implant in the maternal wall |
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Implantation |
Instead of a womb, mice have two uterine hornsinstead of fallopian tubes, which is where the blastocyst will implant After implantation, the blastocyst begins toorganise itself to form a bilaminar blastocyst. It has an outer layer oftrophoblast, then inner layers of epiblast and the primitive endoderm. Theblastocoel still takes up more than half of the inside of the blastocyst The epiblast layer starts moving around the top part of the blastocyst, forming the amniotic cavity, and the primitive endoderm starts to move around the bottom part of the blastocyst, forming the primitive yolk sac. The blastocoele is gone At the same time, the trophoblast cells start projecting out into the endometrium and forming the embryos place of rest within the womb |
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Gastrulation |
On the midline of the top of the epiblast in abilaminar blastocyst, a streak begins to form This is controlled by a signalling region calledthe node At the position of the primitive streak, agroove form Cells from the epiblast move into the groovethrough a process of ingression – cells moving independently of one another.They become interiorised into the embryo These movements eventually completely obliteratethe primitive endoderm The point of gastrulation is to set up the threegerm layers – ectoderm, mesoderm and endoderm |
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Function of germ layers |
Once the germ layers are formed, the cells areonly able to specialise into certain types of cell. The ectoderm can form theepidermis, neurons of the brain and central nervous system and the peripheralnervous system. The mesoderm will form muscle and connective tissues, bone,kidneys and gonads and the tissues of the heart. The endoderm will form theepithelial lining of the digestive system, the stomach, colon and bladder andthe epithelial lining of the respiratory tube |
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Notochord formation |
All of these three layers are moving in throughthe groove, but some pass through the node region. The cells that move throughthe node form a transient structure called the notochord This structure is important for signallingduring neurulation |
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Neurulation |
Occurs in a trilaminar embryo formedafter the development of germ layers Consists of invagination (formation of atube) Initially happens by the formation of aneural groove. Cells on either side of the neural groove foldup, forming a neural fold The sides of the neural fold get closer andcloser together until they join up to form the neuraltube Cells overlying it will become dissociated and become the skin |
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Transgenics |
Nuclei are injected with the DNA at a very early stage when the cell still has 2 pronuclei A holding pipette is used to hold the zygote inplace and an injecting needle is used to inject the plasmids into thepronucleus A female mouse is anaesthetised and the oocytesare placed back into theoviduct, where they will move into the uterine horns Mouse will eventually give birth to pups whichwill hopefully have a phenotype that shows the overexpression of the gene |
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Targeted knockouts |
Embryonic stem cells are collected from the inner cell mass of a blastocyst and gene of interest is removed This is done using a plasmid called a targeting vector |
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Targeting vectors |
Made up of 4 key parts – homologous arms thatflank the gene, a positive selectable marker (e.g. neomycin resistance gene), anegative selectable marker (e.g. thymidine kinase) and a reporter gene (e.g. β-galactosidase) The homologous arms are made up of DNA fromeither side of the gene of interest which are ligated together. This allows theDNA in the stem cells to be found by and replaced with the homologous armsthrough homologous recombination If there is any DNA between the homologous arms, this is also swapped into the DNA in the genome |
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Selectable markers |
The selectable markers make the process moreefficient and the reporter gene allows us to see where the gene of interestwould have normally been expressed The positive selectable marker and the reportergene should be included in the DNA when homologous recombination occurs The negative selectable marker should not beincluded, as if it is it will cause the stem cells to die. This allows us to besure that the homologous recombination has occurred perfectly as cells thathave had recombination carried out wrong will die |
