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66 Cards in this Set
- Front
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what processes/techniques can be used to visualise embryos and tissues studied in developmental studies? (5 types of microscopy) |
Light microscopy Confocal microscopy - uses laserand has uses in immunostaining sheet light microscopy - can makea 3D reconstruction of tissues to look at migration of structures indevelopment High content microscopy - this isa fluorescent microscope that can study multiple samples at one time. Electron microscopy - negative staining at high resolution for cellular structures |
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How are tissues fixed for electron microscopy? |
we keep tissues as they originally are, we fix them in situ to prevent any degradation, extraction and translocation of proteins, lipids and carbohydrates. Fixation of the tissues uses ethanol (for light microscopy) or aldehydes like themaldehyde (for electron microscopy), which cross link proteins so that they do not rot. You then dehydrate the sample. this replaces water in the tissue with organic solvent miscible. this is then made into a tissue block. for electron microscopy, the dehydrated tissue is embedded in plastic. the tissue block is then cut using an untramicrotone with a diamond or glass blade to create sections for studying. these can be stained with heavy metals that are electron dense, before viewing under the electron microscope. |
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how are tissues fixed for electron microscopy? |
we keep tissues as they originally are, we fix them in situ to prevent any degradation, extraction and translocation of proteins, lipids and carbohydrates. Fixation of the tissues uses ethanol (for light microscopy) or aldehydes like themaldehyde (for electron microscopy), which cross link proteins so that they do not rot. You then dehydrate the sample. this replaces water in the tissue with organic solvent miscible. this is then made into a tissue block. for light microscopy, the dehydrated tissue is embedded in paraffin. the tissue block is then cut using a microtone with a metal knife to create sections for studying. these can be stained deferentially (acid-base, histochemical) before viewing under the light microscope. |
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What is cell ablation and how is it done? |
Cell ablation is the controlled removal/destruction of cells to study their effect in model organisms. knock outs can be created like this. in the field, it is used to study regeneration, development and compensation. the removal can be done surgically, but now we usually remove the cells with lasers and then follow the fate of the embryo. This is easy in drosophila, but in vertebrates like mice, it is much more difficult. instead, we link a gene to a toxin and depending on where that gene is expressed, there will be cell death. |
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What is a talpid chick? |
this chick has polydactyl. the digits are fused together due to a mutation in a gene responsible for the sonic hedgehog pathway. they have ataxia, kidney stones, and learning difficulties. these findings can be applied to human cases of the syndrome. |
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what is a mouse piebald? |
these mice have a steak of white or grey hair and a streak on the belly too. pigment cells move faster in mice that have mutated Kit genes. the patterning arises because cells do not multiply as well as they moved through the skin, leaving the underbelly devoid of pigmentation. |
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how is a chick/quail chimera created? |
the nuclei of a chick is transplanted into a quail egg (and visa versa) |
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what methods are used to detect proteins in development? (2) |
immunostaining and western blotting |
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what methods are used to detect mRNA in development? (4) |
Northern blotting, RT-PCR, microarrays, and in situ hybridisation. |
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how can we manipulate genes to create under expression of them? |
Dominant negative proteins - this is used in receptors. you express the mutant. these mutants will usually dimerise with another normal one, so you never get the expression of the normal one, because the mutant one is dominant. Gene targeting by homologous recombination - uses RecA protein in recombination (LINK TO MICROBIOLOGY LECTURES HERE) where sections of important DNA can be removed via targeted recombination so that they are inactivated. RNAi - this stands for RNA interference and is where small sections of siRNA are targetted to known gene sequences that we wish to interrupt Morphilinos - these work in a way very similar to siRNAs. they are small oligonucleotide sections that block expression of genes Mutagenesis screens - the model organisms are exposed to radiation to case mutations and then bred to get full expression of mutations caused Gene editing by CRISPR/Cas9 (SEE LATER LECTURES FOR MORE INFORMATION) |
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What are the four types of blotting (molecular hybridisation) that are used to detect and manipulate gene expression? |
Northern - RNA is put onto a gel and then transferred onto a filter. this is hybridised with labelled DNA. Southern - restriction digested DNA is put on agarose gel. this is denatured and the DNA transferred to a filter to demobilise it. this is then hybridised with an RNA or DNA probe Western - proteins are separated by SDS polysaccharide gel electrophoresis and then transferred to a nitrocellulose membrane. an antibody is then added against the protein of interest, and a HRP coupled secondary antibody to make the reaction. Detection of HRP (light) to gain results South-Western - looks at protein and DNA interaction |
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what is a microarray, what does it measure, and how do we do it? |
In microarrays, you are looking at up and down regulation of mRNA. mRNA is taken from different samples and separated by agarose gel electrophoresis. the mRNA is transferred to a nitrocellulose membrane (immobilisation) and then incubated with a labelled probe of DNA or RNA complementary to the mRNA of interest. you can use more than one probe with different markers for each. you can then use lasers or fluorescence to detect the expression in the samples visually. this is important because the locations and production of mRNA and proteins can be different to each other. |
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What is the method of proteomics? |
1. Preparation of protein extracts 2. isoelectric focusing - separation of proteins due to their isoelectric point by isoelectric focusing. works by applying an electric field to protein with a pH gradient. the proteins separate as they migrate through the pH value that matches its isoelectric point. here, its net electric charge becomes neutral and it stops migrating. 3. Polyacrylamide electrophoresis - proteins move through the gel as a response to an electric field, with smaller ones moving faster than larger ones due to the pore structure of the gel. discontinuous buffer systems are most commonly used for this, where more than one type of buffer is used. |
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what is the purpose preparation of in situ hybridisation? |
1. prevent loss of nucleic acid from the tissue. RNA is unstable, so we need to protect it. we wear gloves and use filtered tips to prevent contamination from RNases 2. Preserve tissue morphology. embryos are small and easily sucked into pipettes, which damages the tissues. 3. allow the probe to penetrate the tissues. this needs to happen so that it can bind with the target mRNA sequence. |
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What are the stages of preparation of an in situ tissue/embryo sample? |
1. rehydrate - the embryos are kept in a freezer at 70% ethanol. to rehydrate them, they are put through an ethanol series (50%, 20%, and then a phosphate buffered solution). this makes the embryo less brittle and easier to work with. it also prepares the tissue for the probe. 2. Permeabilisation - a solution containing proteinase K is used to wash the embryo. this is an indopeoptidase that randomly cleaves peptides. any proteins around the target mRNA can be cleaved for better probe access. the levels of proteinase K must be optimised carefully. in the same way, the time the cells are exposed to the proteinase K must be optimised, because over exposure could lead to tissues disintegrating. 3. Re-fix - now that the contents of the cells have been exposed, a fixative must be used to prevent RNA degradation. this stabilises the RNA 4. Pre-hybridisation - incubate with hybridisation solution that doesn't contain the probe. it binds to non-specific RNA in the cells to reduce background when the probe is added. |
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what types of probe can be used in in-situ hybridisation? |
- oligonucleotides - small, short, chemically synthesised sections of DNA. they are stable and resistant to RNases - single stranded DNA - stable and resistant to RNases. these are longer than oligonucleotides and are created by PCR - RNA probe - forms an RNA-RNA hybrid with the target mRNA. this is stable and resistant to RNases, An antisense and a sense probe are used. antisense probes are complementary to the target RNA and hybridise |
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How are RNA probes synthesised? |
1. choose your probe sequence and insert this into a plasmid vector between two promotor sites 2. linearise the ends of the section by restriction enzymes 2. polymerase will attach to either side and create RNA probes. each promotor has its own polymerase of the same name. depending on which strand it transcribes, it will create either a sense or antisense probe. 3. we used to label probes radioactively, but now, we use digoxigenin (DIG) which is incorporated onto one of the nucleotide bases, e.g. to all of the U bases. |
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How is the antibody applied to the embryo? |
the embryo must undergo extensive washing to get rid of any excess probe that has not been incorporated with the mRNA. the sense probe will be completely removed as it is not complementary to the mRNA (it has the same sequence), but the antisense one will remain as it has bound to the mRNA. no colouration is seen at this point. to detect the target genes, two antibodies are used, the primary one (Sheep anti- DIG) and a secondary one (AP conjugated anti-sheep secondary antibody). the secondary one attaches to an enzymes called alkaline phosphatase for visualisation. |
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how is the in situ hybridisation visualised? |
1. bind the primary antibody 2. washes to remove excess primary antibody 3. bind the secondary antibody 4. wash off the excess secondary antibody 5. add the chromogen - BCIP (5-bromo-4-chloro-3-indolyl-phosphate) or NBT (nitro blue tetrazolium) |
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explain what alkaline phosphatase is and how it is used |
- usually isolated from calf intestine - 140kDa protein - catalyses the hydrolysis of phosphate groups from a substrate molecule, resulting in the release of a coloured product or release of light as a by-product - works best at pH 8-10 - can be inhibited by cyanides, arsenate, inorganic phosphate, and divalent cation chelators such as EDTA. - this is a chromogenic reporter |
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explain what horseradish peroxidase is and how it is used |
- this is a chromogenic reporter - 40kDa protein - catalyses the oxidation of substrates in the presence of hydrogen peroxide, resulting in a coloured product and the release of light as a by product - HRP functions optimally at a near neutral pH and can be inhibited by cyanides and sulphates - antibody-HRP conjugates are superior to antibody-AP conjugates with respect to the specific activity of the enzyme and antibody - has a good turn over rate, good stability, and low cost, making it the enzyme of choice for most applications |
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what is whole mount immunostaining? |
- the use of specific antibodies to detect a single target protein in a sample - it is a method of localising a specific protein either on the surface of or within a cell - protocol may be direct or indirect |
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explain the direct method of immunostaining. |
proteins in the cell are targeted with their corresponding antibodies. the antibodies are already tagged that they can be readily visualised when bound |
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explain the indirect method of immunostaining |
the antibody moves into the cell and attaches to the protein being targeted. the antibody itself has no tag and cannot be visualised. to do so, an additional step is needed in which a tag is used. the tag binds to the antibody as a secondary antibody for visualisation. |
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how are embryos prepared for indirect immunostaining? |
1. block endogenous peroxidase. there will be enzymes that can give a false positive result and lots of background. hydrogen peroxide and methanol are used to reduce this via inhibition 2. rehydrate. this prepares the tissues for incubation with the antibody. this needs to be done step wise to avoid disruption of the tissue architecture. 3. permeabilisation - allows penetration of the antibody. unlike in the other method, you are searching for whole proteins so enzymes that degrade proteins are not used. instead, we incubate the embryo in a detergent that makes it more porous |
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how are the antibodies applied in direct/indirect immunostaining? |
1. block the non specific proteins by incubation in a milk powder that interacts non specifically to reduce background 2. apply the primary antibody which binds to the target protein specifically and directly. the embryo is then washed to remove any unbound antibody 3. apply the secondary antibody. this binds to the primary antibody and gives a means of detection. only seen in the indirect method. the direct method is the same, just without this step. |
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how is staining visualised in immunostaining? |
DAB is added and interacts with HRP (the secondary antibody) to give a brown precipitate. this is irremovable. DAB is a chromogen, just like BCIP and NBT of in situ hybridisation |
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what were aristotles ideas of how different forms of the embryo are formed? |
preformation - all of the structures within the embryo are there to begin with and they just get bigger as the embryo develops (later, there were pictures of a homunculus created by Niklaas Harspeler) epigenesis - the structures arise progressively |
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in what two ways can abnormalities arise? |
- malformations where there is a problem with the GENE - disruptions are where there is something in the ENVIRONMENT that has caused the abnormality |
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what are the invertebrate model organisms? |
- fruit fly - nematodes - sea urchin |
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what are the vertebrate model organisms? |
- frog - chicken - zebrafish - mouse |
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what is a(n) a) anatomical approach? b) experimental approach? c) genetic approach? |
a) observe the embryo as it develops. b) development of high skilled embryo manipulation techniques c) study of the detailed genetics that have an influence upon development (includes the use of knock out models) |
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what are the six practical considerations that we must be aware of when choosing model organisms? |
1. number of embryos 2. cost 3. access 4. micro manipulation ability 5. suitable genetics 6. how much we know about the genetics of the organism - the gene inventory |
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give details of the sea urchin as a model organism |
embryogenesis - 5 days life cycle - 50 days short generation time, fast embryogenesis, large number of progeny difficult to breed in labs transparent embryo which is easy to grow easy to manipulate/perturb embryonic development 21 chromosomes that are diploid has a fully sequenced genome |
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what did hans driesch do in 1885? |
demonstrated that you can grow two full urchins when you separate the two cells of a two cell stage embryo and grow them apart. this showed that each cell in the embryo has its own complete set of genetic instructions that could grow into a full organism |
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give details of drosophila as a model organism |
sperm can be retained in the female for a long time creates a syncytial blastoderm through the nucleus division without cell division easy to make mutants and perform transgenics life cycle - 9 days embryogenesis - 10-12 hours lots of progeny easy and cheap to maintain embryos and larvae are easy to grow polytene chromosomes - giant chromosomes that arise from repeated rounds of DNA replication without cell division. allows multiple copies of the same gene well characterised genes Morgan's experiments with drosophila eye colour provide evidence that heritable factors are located on chromosomes |
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give details of the c elegans nematode as a model organism |
all adults are hemaphrodites - they are females that make sperm for a small amount of time there are four larval stages before sexual maturity embryogenesis - 12 hours life cycle - 50 hours (2 days) short generation time and a large number of progeny they are small and cheap to breed. they can be frozen they are transparent and easy to grow and manipulate easy to disrupt gene function through RNAi 5 pairs of autosomes and 1 pair of sex chromosomes fully sequenced genome |
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what did sydeny brenner discover in c elegans? |
his research led to the mapping of the worms entire nervous system traced each of the somatic cells from a single zygote Brenner saw that although there are 1090 cells in the original cell lineage, adult C elegans would only have 959 cells. he discovered programmed cell death in development. |
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give details of the frog (xenopus) as a model organism |
embryogenesis - 4 days life cycle - 10 months large number of eggs rapid development easy to maintain and relatively low cost large egg embryo that develops outside of the maternal environment easy to manipulate and to make transgenics tetraploid genome - four versions of every gene. genome sequenced |
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what did john gurdon do with xenopus? |
cloned frogs using a nucleus from a somatic cell. awarded the nobel prize for his work demonstrating that adult cells can be converted to stem cells. |
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give details of the chicken as a model organism |
embryogenesis - 6 days life cycle - 21weeks relatively good progeny numbers eggs easy to incubate on a large scale without high cost eggs are large, have external development, and are easy to observe easy to manipulate surgically, not easy to generate transgenics and mutants diploid, 38 chromosomes and one pair of sex chromosomes genome has been sequenced |
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what did nicole le dourain do with chick embryos? |
developed a chick-quail chimera that provided critical insights to neural crest formation during development. determined that the precursors in the neural crest were multipoint. |
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give details and the mouse as a model organism |
embryogenesis - 18 days life cycle - 9 weeks reasonable progeny numbers large colonies easy to maintain but may be expensive relatively short embryogenesis but not easily accessible as they are in the mother mutants/transgenics relatively easy to obtain diploid with 21 chromosomes genome has been sequenced |
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what did martain evans do with mice ? |
the first to generate cultured mouse ES cells first to generate knockout mice by injection of genetically modified ES cells into the mouse blastocyst |
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give details of the zebra fish as a model organism |
embryogenesis - 2 days life cycle - 90 days large number of progeny rapid development potentially expensive to maintain transparent embryo develops outside of the maternal environment easy to manipulate gene expression and can generate transgenics 25 chromosomes genome has been sequenced |
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what is a genetic screen? |
a lab procedure used to create and detect a mutant organism. we observe the phenotypes of mutants to get clues about the function of the mutated genes. |
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what is forward genetics? |
analysis of a biological phenomenon starting from a mutant phenotype. also known as mutagenesis screening. includes chemical and insertional mutagenesis |
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what is reverse genetics? |
investigations on a known gene, especially by creation of loss-of-function mutants. this includes transgenesis, targeted mutagenesis, morphosinos and RNAi (indirect gene disruption), dominant negative reagents and domain swapping. |
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How are forward genetics (a mutagenic screen) performed in zebra fish studies? |
to obtain highly mutated fish, we treat male fish with the chemical ENU (N-ethly-N-nitrosourcea). this chemical is well documented and proven to induce numerous mutations into the genome. we can create a high density of mutations by treating the males with ENU six times - once a week for six weeks. the mutation density of 1.10^5-1.5x10^5 bases is achievable. the chemical is an alkalising agent, which is transferred to thiamine residues. this targets the germ cells (that create sperm). the treated sperm will have mutations on any one of the genes of the genome. the mutated male is then mated with a female wild type fish. most of the F1 progeny will be heterozygous for the mutation. most of the mutations will be different in each of the mutated fish. the F1 progeny are allowed to reach sexual maturity before they are interbred. The F2 progeny that this creates will be around 50% heterozygous for the mutation. these are bred (two heterozygotes are bred together) to get the F3 progeny. this progeny will consist of 50% heterozygotes and 25% homozygotes for the mutation. the other 25% will be wild type. the F3 progeny are then screened for abnormalities. if there is an obvious phenotype of the mutant fish, you can trace the abnormality through development to see the point at which the mutation causes the abnormality. it may well be that homozygous mutants are not found. in this case, the mutation may be lethal during development. alternatively, the original sperm may not have been mutated. the mutants may have more than one mutation. in this case, you subject the mutants to further rounds of breeding to separate these mutations. |
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what is an enhancer trap and how is it performed in zebrafish? |
an enhancer trap is a transgene that is integrated into the genome of the organism. the transgene will have a minimal promotor. this alone won't drive expression. the minimal promotor is upstream from a reporter gene such as GFP. after the GFP is a polyadenilation signal that is important for the stability of the RNA and its processing. the enhancer trap disrupts the coding sequence and creates truncated proteins. this will change the function of the protein whilst also expressing the reporter gene. if the cassette inserted into an intron, the gene expression may not be altered at all. |
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what is a gene trap and how is it performed in zebrafish? |
this is similar to an enhancer trap in its outcome. a transgene is used, but instead of a minimal promotor, a splice acceptor is used. this means it will still behave like an exon during translation when it is inserted. as a result, the intron will still be removed during splicing but GFP will be fused to the endogenous protein, meaning that the gene won't be expressed, but GFP will be. there is no chance of GFP being affected by other regulatory elements. sometimes, it doesn't work and the inserted gene trap is no accepted as an exon. in this case, the trap is inserted, but the endogenous protein is still produced. the gene trap plasmid is injected directly into the zebra fish embryo and the organism is allowed to develop. the result is that the offspring of this fish, when crossed with a wild type fish, may have mutations in any given gene in the genome. if the mutations are in the germline, the offspring will have a heterozygous mutation. if you shine a blue UV light on the fish, the ones with the mutation will glow. this makes it easy to detect mutants. The F1 progeny are cross bred to get homozygous mutant F2 fish. |
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what are morpholinos and how are they used in zebra fish in reverse genetics? |
this is an indirect method that does not directly change the gene sequence morpholinos are short pieces of synthetic nucleic acid. the target gene has coding and non coding regions with the final mRNA being the same as the coding regions. morpholinos have complementary sequences that bind to the RNA strand to stop the resulting protein from being functional. because morphilinos are used to replace the backbone, they are resistant to nucleases and are stable. they are best suited to use in free living organisms that have embryos with a large enough embryo for injection. different types will cause different types of abnormality. |
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how is RNAi used in zebrafish? |
this indirect method relies upon the cell's normal response to infection by RNA viruses. When cells are infected with a virus, double stranded RNA is present in the cytoplasm. it comes into contact with a protein called dicer which breaks down the RNA into small fragments called siRNAs. they are unwound to make single strands. the strands are then combined with a protein complex called Risc (RNA induced silencing complex). the single stranded RNAs guide the risc to strands with complementary sequences. the RNAs attach because there are complementary, and risc degrades the mRNA so that no protein can be formed. in zebra fish, the RNA fragments can be injected directly into the embryo as it is not in the maternal environment |
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mutagenetic screens in mice - forward genetics |
the mutagen ENU (N-ethyl-N-nitrosourea) is injected into the male mice to give them mutations in the germ line cells. this can be used as a model for human disease. ENU screens - identification of mouse mutants that will serve as models of human disease. a male mouse with the mutations is bred with a wild type female to give rise to the F1 progeny which are then screened for abnormalities |
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gene targeting through homologous recombination - mouse models - reverse genetics |
take the gene and clone it. alter it with a drug resistance gene (inserted into the coding region of the clone). introduce the modified DNA to embryonic stem cells in culture. cells may take up the DNA. the drug is applied to see which cells have acquired the resistance. the mutation is inserted via recombination. |
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chimeras in mice |
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how is a mutagenic screen carried out for fruit flies? |
in this screen, the chemical EMS (ethyl-methane- sulphonate) is used to create mutations in male fruit flies. when exposed to the chemical, the males can pick up a high number of mutations, some of which will be in the germ line cells. a female carrying a DTS-dominant temperature sensitive gene (DTS/b) is bred with the mutated male (a*/b or a/a or a/a*). in the next progeny, any fly with the DTS gene will die above 29 degrees. the other gene that the female carries is the 'b' gene. this is embryonically lethal when homologous (so it is a recessive gene). a mutated a*/b is bred with a DTS/b female. this produces progeny where some are killed due to homozygous b/b genotypes (embryonic lethal), and others die due to carrying DTS. then, a a*/b male is bred with an a*/b female to produce a progeny containing a*/a* mutant homozygotes. this genotype is screened for phenotypic abnormalities. you may get a screen where no homozygous mutants are found. this would suggest that the mutation is embryonically lethal. this screen has been carried out for all drosophila genes |
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what is P-element mediated transformation in reverse genetics of fruit flies? |
uses a : - known sequence - transposon - jumping DNA each transposon is surrounded by TIRs (terminal inverted repeats |
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how does P element mediated transformation happen in fruit flies? |
you take two white eyed flies and breed them. inject the P elements into the posterior side of the embryo. insert the reporter gene that gives red eyes. both of these things are on their own plasmids. allow the egg to develop and produce more white eyed ones too. breed the developed embryo with a red eyed fly. any of the offspring could have red eyes. any flies that are transformed will have red eyes. this technique is used to over express a gene and to introduce genes of interest to mutant flies. |
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what is a domain swap? |
replace activating domains in transcription factors with repressor domains and visa versa |
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what is a dominant negative genetic screen?
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over expression of a TF lacking its DNA binding domain |
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what is gametogenesis? |
- The process by which sperm and egg are formed - compartmentalised in mammals - separation of somatic and germ cells (gametes) - gametogenesis in animals includes meiotic cell cycle which is a unique cycle to produce haploid cells. meiosis also occurs in yeast, although no gametes are produced |
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what are the events leading to gametogenesis? |
- Formation of germ plasm (NOT in mammals) - determination of primordial germ cells (PGCs) - migration of PGCs to developing gonads - meiosis - sex-specific differentiation - gametogenesis |
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what is germ plasm? |
it is in the egg, but not the egg of mammals. it has the maternal determinants of PGCs. it is localised to one part of the egg. Differentially inherited by different embryonic cells. some of the cells will get germ plasm when the cell divides, but others will not. the ones with the germ plasm, make germ cells. this gives rise to autonomous specification - they have already inherited all of the determinants that are necessary for gametogenesis. |
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what are PGCs? |
- Primordial germ cells are gamete precursors |
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What are PGCs like in mammals? |
they do not have any germ plasm the PGC genesis is due to exogenous cues - this is epigenesis they do not become germ cells autonomously, they must be told to do so |
