Intro + Methods Flashcards
What are the two general phases of life?
Build up: Embryogenesis,adolescence adulthood
Gradual functional decline: Later adulthood
Who were the IVF pioneers?
- Steptoe, Robert Edwards and Jean Purdy
**Jean Purdy was the first person to observe and describe the human blastocyst stage embryo
What is the epigenesis theory?
- Aristotle
Embryos develop from microscopic entity that lacks any detailed form but somehow becomes more complex, acquires new structures within it as it grows i.e. complexity emerges gradually
What is the Pre-formation theory?
embryo begins life as a perfect microscopic form of what it will become. Proposed by Malphigi. Drew and presented to the world their descriptions of sperm.
Describe the principles of the cell theory
- Proposed in 19th century
- All organisms are composed of one or more cells
- The cell is the most basic unit of structure,function and organisation in all organisms
- All cells arise from pre-existing living cells
What is the Germ plasm determinants theory and who proposed it?
Weismann
- All germ cells contain a full array of determinants of cell identity that then become parcelled out into different lineages within the developing embryo i.e. muscle cell determinants go in to muscle lineages
What is the key thing to remember with mutants and gene expression?
all genes have the mutation but only the cell that require that protein the mutation codes for will be affected
What is paracrine communication?
transmitted from one cell to another which isn’t immediately adjacent over a diffusion gradient
what is autocrine communication?
cell signals to itself at the same time its signalling to other cells
what is juxtacrine communication?
cells physically adjacent contact via surface proteins which remain on the surface (e.g. notch signalling)
What is meant by cell signalling competence?
CELLS MUST HAVE THE CAPACITY TO PERCEIVE THE SIGNAL AND RESPOND APPROPRIATELY
What is the difference between an instructive and a permissive signal?
instructive= initiates new program
permissive = provides favorable environment for a specific program i.e. makes it easier for another cell to bind
Ways at which control of gene expression can be exerted?
Production of mRNA
Processing/stability of mRNA
Production of proteins
Activity of proteins
Why are transcription factors called cis-regulators?
they bid close to genes that they regulate , bind to enhancers upstream of the specific gene
What is Morphogenesis and what does it need?
Cell/tissue movements and changes in cell behaviour that give the developing embryo or organ its shape in 3D
Needs:
- Cell adhesion
- Cell migration
- Cell death
- Cell shape
Throughout differentiation, what increases over time?
potency
What is meant by post-mitotic maturation?
Cells have reached their fate and will stop proliferating
What is embryology?
- Observation and physical experimental manipulation i.e. breaking up embryos and reconstructing them to understand how tissues and cells communicate with eachother
What do all vertebrates used as model organisms have in common?
They all go through pharyngula:
- All have a structure called the notochord
- Neural tube is hollow with lumen
- All have a post-anal tail
- All have pharyngeal clefts in the anterior - portion of the embryo
- All have somites
What is in-situ hybridisation?
- allows you to see gene expression by locating mRNA transcripts for certain genes
- Find a probe complementary to the mRNA transcripts you are looking for
-Can be identified with antibodies to observe location through converting colourless substrate into blue product via enzyme
What are recombinant DNA techniques?
- Modify genome of the organism you’re interested in so the gene you’re interested in studying expresses a fluorescent protein as well to highlight under UV light the cells in which the gene is actually being expressed
- uses GFP
What is RNA-sequencing?
- technique for measuring expression levels of all genes in many different RNA samples
- Extract RNA from material and sequence everything and compare expression levels of every gene to wild type
- level of gene expression looked at using a volcano plot
How is a volcano plot interpreted?
- The greater the fold change either higher or lower means the bigger statistical difference
- The numbers are the measure of transcript abundance
What is the process for single cell RNA sequencing?
- Prepare organ + Dissect intact tissue
- Prepare a dilute suspension of disaggregated single cells
- Sequence the full transcriptome of each single cell separately
- Count transcript numbers for each gene in each cell
- Assign gene expression differences as being relatively high or relatively low for each cell
- mean read count for each gene i.e. in the cell is the gene expression higher or lower than the mean, in order to get a visual representation of the complex abundance of cells and their gene expression
How is a mutation studied?
- Dissect intact brains from WT and mutant, prepare single cell suspension
- Perform sc RNA-seq
- Analyse data
What is immunoflouresence/immunihistochemistry?
- Taking sections of embryo and putting them on a slide using antibodies (immunodetection)
- Antibody recognises PROTEIN of interest
- Secondary antibody attached to a fluorescent label
- Image samples simultaneous with different fluorescently tagged proteins
What is a fusion protein construct?
Fuse protein coding sequence GFP sequence onto interested protein sequence to create a fusion protein so you can visualise and see location
What are some examples of GOF experiments?
- Increase protein levels
- increase expression
- increase length of expression activity
- change location
What is the difference between forward genetics and reverse genetics?
Forward = phenotype- gene conducting random mutations to see what causes the phenotype
Backward = gene-phenotype introducing mutations to post-embryos and analysing them to see if gene is essential in the process
