Intro + Methods

Question 1

What are the two general phases of life?

Answer 1

Build up: Embryogenesis,adolescence adulthood
Gradual functional decline: Later adulthood

Question 2

Who were the IVF pioneers?

Answer 2

• Steptoe, Robert Edwards and Jean Purdy

**Jean Purdy was the first person to observe and describe the human blastocyst stage embryo

Question 3

What is the epigenesis theory?

Answer 3

• 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

Question 4

What is the Pre-formation theory?

Answer 4

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.

Question 5

Describe the principles of the cell theory

Answer 5

• 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

Question 6

What is the Germ plasm determinants theory and who proposed it?

Answer 6

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

Question 7

What is the key thing to remember with mutants and gene expression?

Answer 7

all genes have the mutation but only the cell that require that protein the mutation codes for will be affected

Question 8

What is paracrine communication?

Answer 8

transmitted from one cell to another which isn’t immediately adjacent over a diffusion gradient

Question 9

what is autocrine communication?

Answer 9

cell signals to itself at the same time its signalling to other cells

Question 10

what is juxtacrine communication?

Answer 10

cells physically adjacent contact via surface proteins which remain on the surface (e.g. notch signalling)

Question 11

What is meant by cell signalling competence?

Answer 11

CELLS MUST HAVE THE CAPACITY TO PERCEIVE THE SIGNAL AND RESPOND APPROPRIATELY

Question 12

What is the difference between an instructive and a permissive signal?

Answer 12

instructive= initiates new program
permissive = provides favorable environment for a specific program i.e. makes it easier for another cell to bind

Question 13

Ways at which control of gene expression can be exerted?

Answer 13

Production of mRNA
Processing/stability of mRNA
Production of proteins
Activity of proteins

Question 14

Why are transcription factors called cis-regulators?

Answer 14

they bid close to genes that they regulate , bind to enhancers upstream of the specific gene

Question 15

What is Morphogenesis and what does it need?

Answer 15

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

Question 16

Throughout differentiation, what increases over time?

Answer 16

potency

Question 17

What is meant by post-mitotic maturation?

Answer 17

Cells have reached their fate and will stop proliferating

Question 18

What is embryology?

Answer 18

• Observation and physical experimental manipulation i.e. breaking up embryos and reconstructing them to understand how tissues and cells communicate with eachother

Question 19

What do all vertebrates used as model organisms have in common?

Answer 19

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

Question 20

What is in-situ hybridisation?

Answer 20

• 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

Question 21

What are recombinant DNA techniques?

Answer 21

• 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

Question 22

What is RNA-sequencing?

Answer 22

• 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

Question 23

How is a volcano plot interpreted?

Answer 23

• The greater the fold change either higher or lower means the bigger statistical difference
• The numbers are the measure of transcript abundance

Question 24

What is the process for single cell RNA sequencing?

Answer 24

1. Prepare organ + Dissect intact tissue
2. Prepare a dilute suspension of disaggregated single cells
3. Sequence the full transcriptome of each single cell separately
4. Count transcript numbers for each gene in each cell
5. Assign gene expression differences as being relatively high or relatively low for each cell
6. 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

Question 25

How is a mutation studied?

Answer 25

1. Dissect intact brains from WT and mutant, prepare single cell suspension 2. Perform sc RNA-seq 3. Analyse data

Question 26

What is immunoflouresence/immunihistochemistry?

Answer 26

- 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

Question 27

What is a fusion protein construct?

Answer 27

Fuse protein coding sequence GFP sequence onto interested protein sequence to create a fusion protein so you can visualise and see location

Question 28

What are some examples of GOF experiments?

Answer 28

- Increase protein levels - increase expression - increase length of expression activity - change location

Question 29

What is the difference between forward genetics and reverse genetics?

Answer 29

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