L2 + L3 - Basic Principles and Techniques

Question 1

What is pattern formation?

Answer 1

Process by which cells are organised in space and time to produce a well-ordered structure
Establishment of body plan and the main body axes
Cell has positional information – coordinates for each cell

Question 2

What is morphogenesis?

Answer 2

Cell and tissue movement and changes in cell behaviour that give developing organ its 3D shape

Question 3

What does morphogenesis involve?

Answer 3

Cell adhesion
Cell migration
Cell death – e.g. formation of digits
Cell shape

Question 4

What is cell differentiation?

Answer 4

Process by which cells become different from each other and acquire specialised properties
Governed by changes in gene expression which dictate the repertoire of protein synthesised
Progressive restriction of pluripotency

Question 5

The step wise process of cell differentiation?

Answer 5

Cell specification
Determination
Differentiation
Maturation

Question 6

What is growth?

Answer 6

An increase in mass or size
It is a continuous process
Growth rate varies depending on age and organ
Cell proliferation, cell enlargement, accretion

Question 7

What are the 4 ways development is studies?

Answer 7

Embryology – observed biology and experimental manipulation
Developmental biology – study of genes and proteins
Animal models
Genetics

Question 8

What are the two opposing theories of development?

Answer 8

Funnel model

Hourglass model

Question 9

What is the funnel model?

Answer 9

Haeckel
Diversity occurs at later stages
Later proved to be wrong

Question 10

What is the hourglass model?

Answer 10

Von Baer
Early stages are very similar
Intermediate stages very similar between different animals

Question 11

How to find out where and when the gene is expressed in the embryo?

Answer 11

In situ hybridisation – spatial expression 
Northern blot – spatial expression 
RT-PCR
Micro-array 
Reporter lines – spatial expression

Question 12

How to find out if the protein is expressed with the same timing that the gene?

Answer 12

Western blot

Immunohistochemistry

Question 13

How to find out if the gene/protein is essential for development?

Answer 13

Gain of function

Loss of function

Question 14

How to find out how the gene is regulated?

Answer 14

Embryology – tissue manipulation

Manipulating signalling pathways – drugs, transfection, electroporation, genetics

Question 15

How to find out what the tissues/organs derived from the cells that express this gene?

Answer 15

Embryology – chick chimera, labelling with dye

Genetics – labelling with retrovirus or GFP

Question 16

Study of gene expression is used to?

Answer 16

Establish where and when a gene is expressed

Question 17

How do you study gene expression?

Answer 17

In site hybridisation
Reporter lines
High throughput analyses – microarray, RNAseq

Question 18

In situ hybridisation

Answer 18

1. Get reaction compound
2. Incorporate a probe (anti-DIG-AlkPhos) into the compound
3. Probe enters every cell and form a hybrid wherever there is mRNA
4. Use an antibody this is coupled to the probe to identify where probe is bound to mRNA

Question 19

Reporter lines

Answer 19

Reveals where a gene is expressed
Expression of the gene sequence relies on the expression of regulatory enhancer sequences
Can replace gene sequence with reporter gene
Place regulatory sequence and reporter gene into animal model
Creates a transgene which expresses GFP

Question 20

What do microarrays do?

Answer 20

Try to compare the expression profile of all the genes in the genome between two cell populations

Question 21

Microarrays method

Answer 21

1. Start with two samples and isolate the transcripts
2. Reverse transcribe transcripts to create cDNA
3. Each cDNA labelled with a unique fluorescent dye
4. Hybridise to an array
   - Each spot on the grid has a sequence for 1 gene in the genome
5. The probe forms a hybrid with the cDNA on the array forming a stable complex
6. Plate readers and software read the output of the images produced

Question 22

RNAseq

Answer 22

Sequencing step allows short fragments to be sequenced of each cDNA
Use software to determine which genes are expressed
Can use if only a single cell is available

Question 23

Study of proteins is used to?

Answer 23

Investigate the distribution of proteins

Question 24

How do you study proteins?

Answer 24

Immuno-detection – immune-histochemistry, immune-fluorescence
Fusion protein construct

Question 25

Immunofluorescence

Answer 25

Need prior knowledge of protein Need a specific antibody that can recognise the protein Need a primary antibody – specific to protein of interest

Question 26

Immunofluorescence method

Answer 26

1. Take section of tissue of interest and incubate it with primary antibody 2. To reveal where primary antibody has bound to protein use a secondary antibody (IGg coupled to fluorescent tag) which binds to primary antibody

Question 27

Fusion protein construct

Answer 27

Fusion protein - tagged with GFP | Protein expressed in tissue in which it is normally expressed – can now visualise it

Question 28

Fusion protein construct method

Answer 28

1. Start with a DNA fragment that contains the regulatory sequence of gene of interest 2. Leave gene of interest in place 3. Insert the DNA sequence encoding GFP in frame with the DNA sequence of protein of interest 4. Insert this in 5’ or in 3’ 5. Want to create a single transcript that contains mRNA for GFP and mRNA for protein of interest 6. This is translated creating a single protein with GFP fused to it

Question 29

What are developmental genetics used for?

Answer 29

Determine the function of genes

Question 30

How do you study developmental genetics?

Answer 30

Gain of function – transgenesis | Loss of function – forward and reverse genetics

Question 31

Forward genetics conditions

Answer 31

Animal models – c.elegans, drosophila, zebrafish, mouse Agent – chemical mutagen Selection – based on phenotype of interest ENU – N-ethyl N-nitrosourea

Question 32

Forward genetics - positional cloning of mutated gene

Answer 32

``` Animals randomly mutated Cross mutated and wildtype - Mixed offspring Cross offspring to wild type - If wildtype – wildtype offspring - If heterozygous for mutation – 50% offspring will carry mutation Cross offspring together - 25% homozygous for mutation - 25% wildtype - 50% heterozygous for mutation ```

Question 33

Reverse genetics method

Answer 33

1. Homologous recombination 2. ES cell transfection 3. ES cell selection 4. ES cell injection into blastocyst 5. Implantation into mouse 6. Selection of chimeric mouse 7. Breed

Question 34

Reverse genetics - breeding options

Answer 34

If successful integration of mutation into germline – mouse with mutation in every gene Conventional knockout mouse Conditional knockout mouse – floxed allele

Question 35

What does reverse genetics allow you to test for?

Answer 35

Allows you to test if a gene is essential for the development of the embryo

Question 36

What is the problem with reverse genetics?

Answer 36

If you have a gene coding for a protein with an essential function very early on in embryogenesis, you will never get to the point you can analyse various tissues – embryos die very early on

Question 37

Alternative strategy to reverse genetics?

Answer 37

Generate conditional knockout Allows you to bypass the requirement for a specific gene in early and embryogenesis and study its function in later stages of development

Question 38

What do conditional knouts give you the ability to determine?

Answer 38

In a spatial manor where the knockout will occur | In a temporal manor when the knockout will be carried out

Question 39

Conditional knockout method

Answer 39

1. Flank gene of interest with LOX-P sites which are recognised by Cre recombinase 2. Insert into ES cells 3. Generate a mouse which contains LOX-P sites on either side of gene of interest 4. Cross this mouse with mouse that expresses Cre recombinase gene - Cre recombinase and tissue specific promotor - Cre recombinase and a promoter controlled in a temporal manor

Question 40

What is tissue manipulation used for?

Answer 40

Demonstrate inductive function

Question 41

How do you study tissue manipulation?

Answer 41

Tissue ablation Tissue graft Tissue transplantation Bead/cell implantation

Question 42

Tissue manipulation in chick

Answer 42

John Saunders identified area of limb that had organiser function - Identified using a grafting experiment - Posterior to anterior transplantation - duplicated the chick foot

Question 43

What is observational biology used for?

Answer 43

Fate maps and lineage analysis

Question 44

How do you study observational biology?

Answer 44

Cell/tissue transplantation - Spemann and Mangold Cell/tissue labeling with dye Cell/tissue labeling genetically - electroporation, GFP transgenic lines, brainbow mice and zebrabow

Question 45

Chick and quail studies

Answer 45

Quail and chick embryos are very similar | There are antibodies that recognise proteins at the surface of the nucleus of the quail embryo but not the chick embryo

Question 46

Chick and quail studies method

Answer 46

1. Tissues from quail transplanted early in development into chick host 2. Carry out immunohistochemistry later on to identify location of antibody against quail cells - Quail tissue will now cover a very specific area Cant get single cell resolution

Question 47

Brain bow technique in mouse and zebrafish method

Answer 47

1. Create a transgenic reporter 2. Introduce various genes encoding different fluorescent dyes 3. Each constructs are framed by distinct LOX-P sites - Important as can only have one type of incision 4. Introduce this into the animal model 5. Once you cross the animal with Cre recombinase you have a of having a wide range of combination of colours in each of the cells were recombination has taken place 6. Because of the unique colour of each colour we can follow the future fate of the cells