Lecture 2 and 3 Principles and techniques

Question 1

Ectoderm forms

Answer 1

skin cells
neuron
pigment cell

Question 2

Mesoderm forms

Answer 2

cardiac
skeletal muscle
tubule cell of kidney
RBC 
smooth muscle in gut

Question 3

Endoderm forms

Answer 3

Lung cell
thyroid cell
pancreatic cell

Question 4

Germ cell forms

Answer 4

Sperm

egg

Question 5

How do we study change in cell behaviour or cell-cell communication

Answer 5

Embryology
Developmental biology
Animal models and use of genetics

Question 6

Embryology

Answer 6

• Embryology: observational biology (microscopes) and experimental manipulation (transplants)

Question 7

Developmental biology

Answer 7

study of genes and proteins

Question 8

When a researcher discovers a new gene encoding a TF the questions they need to ask are:

Answer 8

1. Where and when is the gene expressed in the embryo
2. Is the protein expressed with the same timing than the gene
3. How is the gene regulated
4. What are the tissues/organs derived from the cells that express this gene

Question 9

How study where and when gene is expressed in the embryo

Which is not spatial

Answer 9

• in situ hybridization
• northern blot
• RT-PCR (reverse transcriptase PCR)
• micro-array (RNA sequencing) - NOT SPATIAL - unless pick specific place in embryo
• reporter lines (transgenic)

Question 10

Which 2 techniques cannot study location of gene

Answer 10

northern blotting/RT-PCR if the WHOLE cell is used

Question 11

Developmental biology is the study of

Answer 11

gene expression i.e. establishes where and when a gene is expressed

Question 12

Describe process of in situ hybridisation

Answer 12

• Embryo must be fixed (killed)
• Reveals location of target mRNA which you know the identity of
• Probe labelled with DIG which is complementary to the mRNA
• This binds to Anti-DIG (which is an antibody)
• 2 reporter genes used: GFP, beta-galactosidase
• Alkaline phosphatase (coupled to the antibody) gives blue precipitate
• This is seen when anti-DIG hybridises to DIG labelled probe which has hybridised to mRNA i.e. at hybrid DNA:RNA (so when DNA, RNA and AB was present in the embryo)
• Can be temporal as if take embryos at different stages you get diff info. Yet can’t get this from same embryo as it killed in staining. Must be on diff embryos of different stages

Question 13

Describe process of transgenic lines:

1. requires knowledge of
2. expression of a gene is determined by
3. generate a transgene where…
4. reporter gene…
5. introduce this into…
6. GFP is natural fluorescent so preferred as…
7. this is different to fusion of GFP where…

Answer 13

• Requires knowledge of how gene of interest is controlled i.e. what are the promotors and enhancers = regulatory sequences
• Expression of a gene is determined by the regulatory regions
• Generate a transgene where replace gene with a reporter gene e.g. coding sequence for GFP/bGal downstream of regulatory sequences
• Reporter gene i.e. GFP will be expressed wherever the gene is usually expressed when introduced into an animal model
• Introduce this into the animal e.g. mouse, fish, fly
• GFP is naturally fluorescent so preferred – don’t have to fix or kill the embryo so from the same embryo you can generate continuous expression pattern so temporal AND spatial
• This is different to fusion with GFP which is fused in the frame to allow visualisation of the protein, compared to localisation of mRNA here

Question 14

High throughput analyses (microarray, RNAseq) describe

Answer 14

• Simultaneously express 1000s of genes in the embryo in one single experiment
• Looking at the entire genome expressed and not a single gene

Question 15

Microarray is a ‘…’ approach

Answer 15

genome wide

Question 16

What is needed in microarrays

Answer 16

Large amount of mRNA so young/small embryos would be unsuitable

Question 17

Describe microarray technique

Answer 17

o The genome is known and so cDNA for every gene is generated and fixed/placed into a spot on the grid
o mRNA is isolated and labelled from a stage and cell of interest then applied to the grid
o mRNA will hybridise if the gene is present and expressed in that tissue type
o Software reads intensity of fluorescent emitted from the plate
o Can see level of expression of each gene – not necessarily spatial unless can pick specific place in embryo.

Question 18

RNA sequencing describe

Answer 18

o Instead of hybridisation, sequence at the end of fragment to determine the gene
o Can use much less mRNA so advantage vs microarray

Question 19

Why do we see if the protein is expressed with the same timing as the gene

Answer 19

This is done because the production of a mRNA transcript does not mean a functional protein will be produced. The protein may remain untranslated for a period of time to control when and where the gene product functions.

Question 20

Techniques for seeing id the gene is expressed at the same time as the protein

Answer 20

Western blot

immunodetection

Question 21

Western blot describe

Answer 21

• Analyses where and when a gene is expressed if a specific tissue is used to provide the mRNA. If the whole cell is used then it would give no information as to the location of the gene in the embryo

It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide.

Question 22

Immunodetection techniques x2

Answer 22

Immunofluorescene and immunohistochemistry

Question 23

Immunodetection describe

Answer 23

• Investigates the distribution of proteins
• Here you need prior knowledge of the protein and a specific AB to recognise the protein
• This is allows high spatial resolution of sub cellular structures
• If combine with other techniques, get temporal resolution

Question 24

Immunofluorescence describe

Answer 24

• Tissue is fixed for staining so must analyse different tissues at different stages for temporal resolution
• Tissue containing epitope specific to a protein is exposed to a primary antibody
• Primary AB from different species and is raised against secondary AB (anti-IgG coupled to a fluorescent tag)

Question 25

``` Fusion protein construct: Used in which animals Uses Method Example ```

Answer 25

- This is different from a transgenic reporter line - Used in mouse or zebrafish - For live visualising and localisation of protein – temporal and spatial - Coding sequence for GFP is introduced and fused in the coding frame at the future N or C terminus, with the coding sequence of the gene of interest - Gene of interest is left intact - Example: transcript made at nucleus, see diffusion of protein throughout axon of neuron so example where localisation differs between mRNA and protein

Question 26

Gain of function + example

Answer 26

mutation in a gene that confers a gain in the activity of the protein product encoded by the mutated gene e.g. Transgenesis.

Question 27

Loss of function + example

Answer 27

mutation in a gene that disrupts the expression or the function of the protein product encoded by the mutated gene reverse or forward genetics

Question 28

Forward genetics - seeks to identify... - what do you know about the gene - animal models - agent used - leads to...

Answer 28

- Seeks to identify a gene whose mutation caused a particular phenotype - Do not know the gene to begin - C.elegans, drosophila, zebrafish, mouse. - Agent: chemical mutagen – ENU (N-ethyl N-nitrosourea) - Leads to positional cloning of mutated gene

Question 29

E.g. large scale mutagenesis screens for recessive mutations in zebrafish

Answer 29

- Treat male with mutagen x WT female - Male mutagenized as spermatogonia stem cells can still undergo further division but oocytes have already undergone meiotic division - Cross F1 male offspring (+/m) with WT female (+/+) - F2 progeny are 50% WT (+/+), 50% heterozygous for the mutation (+/m) - Sibling cross of F2 progeny means 25% F3 progeny will be homozygous for mutation (i.e. breed to homozygosity)

Question 30

Reverse genetics - seeks to charachterise.. - what do you know about the gene - introduce a mutation so... - Both KO...

Answer 30

- Seeks to characterize the phenotype of particular mutated gene - Already know the gene - Introduce a mutation so no transcript is made or disrupt function of gene product - Both KO eliminate gene function but in all the cells of the embryo vs subset of cells

Question 31

Conventional KO

Answer 31

1. Introduce mutation to disrupt reading frame and so translation of protein by HR 2. Introduce sequence that codes for gene providing resistance to antibiotics by HR 3. ES cell transfection 4. ES cell selection – if HR has taken place, protein translated that confers resistance to AB 5. ES cell injection into inner cell mass of blastocysts 6. Implant this into surrogate mother 7. Chimeric mouse selected (some cells from brown host, some from black ES) 8. Breed to germline transmission 9. Higher degree of chimerism means higher chance ESC have been incorporated into the germ cells so mutation will be translated to the next generation 10. Mutation will be present in every cell of the offspring = issue – cells may not live past post-implantation if the gene has an early function

Question 32

Conditional KO

Answer 32

1. Use if the gene is essential for early development 2. Introduce short sequences flanking gene of interest (has no impact on gene function) 3. Same steps to get mouse where all cells contain floxed version of the gene 4. Generate mouse should have cre expressed under a certain promoter 5. Cross these 2 mice 6. Where conditions of cre promotor met – cre expressed 7. Cre recombinase mouse recognises these sites and so deletes sequence between these 2 sites as cre cleaves inbetween loxP sites and gene is lost 8. So offspring of this cross will contain cells in which gene of interest has been deleted but only in cells expressing cre 9. Bypass any potential function that gene might have had early in development so KO is only in one cell type

Question 33

Methods to see how the gene is regulated?

Answer 33

* Embryology – tissue manipulation (graft, ablation) | * Manipulating signalling pathways – drugs, transfection/electroporation, genetics

Question 34

Tissue manipulation demonstrate

Answer 34

• Demonstrate inductive function

Question 35

Methods of tissue manipulation

Answer 35

- Tissue ablation, graft, transplantation (surgical) | - Bead/cell implantation (signaling molecules, drugs)

Question 36

Spemann and Mangold

Answer 36

e.g. Spemann Mangold 1924, Graft organiser into an ectopic location (on the ventral side), Leads to duplication of the axis, Concluded that the region has an organiser capacity i.e. cells have inductive function and control future development of the embryo

Question 37

John Sanders and Robert Riddle

Answer 37

When ZPA is grafter to ectopic location, get duplication of digits with symmetry

Question 38

What are the tissues/organs derived from the cells that express this gene? (fate mapping)

Answer 38

* Embryology: chick/quail chimera, labeling with dye. | * Genetics: Labeling with retrovirus or GFP, brainbow.

Question 39

Observational biology for/methods

Answer 39

* For fate maps and lineage analyses * Methods: cell/tissue transplantation (Spemann Mangold), label with dye, label genetically (electroporation, GFP, transgenic lines, brainbow, zebrabow)

Question 40

Chick quail chimera - similarities - method - found - cant get

Answer 40

- Both have similar size/differential timing - Took small group of cells from quail embryo and transplanted into chick - Use immunohistochemistry to see where quail contributed as AB recognise proteins at surface of quail but not the chick - Here she found groups located in the gastrulating embryo contribute to dorsal part of neural tube which is a key area where neural crest cells are born - Can’t get single cell resolution

Question 41

Genetic labelling - use... to insert... - which animals - allows...

Answer 41

- Use of homologous recombination to insert various fluorescent genes each of which are flanked by loxP sites - each loxP site is slightly different so is recognised by a different cre recombinase - Can only have one type of excision - this will depend on the cell type and leave a certain set of colors left once crossed with cre recombinase - Introduced into mouse (brainbow), or zebrafish (zebrabow) - Allows single cell resolution as each cell will be labelled with a unique colour of various shades