Lecture 2 - basic principles and techniques

Question 1

Where are nearly all cells of the mammalian body derived from?

Answer 1

3 germ layers that are formed during gastrulation

Question 2

What are the 3 germ layers?

Answer 2

BLASTULA into:
- Ectoderm (e.g. epidermis)
- Mesoderm (e.g. endoskeleton)
- Endoderm (e.g. guts)

Germ cells also set aside - this also occurs in gastrulation.

Question 3

What stage during vertebrate embryo development are remarkable similarities exhibited?

Answer 3

Pharyngula stage

Question 4

What develops during the pharyngula stage?

Answer 4

• Pharyngeal pouches
• Somites
• Notochord
• Hollow neural tube
• Post-anal tail

The Pharyngula stage indicates that developmental processes are highly conserved in evolution.

Question 5

What 4 co-ordinated biological processes underlie embryonic development?

Answer 5

• Pattern formation
• Morphogenesis
• Cell differentiation
• Growth

Question 6

What is pattern formation?

Answer 6

The process by which cells are organized in space and time to produce well-ordered structures within the embryo. ‘identify via coordinates’.

Question 7

What is morphogenesis?

Answer 7

Cell/tissue movement and changes in cell behaviour that gives the developing embryo or organ its shape in 3D.

Cells move and reorganize via morphogenesis

Question 8

What is morphogenesis composed of?

Answer 8

• cell adhesion
• cell migration
• cell death
• cell shape

Question 9

What is cell differentiation?

Answer 9

The process by which cells become different from each other and acquire specialized functional properties.

Governed by changes in gene expression, which dictate the repertoire of proteins synthesized.

Question 10

What is the of specialization?

Answer 10

Pluripotency

Question 11

What is the pathway from stem cell to maturation?

Answer 11

stem cell/progenitor cell
I
specification
I
determination
I
differentiation
I
post-mitotic maturation

Question 12

What is growth?

Answer 12

increase in mass/size
- continuous process (embryonic, foetal, post-natal, adult
- growth rate varies depending on age and organ
- cell proliferation, cell enlargement, ECM production (secretions)

Question 13

What processes underpin developmental processes?

Answer 13

• Changes in individual cell behaviour
• Cell-cell communication
• Gene expression

Question 14

How does analysis of the processes that underpin developmental processes occur?

Answer 14

• animal models and use of genetics
• molecular biology: study of genes and proteins
• embryology: observation and experimental manipulation
• in vitro culture and manipulation of cell or tissue fragments

Question 15

How will research teams study where new genes are expressed in the embryo?

Answer 15

• in situ hybridization
• reporter lines (transgenic)
• RNA sequencing

Question 16

What does in situ hybridisation target?

Answer 16

detects messenger RNA (mRNA) for the gene - not the protein

Question 17

What attaches to the target mRNA?

Answer 17

DIG-labelled probe

Question 18

What attaches to the DIG-labelled probe?

Answer 18

anti-DIG-AlkPhos

Question 19

What attaches to the anti-DIG-AlkPhos?

Answer 19

reaction product

Question 20

What is the process of In situ hybridization?

Answer 20

Probes interact with the target mRNA and recognise the DIG attached. These are recognised by antibodies (anti-DIG). Also has AlkPhos (an enzyme). This enzyme can convert substrate to reactive product.

Complementary sequence to mRNA that mRNA that is produced. Endogenous mRNA (sense strand) is produced then a complementary antisense strand. Also has DIG labels, which are detected by antibody anti-DIG.

Question 21

What is DIG (digoxigenin)?

Answer 21

a molecule that can be conjugated to nucleic acids, such as DNA or RNA probes in order to label them?

Question 22

What is anti-DIG?

Answer 22

The antibody used to detect DIG is anti-DIG, which specifically recognises and binds to the DIG label.

Question 23

What is Alkaline phosphate?

Answer 23

Alkaline phosphatase (AlkPhos) is an enzyme that is often conjugated to the anti-DIG antobody. When the antibody binds to the DIG label, the alkaline phosphatase enzyme can catalyze a reaction that produces a detectable signal, often a colour change, making it possible to visualize the labelled probe.

Question 24

What are reporter lines?

Answer 24

Green Fluorescent Protein

Question 25

Why is fluorescence good for visualization?

Answer 25

Fluorescence gives high contract - good for visualization as there is a large difference

Question 26

How does fluorescence work?

Answer 26

Fluorescence works as an atom becomes excited by light. The electron will then bounce out to a higher energy state, and stays there for a fraction of a second, before returning to its proper level. In the process though, it emits some light, making fluorescence molecules - through the ability to absorb light at a shorter wavelength and emit it at a longer wavelength. Shorter wave length - shorter. Excitation involves heat loss, but there's a shift in the colour (e.g. green to red).

Question 27

What is GFP excited at and emit at?

Answer 27

Excited at 475nm Emits at 510nm

Question 28

Why use fluorescence?

Answer 28

- allows imaging of proteins in live animals - use fluorescence of antibody detection

Question 29

What is needed to make reporter lines?

Answer 29

Entire genomic sequence of gene. GFP made instead of normal gene product. All takes place in test tube then reintroduced into animal.

Question 30

What is RNA-sequencing?

Answer 30

A technique for measuring expression levels of all genes in different samples (e.g. different tissues or with different mutations or with different chemical treatments). 2 samples, hormone present in one and not in another. Extract RNA then perform RNA-seq. Then read counts for all genes and compare gene profiles using volcano plot.

Question 31

How does RNA-sequencing function?

Answer 31

RNA-sequencing functions by isolating RNA (usually from tissue). The extracted RNA is then sequenced to get an idea of all the genes that are expressed in that tissue.

Question 32

What are the applications of RNA-sequencing?

Answer 32

- Exploring function of hormone (in petri-dish with/without hormone) - Comparison of profiles can occur (transcriptional differences between the 2) - can be used to compare disease tissue - e.g. cancer, liver tumour vs normal liver (where are the gene expression differences between the 2) - Mutants - can be compared to wild-type

Question 33

What do volcano plots show?

Answer 33

Plot differences between 2 treatments - V shape - '\' = downregulated genes - '/' = upregulated genes

Question 34

How do in-situ hybridisation and reporter gene constructs differ to RNA-seq analysis?

Answer 34

In-situ hybridisation & Report gene constructs - expression patterns of individual genes. RNA-seq = quantitative information about expression of all genes in the biological sample and identify genes exhibiting differential expression between samples.