Lecture 2 and 3: Principles and techniques

Question 1

Why are animal models used to study developmental biology?

Answer 1

They are a good way of studying cause and effect. All organisms are related and all vertebrates come from the same ancestral organism therefore findings are all relevant. Embryos need to be accessible and manipulable genetically by making mutations or molecularly by modulating protein activity.

Many of the animal models used are cheap, easily reproducible, have a short life cycle and don’t have many ethical regulations in terms of their use.

Due to ethical regulations and increased concern for the use of animals in research, the use of cell culture and synthetic organs e.g., organ-on-a-chip is increasingly on the rise.

Question 2

How do researchers decide which model organism to use?

Answer 2

Depends on the question/process of interest. Different organisms have different advantages.

For example: researching lung development. First use a land vertebrate with a lung. Second use an organism with analogous structure to identify the conserved genes.

Question 3

State and explain the essential developmental processes that occur during formation of the germ layers.

Answer 3

Formation of three layers, endoderm, ectoderm and mesoderm occurs as a result of gastrulation. It embraces four essential processes:

1. PATTERN FORMATION - interactions between cells to produce a structure and the 3D axes.
2. MORPHOGENESIS - change in cell behaviour that lead to changes in 3D structure of the tissue through modulating cell-adhesion, migration and shape etc. Organism becomes more complex.
3. DIFFERENTIATION - intrinsic/extrinsic signals induce cell fates. Governed by changes in gene expression. As cells age, specialisation increases while pluripotency decreases through repression of genes that promote pluripotency. For example, mesenchymal cells can become bone, cartilage, blood, muscle etc.
4. GROWTH - increase in body mass through division, cell enlargement, accumulation of EC matrix deposition, apoptosis repression. This is continuous - it never stops but the rate changes depending on the organ and age.

Question 4

What are the 3D axes? Draw a diagram to show this.

Answer 4

Dorsal-ventral = back-belly

Anterior-posterior = head-tail

Medial-lateral = midline-edge

Question 5

What is in situ hybridisation?

Answer 5

A technique used to identify localisation of mRNA in an organism to see which cells express it and where.

Question 6

What is a northern blot?

Answer 6

A process used to separate mRNA fragments by size.

Question 7

What is RNA sequencing?

Answer 7

A process used to measure expression levels of all genes in an RNA sample. It can be used in bioinformatic analysis to identify molecular phenotypes and define cellular expression.

Question 8

What is principle component analysis?

Answer 8

Clustering cells of similar genetic read counts in WT and mutants to compare the difference.

Question 9

What is immunofluorescence/immunihistochemistry?

Answer 9

Immunofluorescence refers to cells while immunohistochemistry refers to tissue. A technique used to identify proteins via their antigens. It involves a primary antibody which recognises the antigen and a secondary antibody conjugated to an enzyme that converts a colourless substrate to a coloured one. The secondary antibody binds to the primary.

Question 10

What are the two types of genetic analysis? Explain the difference.

Answer 10

Forward: from phenotype to gene. Identify a phenotype and find the genes that cause it when mutated.

Backwards: from gene to phenotype. Mutate a gene and then seek to characterise the phenotype of the mutation. This is often more desirable because in forward genetics you cannot guarantee that all offspring of the mutant will be mutants.

Question 11

What is Cre-LoxP?

Answer 11

Cre-LoxP is a powerful modern tool used in research allowing for spatiotemporal control of a gene deletion, inversion and translocation. It uses cre recombinase to catalyse site specific recombination between two loxP sites.

Question 12

Case study:

‘A new gene has been discovered which encodes a TF. What is the role of the gene in embryonic development’.

Answer 12

1 - where is the gene expressed?

In situ hybridisation:

• synthesise copy of mRNA complementary to the DNA
• chemically label
• Add an antibody which recognises the label and an antibody conjugated to an enzyme that converts a colourless substrate to a coloured
• Introduce construct into the organism
• See where antibody is to read out cells with the gene

2 - what is the genes function?

RNAseq:

• make a mutation or interfere using a drug (genetic/pharma KO)
• extract RNA from WT and KO cells
• perform RNAseq
• read counts for all genes
• volcano plot to identify genes related to the KO - what does exposure to the drug mean?
• overlap the mutant and WT data - the genes that show the biggest changes are more statistically significant

Principle component analysis:

• dissect intact tissue of WT and mutant
• prepare a dilute suspension of disaggregated single cells
• sequence transcriptome of each cell
• compare read count profiles
• compare WT and mutant to identify which cells groups are effected
• repeat across periods of development

Identify the protein using immunofluorescence/immunohistochemistry:

• section tissue
• apply antibodies to detect protein
• use two colours (e.g., GFP and RFP) to see where it is localised relative to another marker

3 - is the gene NECESSARY for a particular function?

LOF - backwards genetics

• from gene to phenotype
• use cre-LoxP to delete a sequence in the gene of interest
• breed mice
• observe the mutant phenotype

4 - is the gene alone SUFFICIENT for the function?

GOF - graft experiments

• implant tissue in an ectopic location and observe the results

5 - what tissues/organs are derived from the cells that express the gene?

Fate map

• use antibody specific for the protein
• transplant into host of a different species in exact same location
• follow where the tissue ends up using the antiobody

Question 13

Give other names for a genetic knock out.

Answer 13

A genetic null mutation or loss of function mutation.

Question 14

What is the most common way to study gene expression and the most common way to study protein expression?

Answer 14

Gene = in situ hybridisation to mRNA

Protein = immunohistochemistry using antibodies that recognise the protein

Question 15

What are transgenic reporter lines and why are antibodies used more than them?

Answer 15

Mice lines that express fluorescent proteins and enable the detection of gene expression. They don’t exist for all genes but there are many antibodies for many proteins. Antibodies are also cheaper and easier to make.

Question 16

What is ENU?

Answer 16

N-ethyl-N-nitrosourea = a mutagenic alkylating agent that cause point mutations by transferring an ethyl group to nucleobases in DNA.

Question 17

Apart from a genetic KO, how else can yo induce complete or partial loss of function?

Answer 17

1. over express dominant negative forms of a protein from a genetically engineered plasmid vector
2. forced expression of mRNA silencing (siRNA)
3. microinjecting or transfection of morpholinos (redesign the nucleic acid sequence)

Question 18

In gene targeting to create KO mice, how is the selection of ES cells in which recombination has occurred carried out?

Answer 18

• the construct containing sequences homologous to the endogenous gene also contains a cassette for a resistance gene to antibiotics e.g., the neo gene resistant to neomycin
• only cells with successful homologous recombination will survive when grown in neo medium