Techniques

Question 1

What are morphological descriptions?

Answer 1

Embryos at different stages being isolated and their appearance being described.

Question 2

What is histology?

Answer 2

Embryos/embryonic organs are isolated and fixed using a fixative (e.g. paraformaldehyde) and embedded in wax, sectioned and put on slides and viewed under the microscope.

Question 3

What stain is used in electron microscopy?

Answer 3

Electron dense staining - heavy metals.

Question 4

What staining is used in light microscopy?

Answer 4

Differential staining - acid-base, histochemical.

Question 5

What happens to a mouse cerebellum as it develops?

Answer 5

It folds more and more extravagantly.

Question 6

What is cell ablation?

Answer 6

Removal of a cell during early development to see the effect on the embryo as it develops. It can be used to study regeneration, development and compensation.

Question 7

What is a modern twist on cell ablation?

Answer 7

Using lasers or toxins to kill cells.

Question 8

What is an example of a natural occurring mutant and what is the effect?

Answer 8

Talpid chick - chicken mutant with abnormal limb patterning.

Question 9

What is another example of a naturally occurring mutation?

Answer 9

The mouse piebald mutation - broad white patches on the belly and head fur. The melanocytes have mutations in a gene called c-kit.

Question 10

What is lineage tracing and why is it useful?

Answer 10

The identification of all progeny of a single cell. It is useful as it is an essential tool for studying stem cell properties in adult mammalian tissues.

Question 11

What are the four methods for fate mapping and lineage tracing of embryos?

Answer 11

Grafting, vital dye marking, fluorescent dye and radioactive labelling, and genetic marking and grafting.

Question 12

What is tissue grafting?

Answer 12

Cells are grafted and allowed to develop to see what position they occupy.

Question 13

What is a problem of using dyes to track development of cells in an early stage embryo?

Answer 13

The dye gets diluted as the cell divides.

Question 14

What are methods to identify genes regulating development?

Answer 14

Homology searching, cloning of cDNA and screening of libraries, screening and sequencing of expressed sequence tags, and mapping and identifying genes responsible for a mutant phenotype.

Question 15

What are the methods to detect gene expression?

Answer 15

mRNA, northern blotting, RT-PCR, microarrays, in-situ hybridisation.

Question 16

What are the methods to detect protein?

Answer 16

Immunostaining, western blotting.

Question 17

What is the process for a southern blot?

Answer 17

DNA is restricted on gel and denatured and transferred to a filter. It is hybridised with a probe - labelled DNA or RNA.

Question 18

What is the process for a northern blot?

Answer 18

RNA on gel is transferred to a filter and hybridised with a probe labelled with DNA.

Question 19

What is the process for a western blot?

Answer 19

There is protein on gel that is transferred to a filter and reacted with a probe (antibody).

Question 20

What can be used for analysis of gene expression?

Answer 20

Northern blotting, polymerase chain reaction, insitu hybridisation and microarray analysis.

Question 21

What is reverse transcriptase?

Answer 21

An RNA-dependent DNA polymerase that forms cDNA from mRNA.

Question 22

How is mRNA removed during RT-PCR?

Answer 22

RNase.

Question 23

What can microarray be used for?

Answer 23

Large scale comparison of gene expression in different samples.

Question 24

How can a microarray be carried out?

Answer 24

Isolate mRNA from the samples, use reverse transcription and fluorescent labelling. Hybridize to microarray.

Question 25

What is in situ hybridisation?

Answer 25

A method of localising either mRNA within the cytplasm or DNA within the chromosomes of the nucleus.

Question 26

How does in situ hybridisation work?

Answer 26

mRNA of interest is detected in cell/tissue using a labelled probe that is complementary to the mRNA of interest.

Question 27

How can gene expression be manipulated?

Answer 27

Overexpression of genes using transgene expression systems, reduced expression of genes using dominant negative proteins, gene targeting by homologous recombination, RNAi, morpholinos and mutageneis screens.

Question 28

What are techniques to analyse gene function?

Answer 28

Introduction of cloned genes into cells by transfection/electroporation. Generation of transgenics by introduction of DNA into fertilized eggs, generation of mutants by homologous recombination in ES cells, regulation of gene expression by transgenic analysis, knockout gene function by antisense RNA, RNA interference.

Question 29

What is the approach using dying of early stage embryos for lineage tracing?

Answer 29

Dye is applied to the early stage embryo which can be seen in later stages.

Question 30

What is the problems of using dyes for lineage tracing?

Answer 30

As the cells divide the dye becomes diluted. There is a limited time frame in which you can do these experiments.

Question 31

How can lineage tracing be conducted in rat retinal cells?

Answer 31

LacZ retroviral vector can be introduced which can then be tracked in the reconstructed retina.

Question 32

What was Spemann and Mangold’s experiment?

Answer 32

Tissues were grafted between amphibian embryos and the development changes were observed - investigating dorsal cells role in gastrulation.

Question 33

What is a commonly used label in In situ hybridisation?

Answer 33

Digoxigenin - a steroid that can be coupled to nucleotides.

Question 34

How does visualisation occur in in situ hybridisation?

Answer 34

The labelled probe is detected using an anti antibody of the probe which contains a visualisation method such as alkaline phosphatase that can cause a colour reaction.