Lecture 3 Flashcards
Describe Haeckels funnel model of development
Embryos start off developing similarly but become progressively different as time goes on
How does the Von Baer Hourglass model differ to the Haeckel funnel model
Embryos start off developing differently, but then converge at gastrulation, showing substantially conserved phenotypes. After this point they then diverge again
How can the spatial and temporal expression of a gene of interest be studied in development, what can you actually measure
You can investigate where and when the mRNA of this gene is transcribed
List the 5 techniques that can be used to determine where/when a gene is expressed in the embryo
In situ hybridisation, northern blotting, reverse transcriptase PCR, micro-arrays and reporter lines
Outline the process of in situ hybridisation
A probe with complementary base pairs to the target gene is synthesised and labelled with a digoxigenin (DIG) immune-tag. This will bind to the target sequence of the gene of interest and then a secondary antibody binds to the DIG label. Binding of the secondary antibody leads to an enzymatic reaction that produces a reaction product that indirectly marks all the cells that produce that specific mRNA.
What is the major disadvantage of in situ hybridisation
You have to kill the embryo in order to carry out the technique
Explain the process of reporter line transgenics
Introduce a coding sequence for a reporter gene i.e. GFP, RFP or ?-gal into the coding sequence of the gene of interest. This will result in the reporter gene being under control of the target genes promoter sequence. Hence, wherever the gene of interest is expressed the reporter will be too. This can be easily visualised or investigated
What methods can be used to determine if a protein is actually expressed at the same time as a gene by investigating if the mRNA produced is actually transcribed
Western blotting, immunohistochemistry
Explain how immunofluorescence can be used to visualise where a protein is expressed during development
Introducing labelled tags to antibodies for various epitopes of a protein of interest allow you to visualise where these epitopes and proteins are expressed
Gain and loss of function experiments are used to investigate if target genes are essential in development, T or F
T
How can loss of function mutations be used to test for essential genes in development
By introducing a premature STOP codon into the target sequence you can investigate how essential a gene is in development. Loss of function mutations results in a disruption in the expression or function of the mutated genes
How can gain of functions mutations be used to investigate essential developmental genes
Mutations in regulatory regions of a DNA sequence can lead to increased levels of transcription. This confers a gain in the activity of the mutated protein
What is the difference between forwards and reverse genetics
Forward genetics investigates the identity of genetic mutations responsible for a specific observed phenotype. In contrast, reverse genetics seeks to characterise the phenotype produced from a particular gene mutation
How can tissue ablation be used in the study of gene regulation during development
Tissue ablation and ectopic grafting experiments can be correlated with levels of transcription factors. You can also investigate is a transcription factor soaked bead is sufficient to direct gene expression
How can the tissues/organs that are derived from cells that express a certain gene be elucidated
This is investigated in order to determine if every single tissue expressing a factor originated from the early structure expressing it. This can be investigated through fate mapping and determination experiments.
