lecture 3 Flashcards
name 3 ways to study development
1) observational biology= manipulation of embryos (embryology)
2) Developmental= study of genes and proteins
3) animal models= many share similar early developmental features
What is the Haeckle funnel model?
Model stating that all organisms begin development with very similar features then gradually begin to show variation
What is the Von Baer Hour glass model?
early stages aren’t so similar but the intermediate stages e.g. gastrulation are and then they begin to differ. Many known genes responsible for disease display similar effects in animal models
How can you identify where and when a gene is expressed?
ISH, northern blot, microarrays, transgenic reporter line, RT-PCR.
How do you carry out ISH and why is it used?
best method to find out where and when a gene is expressed
1) Make an RNA probe that is labelled with DIG
2) harvest and fix the embryo to give it access to the probe and antibodies
3) detect probe using antibody conjigates to alkaline phosphatase= blue precipitate
Describe how to carry out a transgenic reporter line
Need to know the promotors and enhancers involved in the gene of interest’s expression. Replace the gene with GFP
Describe how microarrays can be performed on young embryos
1)Create cDNAs from the RNA transcripts from the 2 tissue types and label with GFP tag
2) allow them to hybridise on the arrays
3) use computer software to observe which genes are expressed
for young embryos use short DNA fragments (RNA seq) which can be used on a single cell
How do you test if the protein is produced at the same time as the gene?
Use western blot or immunohistochemistry (label the protein to observe). Need to study the transcript as well because the protein may not end up where it’s produced e.g. ECM and morphogens
describe immunodetection
Can only be carried out if have prior knowledge of the protein and have an antibody to bind to it. Primary AB binds then secondary which is conjugated fluorescent tag
What is a fusion protein and how does it differ to a reporter?
A tagging protein e.g. GFP is inserted to either the 5’ or 3’ end- produces a single transcript with 2 genes which doesn’t alter protein function. In a reporter, the gene is replaced
How do you test if a gene is essential for development?
1) forward genetics= seeks to identify the gene of a particular phenotype
2) seeks to discover the phenotype of a mutated gene
How is forward genetics carried out and on what organisms?
carried out on drosophila, C. elegans, mouse and zebrafish.
1) mutate the animal using a mutagen e.g. ENU
2) cross with WT then again with WT then interbreed- 1/4 offspring should be homozygous for a mutation
3) carry out a screen and look at organs to identify mutated gene
How is reverse genetics carried out?
Homologous recomb neo into ES cell which has antibiotic resistance. Inject into blastocyst- successful integration indicated by chimerism. If gene is required early on in development, the organism will die- need to carry out a conditional KO
How is a conditional KO carried out?
Flank gene of interest with Lox P site so it can be recognised by cre recombinase when inserted into the ES cell- can be a cre recombinase with a tissue specific promoter or controlled in a certain way. Can view function postnatally.
How are tissues manipulated to test for signalling and what are the requirements?
Tissue grafts- e.g. dorsal tissue placed into ventral tissue= induces a secondary axis in zebrafish (1924). Need to use chicks- drosophila are too small and mammal embryos are inaccessible
