Lecture 2 and 3 Principles and techniques

skin cells neuron pigment cell

cardiac skeletal muscle tubule cell of kidney RBC smooth muscle in gut

Lung cell thyroid cell pancreatic cell

Sperm egg

Embryology Developmental biology Animal models and use of genetics

• Embryology: observational biology (microscopes) and experimental manipulation (transplants)

study of genes and proteins

• Where and when is the gene expressed in the embryo • Is the protein expressed with the same timing than the gene • How is the gene regulated • What are the tissues/organs derived from the cells that express this gene

• in situ hybridization • northern blot • RT-PCR (reverse transcriptase PCR) • micro-array (RNA sequencing) - NOT SPATIAL - unless pick specific place in embryo • reporter lines (transgenic)

northern blotting/RT-PCR if the WHOLE cell is used

gene expression i.e. establishes where and when a gene is expressed

• Embryo must be fixed (killed) • Reveals location of target mRNA which you know the identity of • Probe labelled with DIG which is complementary to the mRNA • This binds to Anti-DIG (which is an antibody) • 2 reporter genes used: GFP, beta-galactosidase • Alkaline phosphatase (coupled to the antibody) gives blue precipitate • This is seen when anti-DIG hybridises to DIG labelled probe which has hybridised to mRNA i.e. at hybrid DNA:RNA (so when DNA, RNA and AB was present in the embryo) • Can be temporal as if take embryos at different stages you get diff info. Yet can’t get this from same embryo as it killed in staining. Must be on diff embryos of different stages

• Requires knowledge of how gene of interest is controlled i.e. what are the promotors and enhancers = regulatory sequences • Expression of a gene is determined by the regulatory regions • Generate a transgene where replace gene with a reporter gene e.g. coding sequence for GFP/bGal downstream of regulatory sequences • Reporter gene i.e. GFP will be expressed wherever the gene is usually expressed when introduced into an animal model • Introduce this into the animal e.g. mouse, fish, fly • GFP is naturally fluorescent so preferred – don’t have to fix or kill the embryo so from the same embryo you can generate continuous expression pattern so temporal AND spatial • This is different to fusion with GFP which is fused in the frame to allow visualisation of the protein, compared to localisation of mRNA here

• Simultaneously express 1000s of genes in the embryo in one single experiment • Looking at the entire genome expressed and not a single gene

genome wide

Large amount of mRNA so young/small embryos would be unsuitable

o The genome is known and so cDNA for every gene is generated and fixed/placed into a spot on the grid o mRNA is isolated and labelled from a stage and cell of interest then applied to the grid o mRNA will hybridise if the gene is present and expressed in that tissue type o Software reads intensity of fluorescent emitted from the plate o Can see level of expression of each gene – not necessarily spatial unless can pick specific place in embryo.

o Instead of hybridisation, sequence at the end of fragment to determine the gene o Can use much less mRNA so advantage vs microarray

This is done because the production of a mRNA transcript does not mean a functional protein will be produced. The protein may remain untranslated for a period of time to control when and where the gene product functions.

Western blot immunodetection

• Analyses where and when a gene is expressed if a specific tissue is used to provide the mRNA. If the whole cell is used then it would give no information as to the location of the gene in the embryo It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide.

Immunofluorescene and immunohistochemistry

• Investigates the distribution of proteins • Here you need prior knowledge of the protein and a specific AB to recognise the protein • This is allows high spatial resolution of sub cellular structures • If combine with other techniques, get temporal resolution

• Tissue is fixed for staining so must analyse different tissues at different stages for temporal resolution • Tissue containing epitope specific to a protein is exposed to a primary antibody • Primary AB from different species and is raised against secondary AB (anti-IgG coupled to a fluorescent tag)