At

E12.5 of mouse development, the lung is composed of two cell types; the

epithelial cells and the surrounding mesenchymal cells. You are interested in

identifying the genes expressed at this stage of development in these two

different cell types at a global level using RNA-seq (RNA-sequencing). Using

the information you have been given in the lectures and this practical session

(both weeks) answer the following questions.

Question 11:  [9 marks].

a) Describe a genetic approach you could use to distinguish and separate these two cell types from the other. (Not simply endogenous gene expression).  Use dot points to outlines the steps involved in making an appropriate mouse model. Hint: knock-in.  (4 marks) 

b) Using the approach you outlined in (a) above, name the specific method that you could use to separate these two cell types from one another so that you can extract RNA from each cell type, to perform your RNA-seq study.  (1 mark).

c) You have performed the RNA-seq and find that a gene called Vegf (Vascular endothelial growth factor) is highly expressed in the lung epithelial cells. Vegf is a secreted signalling molecule (growth factor). Using your RNA-seq data from the two populations, what information could you seek to support the idea that Vegf signaling is involved in lung development?  (Hint: Recall the signaling lectures. What do signalling molecules act on?)  (2 marks).

d) Name a method you could use to validate your finding that Vegf is highly expressed in lung epithelium and to visualise its expression in the embryonic lung.  (2 marks).