Term 2 Practice question: Recombinant DNA technologies

Question 1

Question

Answer 1

You have been given the sequence of a gene that is predicted to encode a protein required for correct cell division in
multicellular organisms.

Your Supervisor has suggested that a transgenic approach using an appropriate model organism would be a good way to understand more about the role of the gene during animal development. You have been asked to design a single gene construct that would allow you to determine both the cell type in which the gene is expressed and the subcellular localisation of the encoded protein.

In your answer you should explain:
a) Your reasoning for the gene construct that you propose.
b) The experimental methods required to make the gene construct.
c) An outline of the transgenic approach you propose, including the model organism used, transformation method and analysis of the transgenic organism.

Question 2

key points

Answer 2

cell division in multicellular organisms
single gene construct
cell structure
subcellular localisation

a) translational fusion construct
b) Reporter gene cloning methods
c) a model organism: C.elegans, Drosophila or mouse, transformation method appropriate fororganism & analysis will depend on the Translational Fusion construct you choose

Question 3

plan your answer part a

Answer 3

Your reasoning for the gene construct that you propose. Translational Fusion Construct
The experimental methods required to make the gene construct.
Reporter Gene – GFP would be a good choice Why?
The promoter region – from the gene

Question 4

plan your answer: part b/c

Answer 4

The experimental methods required to make the gene construct. Reporter Gene, cloning methods

PCR primers can be designed to amplify the gene because the gene sequence is known. The gene would be cloned into an expression vector that would include a reporter gene

you may include an image of a labelled reporter gene expression vector

Steps
1. The Promoter and the coding region could be isolated together by using PCR & template genomic DNA from C.elegans.
2. May decide to use cDNA (RT-PCR) – why? Promoter would then need to be cloned separately.
3. Insert into Expression vector: RE digestions and ligation.
4. RE digests, DNA sequencing to confirm cloning successful (orientation!).5. Transform into model organism.
6. Screen for GFP (or other reporter

Question 5

How to get very high marks

Answer 5

• Include extra cloning details: e.g. ensure open reading frame gene - reporter; add RE sites to PCR primers to aid cloning– how?.
• Include very clear diagrams
• Consideration of different reporter genes/different model organisms – why one better than others (C.elegans a good choice for visualisation)
• Consideration of assay method e.g fluorescence microscopy.

Need to ensure that you are observing dividing cells. Therefore consider the use of a control cell division gene to check co-localisation (examples, research required).

-Consideration of a knockout experiment to test the function of the gene e.g. CRISPR – what happens when the wildtype gene is mutated?