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Gene Expression Systems

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Let's assume that I fould a valuable gene fragment (ex. DNA sequence homology with one of the human myosin II genes)

1. Before I proceed for therapeutic development, How would I know if this gene is actually expressed in humans? What method?

2. Now let's assume it is expressed in human. I wish to express the cDNA corresponding to this gene in order to determine its function. How would you construct an expression vector? Would you use the genomic fragment or a cDNA?

3. How would you transfect your expression plasmid into cells, and what type(s) of cell might you use?

4. How would you confirm expression of your construct and encoded protein? (functions and methods)

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I'm assuming that your gene fragment is from a different organism.
<br>If you want to find it in humans, the first step would be to sequence your gene fragment and then from that information you would want to design primers that would amplify a certain part (or the whole thing depending on how big it is) of the known sequence. Now one thing to think about when designing the primers is that if the gene fragment is from a different species, say drosophilia, the sequence in humans would probably not be the same, so how would you compensate for this? I'm not going to give you all the answers....
<br>So once you've identified the gene in humans, do you use cDNA or genomic DNA for your expression vector? Think about the difference between cDNA and genomic DNA. cDNA is made from an RNA template, where genomic DNA is not. I'll let you know that ...

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