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I- Why use gene vectors?
1- Make commercially useful product (high level expression)
2- Test effect of a gene on a plant, by either expressing or inactivating
II- How does one make a vector?
1- Insert gene into DNA
2- Transcribe the DNA into infectious RNA and inoculate plant
3- Dawson (TMV), Ahlquist (BMV) first to make infectious transcripts
4- Alternatively express DNA in plant to give infectious RNA
III- General strategies
1- Replace or extend an existing gene
2- Add a new gene
3- Use reporter gene (GFP - 240AA, CAT - 300AA, GUS - 600AA)
4- Express from subgenomic replicon
5- Express from subgenomic promoter
6- Construct a unique restriction site at insertion position
7- Fiddle promoter to get maximum expression
IV - TMV as a vector (Dawson)
1- Insert new subgenomic promoter between M and CP
2- Simplest construct is TMV with two CP genes (construct unstable)
3- Next try a heterologous CP (more stable)
4- 3' most CP expressed 10 to 20 the internal CP
5- Replace internal CP with GFP
6- Construct infects N benthamiana, but not N tabacum
V- Optimizing GFP expression from TMV
1- Longer promoter
2- Internal pseudoknots (better expression of internal genes)
3- GFPC3 (improved GFP [more fluorescent])
4- Optimize codon usage (hasn't been done yet)
VI - PVX vector
1- Cut PVX at unique restriction site
2 - Insert new gene (with PVX promoter)
3 - High level expression and active virus (infects tobacco)
4 - Homologous constructs of other potexviruses express poorly
VII - BMV as vector
1- Express genome segments from plant genes
2- Have one inducible gene (so replication can be turned on at will)
3- Replace CP with gene of interest
VIII - Is high level expression of inserts compatible with virus replication?
1- Modified viruses generally replicate poorly
2- DNA shuffling (selective mutagenesis) is a potential solution.