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The plasmid pP2C2S is a PVX expression vector in which the cloning sites are between the ClaI and SalI sites of the enclosed sequence. There is also an EcoRV site that can be used for cloning of blunt ended sequences. We shall soon make available modified plasmids with larger polylinkers for insertion of novel genes. We do have infectious DNA plasmids but these give greater problems with stability of the newly inserted gene. I am therefore reluctant to pass them out.

The DNA should be grown in a dam- strain so that the ClaI site can be cut efficiently. You will need to linearise the plasmid with SpeI (preferably) or other adjacent unique sites before transcription.

The inserted sequence should be as short as possible. The longest gene that we have expressed successfully is GUS. The one other gene that is larger that we have tried was 3.5kb. The virus was not infectious with this long insert. We try to design our constructs so that the initiation codon of the introduced gene is in a reasonable context but I am not convinced that it is crucial. I think that there is some sequence dependent effect on the stability of the inserted sequence.

We normally do our experiments by inoculation of transcripts or DNA (for 35S PVX constructs) to N.clevelandii or N. benthamiana. We use an extract of the inoculated plants for subsequent passage to the experimental material although it is prudent to check the extract for presence of wild type virus before inoculating hundreds of plants: if you take the extract from the transcript inoculated plants within 10d of inoculation you should find (depending on your insert) that the PVX is predominantly the recombinant form. Extracts taken later will have progressively more of the wild type PVX due to loss of the novel gene by recombination. This intrinsic instability of the system is a limitation but, from the other side, is an asset providing a degree of biological containment.


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