Stability of Potato virus X expression vectors is related to insert size: implications for replication models and risk assessment.
We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert-deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.
Avesani
Linda
L
Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada Le Grazie 15, 37134 Verona, Italy.
Marconi
Giampiero
G
Morandini
Francesca
F
Albertini
Emidio
E
Bruschetta
Matteo
M
Bortesi
Luisa
L
Pezzotti
Mario
M
Porceddu
Andrea
A
D000818:Animals; D001483:Base Sequence; D015693:Gene Products, nef / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors; D005968:Glutamate Decarboxylase / Q000235:genetics; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics; D017863:Potexvirus / Q000378:metabolism*; D011384:Proinsulin / Q000235:genetics; D018570:Risk Assessment; D012689:Sequence Homology, Nucleic Acid
D015693:Gene Products, nef; D010940:Plant Proteins; C095825:expansin protein, plant; D016753:Interleukin-10; D011384:Proinsulin; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2