Generation of maize cell lines containing autonomously replicating maize streak virus-based gene vectors

Virion sense gene replacement derivatives of maize streak virus (MSV) were constructed with selectable marker expression cassettes based on the bialap hos resistance gene (bar) and the CaMV 35S promoter. The effect on replicat ion of increasing the genomic size was tested by including: (1) the 550-bp maize adh I intron and 68-bp TMV Omega RNA leader sequences upstream of the bar genes; and (2) a fusion between the bar and E. coli glutathione reduct ase (gor) genes. Three recombinant viral vectors ranging in size from 2.7 k b to 4.8 kb replicated efficiently in biolistically transfected cells of su spension cultured Black Mexican sweetcorn (BMS) cells. Deletions greater th an 39 bp 3' of the stemloop sequence in the LIR adversely affected replicon release. Transformed bialaphos-resistant EMS cell lines were generated wit h all three vectors containing the bar gene: between 38 and 60% of cell lin es contained replicating viral episomes. The replicons were structurally st able, replicated to copy numbers of over 500 per haploid genome, and were d etected for more than one year after introduction. We noted significant enh ancement of bar gene expression, both at the protein and RNA levels, associ ated with the presence of episomal vector DNA. The maize adhI intron and TM V Omega RNA leader sequences did not seem to have a significant effect on b ar gene expression from replicating constructs, although expression from co ntrols was enhanced. The results suggest that MSV-based constructs would pr ovide a useful system for long-term gene amplification in cereal cell cultu re systems.