Figure 2

MMTV transfer vectors RNA can be cross-packaged by MPMV proteins. (A) PCR amplification of cytoplasmic RNAs treated with DNase to ensure the absence of any contaminating DNA in the RNA preparations using primers OTR537 and OTR538. (B) Control for nucleocytoplasmic RNA fractionation technique to ensure that the transfer vector RNAs were properly transported to the cytoplasm. Upper panel represents the multiplex RT-PCR for unspliced β-actin mRNA (found exclusively in the nucleus) and 18S ribosomal RNA as a control for the presence of amplifiable cDNA in the PCR reactions as described in the materials and methods and results sections. Unspliced β-actin was not detected in the cytoplasmic RNA fraction, ensuring that the transfer vector RNAs were properly transported to the cytoplasm without any compromise on the integrity of the nuclear membrane. The lower panel represents RT-PCR on cytoplasmic RNA for spliced β-actin mRNA and should be present in both nuclear and cytoplasmic fractions. (C) RT-PCR of cytoplasmic cDNA amplified using MMTV specific primers to ensure that the transfer vector RNAs were stably expressed. (D) PCR amplification of DNase treated viral RNAs to confirm the absence of any contaminating plasmid DNA carried over from the transfected cultures. (E) RT-PCR of viral cDNAs amplified using virus specific primers and probed with the PCR product amplified using the same set of primers and HYB MTV as a template. Amplifications were conducted for 15, 20, and 25 cycles and, in addition to agarose gel, Southern blots are also shown. For this set of experiment, while amplifying DNase treated viral RNAs, cytoplasmic and viral cDNAs, primers OTR643 and OTR676 were used and should amplify 585 bp fragment.