Retroviral-mediated Transfer Of Antisense Human C-myc Gene Into Mammalian Cells

Retroviruses have become important tools for efficient transfer of genes into eukaryotic cells. Retrovirus vectors can be made in the absence of helper virus by using retrovirus packaging cell lines. Successful gene transfer by viral infection has been demonstrated for a variety of genes inserted into retroviral vectors. To investigate the biological functions associated with a gene sequence, a valuable genetic approach is to introduce cloned DNA encoding antisense RNA into cells to specifically eliminate the gene product of interest or to block its function. A retroviral vector, called pDAM3, containing the neomycin resistant gene and the antisense human c-myc gene fragment (the third exon and 3'flanking sequence) has been constructed. pDAM3 was introduced into PA317 amphotropic packaging cells by the calcium phosphate precipitation method. Individual G418-resistant PA317 colonies were isolated. The virus titer of several cell lines were determined by infection of NIH/3T3 cells, followed by selection in G418. The highest titer obtained was 8×10~5 G418-resistant colony forming units/ml. Single and pooled G418-resistant PA317 colonies with high titers were expanded and analyzed by Southern blot for the presence of intact viral sequences. All cell lines were found to transfer the internal sequences of the pDAM3 vector without aberrant rearrangement. DAM3 recombinant virus infected human esophegeal cancer cell line EC8712 efficiently. DAM3 virus infected EC8712(called EC-DAM3)...