Cytoplasmic transfer of DNA containing simian virus 40 sequences into mouse 3T3 cells.

Abstract

This paper describes the rare cytoplasmic transmission of defective simian virus 40 (SV40) viral DNA from enucleated cells (i.e., cytoplasts) of the SV40-transformed mouse cell line SVT2 (chloramphenicol-resistant) into cybrid cells formed by fusion of these cytoplasts with BALB/c 3T3 cells (thymidine kinase-deficient). The cybrids were selected in medium containing 1% serum, bromodeoxyuridine, and chloramphenicol. They were identified by their 3T3 chromosome content, by the instability of tumor (T)-antigen expression, by their transformed phenotype, and by their drug resistance. The yield of rare cybrids was about 5 x 10(-7) 0.1% of the yield on medium with 10% serum. The presence of the SV40 genome was detected by the expression of SV40-specific T antigen and confirmed (unpublished data) by hybridization of viral DNA probes with restriction enzyme fragments of nuclear DNAs from cybrid clones. Restriction site mapping (unpublished data) showed that at least 1 kilobase of host flanking DNA on each side of the SV40 DNA was included in the transferred segment. The transforming DNA was not stably integrated initially, as judged by cellular heterogeneity in T-antigen expression. Stable T-antigen-positive and negative subclones were recovered in 10% serum; instability could be retained for at least 30 doublings during growth in 1% serum. The instability is interpreted as evidence of non-integration or unstable integration of the transferred DNA into the host genome. The cytoplasmic transfer is interpreted as evidence that chromosomal fragments or intact chromosomes can be transferred rarely through the cytoplasm in cybrid crosses.