Calcium phosphate-mediated DNA transfection and retroviral infection are two alternative gene transfer techniques designed to introduce specific DNA fragments into the chromosomes of recipient cells. To compare the efficiency of expression of genes introduced into cells by either of these two techniques, a retrovirus-derived vector was constructed from the genome of Moloney murine leukemia virus by replacing the coding sequences of the envelope gene with the bacterial Neo(r) gene derived from Tn5, termed rEnv-Neo(r). Expression of the hybrid Neo(r) gene in NIH 3T3 cells after DNA transfection or retroviral infection was determined by measuring the steady-state levels of the corresponding cytoplasmic polyadenylated RNA species. Cells containing one copy of the integrated rEnv-Neo(r) DNA introduced into cells by retroviral infection expressed 10- to 50-fold-higher levels of vector-specific RNA compared with cells harboring one copy of the same DNA derived by DNA transfection. Analysis of the integrated rEnv-Neo(r) DNA with the methylation-sensitive restriction enzyme SmaI has shown that DNA integrated after DNA transfection but not after viral infection is partially methylated, predominantly in the 5' long terminal repeat, the region involved in initiation of transcription.
ASJC Scopus subject areas
- Insect Science