635. Macrophage Colony Stimulating Factor Expression in Retrovirally Transfected Cells Is Dependent upon the Adherence Status of the Target Cells

Numerous cell types including both normal and cancerous cells retrovirally transduced with either the membrane or the secreted forms of macrophage colony stimulating factor (mM-CSF and sM-CSF, respectively) using LXSN-based vectors showed a variable expression of the transgene. Expression of either form of M-CSF correlated with the cells' adhesion to culture dishes. Transduced adherent cells produced more sM-CSF by ELISA and mM-CSF by flow cytometry; whereas, the non-adherent cells synthesized little M-CSF. Use of a granulocyte-macrophage colony stimulating factor (GM-CSF) LXSN-based retrovirus failed to show this dichotomy. Real-time polymerase chain reaction analysis using the ΔΔCT method showed that adherent cells produced more M-CSF-specific mRNA than the non-adherent cells, while maintaining similar mRNA levels for the β-actin and neomycin resistance genes. Examination of the M-CSF genes used for creating these M-CSF retroviral vectors indicated that 170 base pairs of the 5′ flanking untranslated region of the M-CSF gene were present in the M-CSF retroviral constructs. Ligation of this 5′ region of the M-CSF gene to the 5′ proximal end of the enhanced green fluorescent protein gene (EGFP) caused the expression of EGFP to show the same dichotomy as previously seen with the M-CSF. This work suggests that this 5′ flanking region of the M-CSF gene could be an important way to get transgenic expression within adherent cells, but not in non-adherent cells, when differential gene expression is needed.