A universal retroviral vector for efficient constitutive expression of exogenous genes.

We have constructed a plasmid pXTl which can be used to derive replication-defective retroviruses capable of efficient constitutive expression of foreign genes in embryonic as well as adult cells. This plasmid is based on pXm5 (used to derive the retrovirus N2 (ref. 1)), and has the LTRs and part ofthe gag region of Moloney Murine Leukaemia Virus. A selectable neomycin (neo) gene, conferring G418 resistance, is under the control of the LTR, which although normally repressed in embryonic cells (2), can become transcriptionally active under selective conditions (3). An internal herpes simplex virus thymidine kinase (TK) promoter is used for expression ofthe exogenous gene; this promoter is active in embryonic stem cells (4) and in a wide variety oftissues in transgenic mice (5). Infectious, helper-free replication-defective retroviruses can be obtained either by direct transfection onto the P2 packaging cell line (6), or via transfection ofthe amphotrophic cell line PA317 (7), and infection of T2 cells . Titres of viral supematants on NIH3T3 fibroblasts range from 1-5x105 G418R cfu/ml ('2 transfection) to 1-5x106 G418R cfu/ml ('2 infection). Titres on embryonic stem (EC or ES) cells are 103-105 fold lower (depending on cell line) due to repression of the viral LTR. Viruses based on pXTI, carrying a transforming gene under the control of the TK promoter, have been used to infect fibroblasts, EC and ES cells, bone marrow and primary embryonic cerebellum cultures; in all cases both neo and exogenous genes are efficiently expressed.