Infection by murine retroviruses in embryonic carcinoma (EC) and embryonic stem cells is highly restricted. The transcriptional unit of the Moloney murine leukemic virus (MoMuLV) long terminal repeat (LTR) is inactive in EC and embryonic stem cells in association with increased proviral methylation. In this study, expression in F9 EC cells was achieved from novel retroviral vectors containing three modifications in the MoMuLV-based retroviral vector: presence of the myeloproliferative sarcoma virus LTR, substitution of the primer binding site, and either deletion of a negative control region at the 5 end of the LTR or insertion of a demethylating sequence. We conclude that inhibition of expression from the MoMuLV LTR in EC cells is mediated through the additive effects of multiple cis-acting elements affecting the state of methylation of the provirus.), and the G1Na plasmid was provided by P. Tolstechev (Genetic Therapy, Inc., Gaithersburg, Md.). The LN vector was constructed and packaged in the laboratory of A. Dusty Miller (Fred Hutchinson Cancer Center, Seattle, Wash.).The MPSV LTR was used to replace the 3Ј MoMuLV LTR of G1Na to make MPneo. The NCR was removed from the MPSV LTR as an NheI (at nucleotide 33 in the LTR)-to-Sau3a (at nucleotide 97 in the LTR) fragment. The cut ends of the LTR were ligated together after fill-in by Klenow DNA polymerase to make the MPncr 3Ј LTR; this was then used to replace the 3Ј LTR of G1Na, yielding MPncrneo. The Thy-1 fragment in the plasmid Bluescript was opened at the SmaI site immediately 3Ј of the insert, and a synthetic oligonucleotide encoding an XbaI site (New England Biolabs, Beverly, Mass.) was ligated in place. The Thy-1 piece was isolated as an XbaI-XbaI fragment and cloned into the NheI site of the MPSV LTR in Bluescript. The Thy-1-substituted MPSV LTR was inserted in place of the 3Ј LTR in G1Na to make MPthyneo.The 5Ј LTR and psi region from plasmid LN was subcloned as an EcoRI-EcoRI fragment. The KpnI-SpeI fragment encompassing the PBS was removed and replaced by the KpnI-SpeI fragment from dl587rev. The 5Ј LTR/leader region fragment containing the dl587rev PBS was then returned to the MPneo, MPthyneo, and MPncrneo plasmids to produce MPdlneo, MPthydlneo, and MPncrdlneo.To make LNncrneo, the NCR (NheI at position 33 to Sau3a at position 97; Fig.

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