pLNCX2 contains elements derived from Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma virus (MoMuSV), and is designed for retroviral gene delivery and expression (1– 3). Upon transfection into a packaging cell line, pLNCX2 can transiently express, or integrate and stably express, a transcript containing Y+ (the extended viral packaging signal) a selectable marker, and the gene of interest. The 5' viral LTR in this vector contains viral promoter/enhancer sequences that control expression of the neomycin resistance (Neor) gene for antibiotic selection in eukaryotic cells. A gene of interest can be cloned into the multiple cloning site immediately downstream of the human cytomegalovirus (CMV) immediate early promoter (PCMV). pLNCX2 also includes the Col E1 origin of replication and E. coli Ampr gene for propagation and antibiotic selection in bacteria.

載體應(yīng)用

pLNCX2 can be transfected into a packaging cell line such as the RetroPackTM PT67 Cell Line (#K1060-D). Once in the cell, RNA from the vector is packaged into infectious, replicationincompetent retroviral particles. pLNCX2 does not contain the structural genes (gag, pol, and env) necessary for particle formation and replication; these genes are stably integrated into PT67 (4–7). Subsequent introduction of pLNCX2, containing Y+, transcription and processing elements, and the gene of interest produces high-titer, replication-incompetent infectious virus. These retroviral particles can infect target cells and transmit the gene of interest (which is cloned between the viral LTR sequences), but cannot replicate within these cells since the cells lack the viral structural genes. The separate introduction and integration of the structural genes into the packaging cell line minimizes the chances of producing replication-competent virus due to recombination events during cell proliferation.