pLXIN contains elements derived from Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma virus (MoMuSV), and is a bicistronic retroviral vector designed for retroviral gene delivery and expression (1–3). This vector contains an internal ribosome entry site (IRES) between the MCS and the neomycin resistance (Neor) gene. Thus, the 5' viral LTR promoter in this vector controls expression of both the gene of interest and Neor. pLXIN also possesses a Col E1 origin of replication and E. coli Ampr gene for propagation and antibiotic selection in bacteria.
The use of this bicistronic vector poses two primary advantages: Upon transfection into a packaging cell line, selection for antibiotic resistance allows for the simultaneous selection of high-titer, virus-producing lines. Later, upon infection of target cells with this virus, neomycin resistance correlates to expression of the gene of interest.
pLXIN can be transfected into a packaging cell line such as the RetroPack PT67 Cell Line (Cat. No. 631510). Once in the cell, RNA from the vector is packaged into infectious, replicationincompetent retroviral particles. pLXIN does not contain the structural genes (gag, pol, and env) necessary for particle formation and replication; however, these genes are stably integrated into PT67 (4–7). Subsequent introduction of pLXIN, containing Ψ+ (psi; the extended viral packaging signal), transcription and processing elements, and the gene of interest produces high-titer, replication-incompetent infectious virus. That is, 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.
