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Typical vector for expression of antibody product in mammalian cells. This representative plasmid contains both the heavy-chain (VH, CH1, CH2, CH3) and light-chain (VL, CKappa) genes of the IgG gene. Each is driven by its own promoter (PCMV) and has a polyadenylation signal (PolyA) downstream. A leader sequence (L) which serves as a signal for secretion is present for each IgG gene. A mammalian selectable marker, Neo in this case, is included with its own promoter (PSV40) and polyadenylation signal (SV40 pA). Sequence elements for replication and selection in E. coli are also included, namely, an origin of replication (pUC ori) and selectable marker (Amp).
Overview of selection of stable and amplified clones.
Overview of production of replication-defective retroviral particles using packaging cells and subsequent transduction of target cells. A replication-incompetent vector, which includes the 5′ and 3′ LTR regions, the packaging sequence (ψ), a gene of interest (Gene X), and a mammalian selectable marker (neo) with its own promoter (pro), is introduced into a packaging cell line which supplies the required viral accessory proteins, namely Gag, Pol, and Env. This bacterial plasmid also contains the required genetic elements for replication (ori) and selection (Amp) in E. coli. Upon entry into the packaging cell line (via any desired transfection methodology), the plasmid is transported into the nucleus, where it is transiently transcribed. The viral transcript initiates at the 5′ LTR and terminates at the 3′ LTR, generating a full-length viral transcript. It contains the packaging sequence (ψ), which allows packaging into viral particles. Fully assembled viral particles subsequently bud out of the packaging cell line. These viral particles are harvested from the supernatant 48 h after transfection. The virus-containing supernatant is subsequently incubated with the target cells, and entry of the viral particles occurs through interaction with host-cell receptors. Once the viral core is released into the cytoplasm, reverse transcription of the RNA viral genome begins using reverse transcriptase contained within the viral particle. The DNA copy of the viral genome then integrates into the host genome, and the gene of interest and selectable marker can be expressed. One infectious virion produces one integrated copy of the viral genome. Figure adapted from reference 20 .
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