We have used the lentivirus gene expression vector pLV[Exp]-EGFP/Neo-EF1A>hITGA5[NM_002205.5] with a vector ID of VB900124-1009rmb. It was purchased from a vector company. Based on the company description, the lentiviral vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells together with a number of helper plasmids. Inside the packaging cells, vector DNA located between the two long terminal repeats (LTRs) is transcribed into RNA, and viral proteins expressed by the helper plasmids further package the RNA into virus. Active virus is then released into the supernatant, which can be used to infect target cells directly or after concentration.
Vector Components:
- RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into a live virus.
- 5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype viruses, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript.
- Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.
- RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.
- cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.
- EF1A: Promoter in mammalian, driving the alpha 5 integrin gene.
- Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.
- hITGA5[NM_002205.5]: The open reading frame of alpha 5 integrin gene. WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.
- CMV: A gene promoter. It drives the ubiquitous expression of the downstream marker gene.
- EGFP/Neo: A dual-reporter gene for a marker. This allows cells transduced with the vector to be selected and/or visualized.
- 3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration).
- SV40 early pA: Simian virus 40 early polyadenylation signal. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.
- Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
- pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
