RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into 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 virus, 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. On our vector, Δ5' LTR is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of Δ5' LTR.
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.
U6/2xLacO: Modified human U6 small nuclear 1 promoter containing two repeats of the lac operator sequence. Pol III promoter which suppresses small RNA expression in the presence of LacI.
Sense, Antisense: These sequences are derived from your target sequence and are transcribed to form the stem portion of the “hairpin” structure of the shRNA.
Loop: This optimized sequence is transcribed to form the loop portion of the shRNA “hairpin” structure.
Terminator: Terminates transcription of the shRNA.
LacO: Lac operator. In the absence of IPTG, it is bound by lac repressor (LacI), leading to transcriptional repression of the upstream shRNA. In the presence of IPTG, LacI can no longer bind to LacO, thus allowing upstream shRNA to be transcribed.
hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream ORF.
Regulatory protein: Lac repressor (LacI) and a drug selection gene (such as puromycin resistance gene) linked by T2A linker. Allows cells to express LacI protein and be resistant to the corresponding drug. In the absence of IPTG, LacI binds to LacO to repress transcription of downstream genes or small RNAs. In the presence of IPTG, LacI undergoes a conformational change and is no longer able to bind to LacO, thus allowing downstream genes or small RNAs to be transcribed.
WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.
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). The polyadenylation signal contained in ΔU3/3' LTR serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.
SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. 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.