Lentiviral vector system is an efficient vector, which can introduce genes into mammalian cells permanently. Lentiviral flex conditional gene expression vector (Cre-on) combines Creative BioMart's third-generation lentiviral vector system with Cre responsive flex conditional gene expression system, which can help you realize the permanent integration of CRE responsive flex switch and host genome.
Cre-on system uses two pairs of loxP mutation recombination sites, located on both sides of the target gene (GOI). This structure can completely inhibit gene expression in the absence of Cre, and activate gene expression when GOI depends on Cre inversion.
Flex Cre-on switch consists of two pairs of heteromorphic loxP mutation recombination sites, namely loxP with wild-type sequence and lox2272 with mutation sequence. Its flanking is ORF with opposite (antisense) direction to the promoter. Cre can recognize these two loxP variants, but only the same loxP site pair can be reorganized with each other, not with any other variants. LoxP and lox2272 sites are organized alternately, with each pair in reverse parallel. In the absence of Cre recombinase, ORF is not expressed because of its antisense orientation relative to the promoter. In the presence of CRE, loxP and lox2272 sites are recombined with other loxP and lox2272 sites respectively, resulting in ORF transformation into a meaningful direction, and one of them is removed from each pair of the same recombination sites. This allows the promoter selected by the user to drive the transcription of the target gene. Since there are two different loxP mutation sites on both sides of ORF, no further recombination will occur even if Cre exists.
| Step | Description |
| 1 | The expression vector of the lentiviral flex Cre-on gene is constructed in E. coli, and the flex Cre-on switch is placed between two long terminal repeat (LTR) sequences. |
| 2 | It is then transfected into packaging cells with several helper plasmids. In the packaging cell, the vector DNA between the two LTRs is transcribed into RNA, and the viral protein expressed by the auxiliary plasmid further packages the RNA into the virus. |
| 3 | The live virus is then released into the supernatant, which can be used to infect the target cells directly or after concentration. |
| 4 | When the virus is added to the target cell, the RNA cargo is shuttled into the cell, where it is reverse transcribed into DNA and randomly integrated into the host genome. |
| 5 | During the construction of the vector, the flex Cre-on switch between the two LTRs is permanently inserted into the host DNA together with the rest of the virus genome. |
| 6 | After Cre mediated sequence inversion, Cre recombinase can activate gene expression |
Customers who need our assistance are more than welcome to contact us or submit an online inquiry. Our professional team will provide you with detailed guidance and solution ASAP.
