LoxP GFP/RFP ColorSwtich
CRE recombination provides an excellent tool for conditional gene targeting by allowing transgenic animal modeling that links genotypes (alterations in genomic DNA) to biological outcomes (phenotypes). CRE reporting lentivirus is a very effective method for verifying the performance of CRE-mediated recombination in vivo. It is also a control indicator for verification of CRE lox based systems.
CRE recombination activity detection:
GenTarget LoxP ColorSwitch CRE reporting lentiviral particles provide a convenient, easy, and fast indicator of CRE recombination efficacy in vivo or in cell culture. These lentiviral particles have been engineered to constitutively express the “LoxP-GFP-stop-LoxP-RFP-Stop” cassette under either an enhanced CMV promoter or EF1a promoter, reporting the presence of CRE enzyme via a color switch mechanism (see the expression cassette scheme below).
How it works:
This lentivirus demonstrates a strong GFP fluorescence after infection into any mammalian cells but does not show an RFP fluorescence signal. Once the CRE protein is present in the nucleus, CRE excises/deletes the DNA fragment between two loxP sites. As a result, the GFP is removed and RFP is expressed, with a resulting switch to RFP fluorescence. The ratio of RFP-expressing to GFP-expressing cells can be easily monitored via fluorescence cell sorting, visualized by microscopy, or the fluorescence intensity may be measured by a fluorometer with GFP and RFP filter sets. See the sample results below.
Images taken before and 48 hours after the CRE expression particles (CAT#: LVP339) was added into cell culture.
GenTarget provides the CRE reporting lentivirus with different antibiotic selection markers as puromycin, neomycin, or blasticidin. The selection markers are expressed under an RSV promoter (not shown in the scheme above). Therefore, you can easily select the positive transduced cells (expressing the CRE detection cassette) via a specific antibiotic marker, blasticidin (Bsd), Puromycin (puro), or Neomycin (Neo).
Lentivirus are provided as in two formats:
- Crude Lentivirus: 200ul /vial in DMEM containing 10 x Polybrene;
- Concentrated lentivirus: 200ul PBS solution which is better for hard-to-transduced cell types or for serum-free culture.
Please see Product manual for details.
See also our Pre-made Color-Swtich CRE reporter cell lines that transformed using this LoxP GFP/RFP lentivirus.