Biomedical research is becoming an increasingly open and sharing environment; here, in The Grove Lab, we're all for this! We have recently been preparing a manuscript for the new open access platform created the Wellcome Trust and F1000 research, which will we post on in the future. We have also set up a lab page at Addgene, the online portal for the distribution of plasmids.
In time, we will use this platform to share all the plasmids we generate that may be useful to other researchers. To start with we've deposited a handful of plasmids encoding the core entry receptors for hepatitis C virus (HCV): CD81, scavenger receptor-B I, claudin-1 and occludin, you can read more about these in various review articles, like this or this. We use these plasmids as tools to investigate virus entry, however, these proteins also have important physiological functions, so these plasmids may be of use to others outside of HCV research.
These particular 'pDUAL' plasmids are lentiviral expression vectors that express both the gene of interest and green fluorescent protein from separate promoters. The genes were PCR cloned by Lucas Walker (Research Assistant) from cDNA extracted from human hepatoma cells. For more on the use of lentiviral vectors see this excellent guide from Addgene.
Here is some example data using one of these vectors. We generated lentivirus encoding human CD81 and then transduced some CD81 KO Huh-7 cells. We then assessed their expression of GFP and CD81 using flow cytometry. What should be clear from the plot below is that the untransduced cells are resolutely negative, whereas the cells that received the lentivirus have GFP and CD81 galore! We're currently using this system to characterise some CD81 mutants, which we will also share soon enough.
So, please, help yourself to the fruits of our cloning!