I need to check the binding of antibodies against HIV-1 Envelope expressed on cell surface (293T). I have several mutant HIV-1 envelope in a plasmid which do not express EGFP. So, I need to clone the envelopes to a new plasmid which will co-express EGFP. I tried with pcDNA3.1-IRES-EGFP plasmid but the expression is very low. I have done experiment with highly potent antibodies like VRC01, b12. Now, I want to use new plasmid for expression of HIV-1 envelope + EGFP to use in FACS analysis. Please suggest me something or any possible ways to increase the expression of HIV-1 Env gene (my wild type is JRFL).
Plasmids that have the hCMV IE-1 promoter, and using the human t-PA signal sequence work well. Are you using a native signal from HIV or another signal? I recommend using pEMC* made by Vicente Planelles based on a vector that we made about 20 years ago. We have a modified version that we send to any academic lab and have gotten good results expressing lab Envs as well as cloned Envs from plasma of subjects (gp160).
Dear Nancy thanks for your valuable answer. I have another question does the pEMC plasmid express reporter genes like EGFP? In addition, I do not know about signals. Will you please give me some idea so that I can dig more.
1. Planelles, V., N. L. Haigwood, M. L. Marthas, K. A. Mann, C. Scandella, W. D. Lidster, J. R. Shuster, R. Van Kuyk, P. A. Marx, M. B. Gardner, and et al. 1991. Functional and immunological characterization of siv envelope glycoprotein produced in genetically engineered mammalian cells. AIDS research and human retroviruses 7:889-898, 1760229
This publication describes the modified vector for SIV Env expression and the Chapman article describes the original vector construct. We typically do not express EGFP along with the native Env genes, but I can look at a few more publications to see whether some of our collaborators may be using these vectors this way.
Our lab really likes using pFIN expression vectors for equimolar expression of a reporter and a gene. These are much better than IRES constructs in my opinion. Here is the original reference:
Expression characteristics of dual-promoter lentiviral vectors targeting retinal photoreceptors and Muller cells.
Semple-Rowland SL, Coggin WE, Geesey M, Eccles KS, Abraham L, Pachigar K, Ludlow R, Khani SC, Smith WC
Mol Vis. 2010 May 27;16:916-34.
You can clone Env into one of the pFIN GFP vectors found on ADDGENE. This should be pretty straightforward. We have lots of pFIN clones with HIV genes, but not env. Good luck.
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