Human T-lymphocytes (Jurkat cells) expressing TAT-72: LM and TEM

Abstract

The human immunodeficiency virus (HIV-1) is dependent upon the synthesis of TAT for efficient transcriptional activation and replication1. TAT serves to transactivate the HIV-LTR which leads to an increased transcriptional activation of approximately 1000 fold.We have transfected human T-lymphocytes with an expression vector containing the HIV-1 LTR driving the synthesis of TAT-72. A stable cell line was generated by selection with G418.Cells were centrifuged with a cytospin and stained for nucleic acids with methyl green and pyronin Y in order to visualize and to localize DNA and RNA respectively in each preparation examined by light microscopy (LM). In addition, cells were fixed with a 3.5% glutaraldehyde buffered solution (0.1M Na cacodylate) for 5 min at room temperature, 35 min at 4°C, centrifuged in a microfuge (400 rpm, 3 min) to obtain a cellular pellet. Cell pellets were washed in a buffer-sucrose solution and postfixed in a 1.5% aqueous solution of OsO4. Dehydration and epoxy embedding was followed by sectioning for TEM, following treatment with uranyl and lead salts.