For the production of lentiviral particles, three components are generally required: 1) a lentiviral vector containing your inserts of interest (cDNA, shRNA, or miRNA), 2) one or two packaging vectors which contain all necessary viral structure proteins, 3) an envelope vector expressing Vesicular Stomatitis Virus (VSV) glycoprotein (G). In general, lentiviral vectors with a wildtype 5′ LTR need the 2nd generation packaging system because these vectors require TAT for activation. The 3rd generation lentiviral packaging mix will only support lentiviral expression vectors with a chimeric 5′ LTR in which the HIV promoter is replaced with CMV or RSV, thus making it TAT-independent.