Generation of stable cell knockdown cell lines by lentiviral transduction

Lentiviral GLUD1 short hairpin RNA (shRNA) was purchased from Open Biosystems in the pLKO vector. ShRNA #1 was Open Biosystems catalogue number RHS3979-201758959 and shRNA#2 was RHS3979-201758962. pLKO scramble control vector was generously contributed by the laboratory of Dr. Scott Weed (WVU). Lentiviral constructs were packaged and amplified in 293T cells by transfection of 3.5 μg shRNA vector, 2.3 μg sPAX2, and 1.2 μg CMV-VSV-G on 60 mm² dishes that were pre-coated for >1 hr with 50 μg/mL collagen using Lipofectamine 2000. GRHL2 shRNA was described previously and was shown to duplicate the biologic effects of transfected siRNAs (15).