BACKGROUND: Design of efficient nonviral gene delivery systems is limited as a result of the rudimentary understanding of the specific molecules and processes that facilitate DNA transfer. METHODS: Lipoplexes formed with Lipofectamine 2000 (LF2000) and plasmid-encoding green fluorescent protein (GFP) were delivered to the HEK 293T cell line. After treating cells with lipoplexes, HG-U133 Affymetrix microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP-) at 8, 16 and 24 h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. RESULTS: Relative to untreated cells 2 h after lipoplex treatment, only downregulated genes were identified ≥ 30-fold: ALMS1, ITGB1, FCGR3A, DOCK10 and ZDDHC13. Subsequently, relative to GFP- cells, the GFP+ cell population showed at least a five-fold upregulation of RAP1A and PACSIN3 (8 h) or HSPA6 and RAP1A (16 and 24 h). Pharmacologic studies altering endogenous levels for ALMS1, FCGR3A, and DOCK10 (involved in filopodia protrusions), ITGB1 (integrin signaling), ZDDHC13 (membrane trafficking) and PACSIN3 (proteolytic shedding of membrane receptors) were able to increase or decrease transgene production. CONCLUSIONS: RAP1A, PACSIN3 and HSPA6 may help lipoplex-treated cells overcome a transcriptional shutdown due to treatment with lipoplexes and provide new targets for investigating molecular mechanisms of transfection or for enhancing transfection through cell priming or engineering of the nonviral gene delivery system.
BACKGROUND: Design of efficient nonviral gene delivery systems is limited as a result of the rudimentary understanding of the specific molecules and processes that facilitate DNA transfer. METHODS: Lipoplexes formed with Lipofectamine 2000 (LF2000) and plasmid-encoding green fluorescent protein (GFP) were delivered to the HEK 293T cell line. After treating cells with lipoplexes, HG-U133 Affymetrix microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP-) at 8, 16 and 24 h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. RESULTS: Relative to untreated cells 2 h after lipoplex treatment, only downregulated genes were identified ≥ 30-fold: ALMS1, ITGB1, FCGR3A, DOCK10 and ZDDHC13. Subsequently, relative to GFP- cells, the GFP+ cell population showed at least a five-fold upregulation of RAP1A and PACSIN3 (8 h) or HSPA6 and RAP1A (16 and 24 h). Pharmacologic studies altering endogenous levels for ALMS1, FCGR3A, and DOCK10 (involved in filopodia protrusions), ITGB1 (integrin signaling), ZDDHC13 (membrane trafficking) and PACSIN3 (proteolytic shedding of membrane receptors) were able to increase or decrease transgene production. CONCLUSIONS:RAP1A, PACSIN3 and HSPA6 may help lipoplex-treated cells overcome a transcriptional shutdown due to treatment with lipoplexes and provide new targets for investigating molecular mechanisms of transfection or for enhancing transfection through cell priming or engineering of the nonviral gene delivery system.
Authors: Tyler Kozisek; Andrew Hamann; Albert Nguyen; Michael Miller; Sarah Plautz; Angela K Pannier Journal: J Biol Eng Date: 2020-05-19 Impact factor: 4.355