Rapid Production of Retroviruses for Efficient Gene Delivery to Mammalian Cells Using 293<scp>T</scp> Cell–Based Systems

Swift, Susan; Lorens, James B.; Achacoso, Philip; Nolan, Garry P.

doi:10.1002/0471142735.im1017cs31

Cited by 293 publications

(261 citation statements)

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“…Phoenix™ Amphotropic packaging cell lines are second generation retrovirus producer lines that were generated from human embryonic kidney (HEK) 293T cells 33,34 . In addition to the temperature sensitive T antigen co-selected with neomycin already present in HEK293T Note, the mitochondrial uncoupler dose and treatment duration should be tested for each cell line in order to efficiently induce widespread mitochondrial depletion.…”

Section: Experimental Designmentioning

confidence: 99%

Depletion of mitochondria in mammalian cells through enforced mitophagy

et al. 2016

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Mitochondria are not only the "power-house" of the cell, but are also involved in a multitude of processes that include calcium storage, cell-cycle and cell-death. Traditional means to investigate mitochondrial importance in a given cellular process have centred upon depletion of mitochondrial DNA (mtDNA) through chemical or genetic means. While these methods severely disrupt mitochondrial electron transport chain, mtDNA-depleted cells still maintain mitochondria and many mitochondrial functions. Here, we describe a straightforward protocol to generate mammalian cell populations with low to non-detectable levels of mitochondria. Ectopic expression of the ubiquitin E3 ligase Parkin, combined with short-term mitochondrial uncoupler treatment, engages widespread mitophagy, and effectively eliminates mitochondria. In this protocol, we explain how to generate mitochondria-depleted cells and a variety of methods to confirm mitochondrial clearance. Furthermore, we describe culture conditions to maintain mitochondrial-depleted cells with minimal loss of viability for longitudinal studies. This method should prove useful to investigate the importance of mitochondria in a variety of biological processes.

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Section: Experimental Designmentioning

confidence: 99%

Depletion of mitochondria in mammalian cells through enforced mitophagy

et al. 2016

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“…Melan-mu3 (referred to as melan-mu), melan-rp2 (Gwynn et al, 2004) (referred to as melan-rp; Bloc1s3 rp/rp ), melanpa1 (referred to as melan-pa), melan-coa2 (Suzuki et al, 2001) (referred to as melan-coa; Hps3 coa/coa ), and melan-a (Bennett et al, 1987) were maintained as described previously (Sviderskaya et al, 2002). Retrovirus production from transiently transfected 293T cells and transduction of primary melanocytes and cell lines were carried out as described previously (Swift et al, 1999). Stable transductants were selected in medium containing 200 -400 g/ml hygromycin B.…”

Section: Cell Culture and Transgene Expressionmentioning

confidence: 99%

BLOC-1 Is Required for Cargo-specific Sorting from Vacuolar Early Endosomes toward Lysosome-related Organelles

Setty

Tenza

Truschel

et al. 2007

MBoC

201

342

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Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function.Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1-deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS. INTRODUCTIONHermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by hypopigmentation, prolonged bleeding, and sometimes ceroid accumulation, lung fibrosis, and/or immune defects leading to premature death Wei, 2006). HPS or a similar disorder in mice results from mutations in any of at least 15 genes (Wei, 2006). All of these genes are ubiquitously expressed, but their mutation in HPS affects mainly the generation and function of selected tissuespecific lysosome-related organelles (LROs;Bonifacino, 2004;Di Pietro and Dell'Angelica, 2005). Those LROs that are most severely affected in all forms of HPS-pigment cell melanosomes, platelet dense granules, and lung lamellar bodies-are unique in that they coexist with bona fide lysosomes in their respective cell types (Dell'Angelica et al., 2000;Marks and Seabra, 2001). The 15 known HPS-associated genes have been identified, and although the products of most are thought to participate in trafficking events that are uniquely required to form this class of LRO, the function of only a few is understood in detail.The genes disrupted in human HPS-7 (Li et al., 2003) and HPS-8 (Morgan et al., 2006) and in the mouse HPS models pallid, muted, reduced pigmentation (rp), cappuccino, and sandy encode five of the eight known subunits of a stable protein complex known as biogenesis of lysosome-related organelles complex (BLOC)-1 (Falcon-Perez et al., 2002;Moriyama and Bonifacino, 2002;Ciciotte et al., 2003;Li et al., 2003;Gwynn et al., 2004;Starcevic and Dell'Angelica, 2004). To date, no specific subcellular function has been assigned...

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“…Expression of proteins in cells was performed by transfection of a 293 T packaging cell line with a retroviral expression vector containing the DNA of interest (14). Virus was harvested 24-48 h after transfection in medium suited for the target cells.…”

Section: Dstomato/gfp-expressionmentioning

confidence: 99%

A novel imaging‐based high‐throughput screening approach to anti‐angiogenic drug discovery

et al. 2009

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The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting surge in available candidate therapeutics highlights the need for robust, highthroughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-throughput/high-content image screening. Co-culture of primary human endothelial cells (EC) and vascular smooth muscle cells (vSMC) results in assembly of a network of tubular endothelial structures enveloped with vascular basement membrane proteins, thus, comprising the three main components of blood vessels. Initially, EC are dependent on vSMC-derived VEGF and sensitive to clinical anti-angiogenic therapeutics. A subsequent phenotypic VEGFswitch renders EC networks resistant to anti-VEGF therapeutics, demarcating a mature vascular phenotype. Conversely, mature EC networks remain sensitive to vascular disrupting agents. Therefore, candidate anti-angiogenic compounds can be interrogated for their relative potency on immature and mature networks and classified as either vascular normalizing or vascular disrupting agents. Here, we demonstrate that the EC-vSMC co-culture assay represents a robust high-content imaging high-throughput screening system for identification of novel anti-angiogenic agents. A pilot highthroughput screening campaign was used to define informative imaging parameters and develop a follow-up dose-response scheme for hit characterization. High-throughput screening using the EC-vSMC co-culture assay establishes a new platform to screen for novel anti-angiogenic compounds for cancer therapy. ' 2009 International Society for Advancement of Cytometry Key termshigh-throughput screening; endothelial/mural cell co-culture; angiogenesis INHIBITING angiogenesis is a validated therapeutic modality for cancer (1). Hence, new agents that potently inhibit pathological angiogenesis are a critical component of future combination cancer therapies (2). The identification of candidate therapeutics relies on current in vitro angiogenesis assays that measure effects on endothelial cell migration, proliferation, apoptosis and tube formation, and in vivo models such as the chick chorioallantoic membrane assay, corneal neovascularization assay, and Matrigel plug assays (3-5). Current in vitro angiogenesis assays are generally focused on specific behaviors of monocultured endothelial cells (EC) (e.g., migration, proliferation) and fail to model heterotypic perivascular interactions, whereas in vivo systems are not compatible ...

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Rapid Production of Retroviruses for Efficient Gene Delivery to Mammalian Cells Using 293T Cell–Based Systems

Cited by 293 publications

References 41 publications

Depletion of mitochondria in mammalian cells through enforced mitophagy

Depletion of mitochondria in mammalian cells through enforced mitophagy

BLOC-1 Is Required for Cargo-specific Sorting from Vacuolar Early Endosomes toward Lysosome-related Organelles

A novel imaging‐based high‐throughput screening approach to anti‐angiogenic drug discovery

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