Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications

Dalby, Brian

doi:10.1016/j.ymeth.2003.11.023

Cited by 507 publications

(374 citation statements)

References 10 publications

(12 reference statements)

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“…It was observed that there is only a slight reduction in efficiency following the 2 h incubation of lipoplex both in presence and absence of shear stress. Such slight loss of transfection efficiency was previously reported where the DNA-Lipofectamine 2000 complexes were relatively stable and continued to provide high transfection efficiency even up to 2 h after complexation (Dalby et al 2004). This however does not preclude a change in shape and therefore increased toxicity when lipoplex is sheared along with cells.…”

Section: Discussionsupporting

confidence: 69%

Shear stress increases cytotoxicity and reduces transfection efficiency of liposomal gene delivery to CHO-S cells

Rawat

Gadgil

2016

Cytotechnology

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Animal cells in suspension experience shear stress in different situations such as in vivo due to hemodynamics, or in vitro due to agitation in large-scale bioreactors. Shear stress is known to affect cell physiology, including binding and uptake of extracellular cargo. In adherent cells the effects of exposure to shear stress on particle binding kinetics and uptake have been studied. There are however no reports on the effect of shear stress on extracellular cargo delivery to suspension cells. In this study, we have evaluated the effect of shear stress on transfection of CHO-S cells using Lipofectamine 2000 in a simple flow apparatus. Our results show decreased cell growth and transfection efficiency upon lipoplex assisted transfection of CHO-S while being subjected to shear stress. This effect is not seen to the same extent when cells are exposed to shear stress in absence of the lipoplex complex and subsequently transfected, or if the lipoplex is subjected to shear stress and subsequently used to transfect the cells. It is also not seen to the same extent when cells are exposed to shear stress in presence of liposome alone, suggesting that the observed effect is dependent on interaction of the lipoplex with cells in the presence of shear stress. These results suggest that studies involving liposomal DNA delivery in presence of shear stress such as large scale transient protein expression should account for the effect of shear during lipoplex assisted DNA delivery.

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Section: Discussionsupporting

confidence: 69%

Shear stress increases cytotoxicity and reduces transfection efficiency of liposomal gene delivery to CHO-S cells

Rawat

Gadgil

2016

Cytotechnology

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“…For each transfection 30 pmol of the appropriate siRNA and 2 l of Lipofectamine 2000 TM (Invitrogen) were diluted separately in Opti-MEM TM (Invitrogen) and mixed together after 5 min. All of the subsequent steps were carried out as per the manufacturer's published method for transfection of siRNA (18). Knockdown efficiency was validated by Western blotting for both PS1 and PS2.…”

Section: Methodsmentioning

confidence: 99%

Presenilins Promote the Cellular Uptake of Copper and Zinc and Maintain Copper Chaperone of SOD1-dependent Copper/Zinc Superoxide Dismutase Activity

Greenough

Volitakis

et al. 2011

Journal of Biological Chemistry

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Dyshomeostasis of extracellular zinc and copper has been implicated in ␤-amyloid aggregation, the major pathology associated with Alzheimer disease. Presenilin mediates the proteolytic cleavage of the ␤-amyloid precursor protein to release ␤-amyloid, and mutations in presenilin can cause familial Alzheimer disease. We tested whether presenilin expression affects copper and zinc transport. Studying murine embryonic fibroblasts (MEFs) from presenilin knock-out mice or RNA interference of presenilin expression in HEK293T cells, we observed a marked decrease in saturable uptake of radiolabeled copper and zinc. Measurement of basal metal levels in 6-monthold presenilin 1 heterozygous knock-out (PS1 ؉/؊ ) mice revealed significant deficiencies of copper and zinc in several tissues, including brain. Copper/zinc superoxide dismutase (SOD1) activity was significantly decreased in both presenilin knock-out MEFs and brain tissue of presenilin 1 heterozygous knock-out mice. In the MEFs and PS1 ؉/؊ brains, copper chaperone of SOD1 (CCS) levels were decreased. Zinc-dependent alkaline phosphatase activity was not decreased in the PS null MEFs. These data indicate that presenilins are important for cellular copper and zinc turnover, influencing SOD1 activity, and having the potential to indirectly impact ␤-amyloid aggregation through metal ion clearance.

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“…Either RanBP9-specific or single strand sense control siRNAs were transiently transfected at 50 -100 nM final concentrations using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Hippocampal neurons were grown for 7-10 days until a robust network of processes formed before transfections, and the neurons were similarly transfected with siRNA as previously described (17,18). After 24 h of the first transfection (17,18), a second transfection was performed to enhance the knock-down effect of the siRNA.…”

Section: Methodsmentioning

confidence: 99%

Novel Role of RanBP9 in BACE1 Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation

Lakshmana

Yoon

Chen

et al. 2009

Journal of Biological Chemistry

144

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Accumulation of the amyloid ␤ (A␤) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted A␤ generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and A␤ generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced ␤-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced A␤ generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease. The major defining pathological hallmark of Alzheimer disease (AD)2 is the accumulation of amyloid ␤ protein (A␤), a neurotoxic peptide derived from ␤-and ␥-secretase cleavages of the amyloid precursor protein (APP). The vast majority of APP is constitutively cleaved in the middle of the A␤ sequence by ␣-secretase (ADAM10/TACE/ADAM17) in the non-amyloidogenic pathway, thereby abrogating the generation of an intact A␤ peptide. Alternatively, a small proportion of APP is cleaved in the amyloidogenic pathway, leading to the secretion of A␤ peptides (37-42 amino acids) via two proteolytic enzymes, ␤-and ␥-secretase, known as BACE1 and presenilin, respectively (1).The proteolytic processing of APP to generate A␤ requires the trafficking of APP such that APP and BACE1 are brought together in close proximity for ␤-secretase cleavage to occur. We and others have shown that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytosis receptor (2), binds to APP and alters its trafficking to promote A␤ generation. The loss of LRP substantially reduces A␤ release, a phenotype that is reversed when full-length (LRP-FL) or truncated LRP is transfected in LRP-deficient cells (3, 4). Specifically, LRP-CT lacking the extracellular ligand binding regions but containing the transmembrane domain and the cytoplasmic tail is capable of rescuing amyloidogenic processing of APP and A␤ release in LRP deficient cells (3). Moreover, the LRP soluble tail (LRP-ST) lacking the transmembrane domain and only containing the cytoplasmic tail of LRP is sufficient to enhance A␤ secretion (5). This activity of LRP-ST is achieved by promoting APP/BACE1 intera...

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Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications

Cited by 507 publications

References 10 publications

Shear stress increases cytotoxicity and reduces transfection efficiency of liposomal gene delivery to CHO-S cells

Shear stress increases cytotoxicity and reduces transfection efficiency of liposomal gene delivery to CHO-S cells

Presenilins Promote the Cellular Uptake of Copper and Zinc and Maintain Copper Chaperone of SOD1-dependent Copper/Zinc Superoxide Dismutase Activity

Novel Role of RanBP9 in BACE1 Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation

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