Lipofectamine RNAiMAX: An Efficient siRNA Transfection Reagent in Human Embryonic Stem Cells

Zhao, Ming; Yang, Hong; Jiang, Xingjun; Zhou, Wen; Zhu, Bin; Zeng, Ying; Yao, Kai-Tai; Ren, Chao

doi:10.1007/s12033-008-9043-x

Cited by 66 publications

(53 citation statements)

References 20 publications

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“…AllStars siRNA was used as a negative control. NECs were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol (42). In brief, cultured NECs (day 4) were incubated in Hanks' balanced salt solution for 10 min at 37°C and then carefully collected by pipetting.…”

Section: Methodsmentioning

confidence: 99%

Involvement of β1-Integrin Up-regulation in Basic Fibroblast Growth Factor- and Epidermal Growth Factor-induced Proliferation of Mouse Neuroepithelial Cells

Suzuki

Yanagisawa

Yagi

et al. 2010

Journal of Biological Chemistry

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In neural stem cells, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote cell proliferation and self-renewal. In the bFGF-and EGF-responsive neural stem cells, ␤1-integrin also plays important roles in crucial cellular processes, including proliferation, migration, and apoptosis. The cross-talk of the signaling pathways mediated by these growth factors and ␤1-integrin, however, has not been fully elucidated. Here we report a novel molecular mechanism through which bFGF or EGF promotes the proliferation of mouse neuroepithelial cells (NECs). In the NECs, total ␤1-integrin expression levels and proliferation were dose-dependently increased by bFGF but not by EGF. EGF rather than bFGF strongly induced the increase of ␤1-integrin localization on the NEC surface. bFGF-and EGF-induced ␤1-integrin up-regulation and proliferation were inhibited after treatment with a mitogen-activated protein kinase kinase inhibitor, U0126, which indicates the dependence on the mitogen-activated protein kinase pathway. Involvement of ␤1-integrin in bFGF-and EGF-induced proliferation was confirmed by the finding that NEC proliferation and adhesion to fibronectin-coated dishes were inhibited by knockdown of ␤1-integrin using small interfering RNA. On the other hand, apoptosis was induced in NECs treated with RGD peptide, a small ␤1-integrin inhibitor peptide with the Arg-Gly-Asp motif, but it was independent of ␤1-integrin expression levels. Those results suggest that regulation of ␤1-integrin expression/localization is involved in cellular processes, such as proliferation, induced by bFGF and EGF in NECs. The mechanism underlying the proliferation through ␤1-integrin would not be expected to be completely identical, however, for bFGF and EGF.Neural stem cells (NSCs) 3 are defined as undifferentiated neural cells with a high potential for proliferation and the capacity for self-renewal with retention of multipotency (1-4). During development, NSCs have the capability to generate brain-forming cells, such as neurons, astrocytes, and oligodendrocytes. The application of NSCs to cell-based transplantation is a very attractive and promising strategy for regenerative and restorative medicine (5-7).The fate of NSCs (self-renewal, proliferation, differentiation, survival, and death) is regulated by intracellular programs mediated by a number of transcription factors or epigenetic modifications, including DNA methylation, histone modification, and non-coding RNA expression (8 -11). Furthermore, the signaling pathways of various growth factors, the extracellular matrix, and cell adhesion molecules present in specific microenvironments or niche in NSCs are also known to play important roles in maintenance of the stem cell population (12-18). In particular, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) are primary mitogens to promote proliferation of NSCs through the mitogen-activated protein kinase (MAPK) pathway in vitro (19 -22). The responses of NSCs to those growth factors are thought to ...

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Section: Methodsmentioning

confidence: 99%

Involvement of β1-Integrin Up-regulation in Basic Fibroblast Growth Factor- and Epidermal Growth Factor-induced Proliferation of Mouse Neuroepithelial Cells

Suzuki

Yanagisawa

Yagi

et al. 2010

Journal of Biological Chemistry

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“…RNAiMAX has been used in multigene transfection and is considered an effective tool for gene delivery. As a new cationic liposome agent, RNAiMAX has a higher transfection efficiency compared with several other transfecting agents such as Oligofectamine and Lipofectamine 2000 (Zhao et al, 2008;Kamaci et al, 2011). Then, we found that RNAiMAX was effective for delivering FAM-miR-21 agomir into cortical neurons.…”

Section: Discussionmentioning

confidence: 90%

“…Lipofectamine RNAiMAX (RNAiMAX), a new cationic lipofection reagent, has shown higher efficiency in delivering miRNA into many stem cells and cell lines (Zhao et al, 2008;Malamas et al, 2013), although its transfection efficiency is still unclear in most primary cells, especially neurons. Here, we sought to establish a lipofection protocol to upregulate the expression level of miR-21 in neurons under different transfection conditions, including different transfection solvents (DMEM or Neurobasal), pretreatments of transfection (with starvation or not), and different reagent ratios, while maintaining viability and functional abilities of neurons.…”

mentioning

confidence: 99%

Establishment of Lipofection Protocol for Efficient miR-21 Transfection into Cortical Neurons In Vitro

Han

Tan

et al. 2015

DNA and Cell Biology

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Dysregulated microRNAs in neurons could cause many nervous system diseases. The therapeutic manipulation of these pathogenic microRNAs necessitates novel, efficient delivery systems to facilitate microRNA modulators targeting neurons with minimal off-target effects. The study aimed to establish a lipofection protocol to upregulate expression levels of miR-21 in neurons under different conditions, including different serum-free medium, transfection conditions, and reagent concentration, by evaluating the expression levels of miR-21 and neuron injury. The expression levels of miR-21 were higher in neurons transfected by Neurobasal-A than by DMEM. Expression levels of miR-21 were already the highest at the ratio RNAiMAX:miR-21 = 3:5, but the increase of RNAiMAX's concentration had not caused the further upregulation of expression level of miR-21. Neuron injury was condition dependent and dose dependent after transfection. Compared to S-Neurobasal groups, neurons have a smaller injury in N-Neurobasal groups, and compared to ratios RNAiMAX:miR-21 = 4:5, 5:5, neuron injury was smaller at ratios of RNAiMAX:miR-21 = 1:5, 2:5, 3:5. Without the pretreatment of starvation in vitro, the lipofection protocol was that RNAiMAX/miR-21 agomir complexes were diluted in Neurobasal-A at the ratio of RNAiMAX:miR-21 = 3:5.

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“…Cell culture H9 and H1 hES cell (WiCell Research Institute, Madison, WI, USA) culture was performed as described previously [24,34]. Briefly, H1 and H9 cells were cultivated on mouse embryonic fibroblasts (MEFs) in DMEM/F12 medium with 20% knockout serum replacement (KSR), 1 mM L-glutamine, 1% nonessential amino acids and 4 ng/ml basic fibroblast growth factor (all purchased from Invitrogen).…”

Section: Tissue Slidesmentioning

confidence: 99%

HESRG: a novel biomarker for intracranial germinoma and embryonal carcinoma

Wanggou

Jiang

et al. 2011

J Neurooncol

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The novel stem cell-related gene, HESRG, was first identified by our group, and its expression pattern in human tumors remains unknown. In this study, we used RT-PCR to systematically investigate the expression of HESRG in various types of intracranial tumors and found that HESRG was expressed only in germinoma and embryonal carcinoma, but hardly at all in other types of brain tumors. Real-time PCR results further confirmed this expression pattern. Subsequently, we tested 134 intracranial non-germ cell tumors and 64 intracranial germ cell tumors by immunohistochemistry. Our results showed that HESRG was expressed strongly and diffusively in the nuclei of tumor cells in intracranial germinoma and embryonal carcinoma as well as in human embryonic stem cells. No positive staining signal was observed in any other type of intracranial tumors. In germinomas, 25 of 31 showed intensive (3+) expression, four cases showed moderate (2+) immunostaining and the remaining 2 cases showed weak (1+) immunostaining. In embryonal carcinoma, 6 of 9 showed intensive (3+) immunostaining and 3 of 9 showed moderate (2+) immunostaining. These results suggest that HESRG is a novel, sensitive and specific biomarker for intracranial germinoma and embryonal carcinoma.

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Lipofectamine RNAiMAX: An Efficient siRNA Transfection Reagent in Human Embryonic Stem Cells

Cited by 66 publications

References 20 publications

Involvement of β1-Integrin Up-regulation in Basic Fibroblast Growth Factor- and Epidermal Growth Factor-induced Proliferation of Mouse Neuroepithelial Cells

Involvement of β1-Integrin Up-regulation in Basic Fibroblast Growth Factor- and Epidermal Growth Factor-induced Proliferation of Mouse Neuroepithelial Cells

Establishment of Lipofection Protocol for Efficient miR-21 Transfection into Cortical Neurons In Vitro

HESRG: a novel biomarker for intracranial germinoma and embryonal carcinoma

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