Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons

Guerra-Crespo, Magdalena

doi:10.1016/s0165-0270(03)00125-0

Cited by 36 publications

(30 citation statements)

References 44 publications

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“…COS-7 cells were transfected with plasmids (500 ng) coding for different Stau1 mutants using PEI as previously described (Guerra-Crespo et al 2003). Cells were fixed in a freshly made 4% paraformaldehyde solution in PBS for 20 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS.…”

Section: Microscopymentioning

confidence: 99%

Multimerization of Staufen1 in live cells

Martel

Dugré-Brisson²,

Boulay³

et al. 2010

RNA

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Transport of mRNA is an efficient mechanism to target proteins to specific regions of a cell. Although it is well documented that mRNAs are transported in ribonucleoprotein (RNP) complexes, several of the mechanisms involved in complex formation and localization are poorly understood. Staufen (Stau) 1, a double-stranded RNA-binding protein, is a well accepted marker of mRNA transport complexes. In this manuscript, we provide evidence that Stau1 self-associates in live cells using immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays. The double-stranded RNA-binding domains dsRBD3 and dsRBD4 contributed about half of the signal, suggesting that Stau1 RNA-binding activity is involved in Stau1 selfassociation. Protein-protein interaction also occurred, via dsRBD5 and dsRBD2, as shown by in vitro pull-down, yeast twohybrid, and BRET assays in live cells. Interestingly, Stau1 self-association contributes to the formation of oligomeric complexes as evidenced by the coexpression of split Renilla luciferase halves covalently linked to Stau1 in a protein complementation assay (PCA) combined with a BRET assay with Stau1-YFP. Moreover, we showed that these higher-order Stau1-containing complexes carry RNAs when the RNA stain SYTO 14 was used as the energy acceptor in the PCA/BRET assay. The oligomeric composition of Stau1-containing complexes and the presence of specific mRNAs have been confirmed by biochemical approaches involving two successive immunoprecipitations of Stau1-tagged molecules followed by qRT-PCR amplification. Altogether, these results indicate that Stau1 self-associates in mRNPs via its multiple functional domains that can select mRNAs to be transported and establish protein-protein interaction.

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Section: Microscopymentioning

confidence: 99%

Multimerization of Staufen1 in live cells

Martel

Dugré-Brisson²,

Boulay³

et al. 2010

RNA

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“…In transient gene expression studies, cells are often cotransfected with the gene under study together with a GFP expression vector in order to sort transfected from untransfected cells by FACS. For example Guerra-Crespo et al (2003) cotransfected primary rat hypothalamic neurones with a GFP reporter under the transcriptional control of the thyrotrophin-releasing hormone (TRH) promoter. FACS was applied to isolate TRH neurons for further study.…”

Section: Fluorescence-activated Cell Sortingmentioning

confidence: 99%

Pharmaceutical Applications of GFP and RCFP

Bevan

Rees

2005

Green Fluorescent Protein

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“…33 In addition, PEI was concluded to effectively enhance the DNA-transfection efficiency and enzyme immobilization. [34][35][36][37][38] Although the composite PEO/chitin/chitosan scaffolds are very promising in cartilage regeneration, adhesion of chondrocytes on the scaffold surfaces can be improved. Note that PEI was concluded to effectively enhance cell adhesion.…”

Section: Introductionmentioning

confidence: 99%

Application of polyethyleneimine‐modified scaffolds to the regeneration of cartilaginous tissue

Kuo

2009

Biotechnology Progress

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In this study, we analyzed the physicochemical and biophysical properties of three-dimensional scaffolds modified using polyethyleneimine (PEI) and applied these scaffolds to the cultivation of bovine knee chondrocytes (BKCs). PEI was crosslinked in the bulk or on the surface of the ternary scaffolds comprising polyethylene oxide, chitin and chitosan. The results revealed that when the concentration of PEI was less than 300 microg/mL, the cytotoxicity of a scaffold was on the same order in the two method of modification. An increase in the concentration of PEI favored the adhesion of BKCs. When the amount of PEI in scaffolds is fixed, the surface-modified scaffolds exhibited a higher adhesion efficiency of BKCs than the bulk-modified scaffolds. For the regeneration of cartilaginous components, a higher amount of PEI in a scaffold yielded larger amounts of proliferated BKCs, secreted glycosaminoglycans, and produced collagen. In addition, the formation of neocartilage in the surface-modified scaffolds was more effective than that in the bulk-modified scaffolds. These tissue-engineered scaffolds, modified by an appropriate concentration of PEI, can be potentially applied to cartilage repair in clinical trials.

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Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons

Cited by 36 publications

References 44 publications

Multimerization of Staufen1 in live cells

Multimerization of Staufen1 in live cells

Pharmaceutical Applications of GFP and RCFP

Application of polyethyleneimine‐modified scaffolds to the regeneration of cartilaginous tissue

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