High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment

Lopata, Margaret A.; Cleveland, Don W.; Sollner‐Webb, Barbara

doi:10.1093/nar/12.14.5707

Cited by 840 publications

(395 citation statements)

References 13 publications

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“…Transfection into COS7 Cells-COS7 cells were cultured in DMEM with 10% FBS. Transfection of plasmids into COS7 cells was carried out by the standard DEAE-dextran method (36). Cells were seeded on a 60-mm dish at a cell density of 5 ϫ 10 5 cells/dish in DMEM with 10% FBS and cultured overnight.…”

Section: Methodsmentioning

confidence: 99%

Cytoskeletal Rearrangements and Transcriptional Activation of c-fos Serum Response Element by Rho-kinase

Chihara

Amano

Nakamura

et al. 1997

Journal of Biological Chemistry

168

134

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The small GTPase Rho is implicated in cytoskeletal rearrangements including stress fiber and focal adhesion formation and in the transcriptional activation of c-fos serum response element. In vitro, Rho-kinase, which is activated by Rho, phosphorylates not only myosin light chain (MLC) (thereby activating myosin ATPase) but also myosin phosphatase, thus inactivating myosin phosphatase. Rho-kinase is involved in the formation of stress fibers and focal adhesions in fibroblasts. Here we show that the expression of constitutively active Rho-kinase increased the level of MLC phosphorylation. The activity of Rho-kinase was necessary for maintaining the vinculin-containing focal adhesions, whereas organized actin stress fibers were not necessary for this. The microinjection of constitutively active Rho-kinase into fibroblasts induced the formation of focal adhesions to some extent under the conditions where organized actin stress fibers were disrupted. The expression of constitutively active Rhokinase also stimulated the transcriptional activity of c-fos serum response element. These results suggest that Rho-kinase has distinct roles in divergent pathways downstream of Rho, which include MLC phosphorylation leading to stress fiber formation, focal adhesion formation, and gene expression.

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Section: Methodsmentioning

confidence: 99%

Cytoskeletal Rearrangements and Transcriptional Activation of c-fos Serum Response Element by Rho-kinase

Chihara

Amano

Nakamura

et al. 1997

Journal of Biological Chemistry

168

134

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show abstract

“…Cell Transfection and Analysis-COS-1 and H-35 cells were transfected by the DEAE-dextran method (20) and COS-7 cells by the lipofectamine method (4). For analysis of STAT protein activation, COS cells were maintained for 16 h in serum-free medium, followed by treatment of cells with 100 ng/ml leptin or G-CSF for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Leptin Receptor (OB-R) Signaling

White

Kuropatwinski

Devos

et al. 1997

Journal of Biological Chemistry

260

101

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The leptin receptor (OB-R) mediates the weight regulatory effects of the adipocyte secreted hormone leptin (OB). Previously we have shown that the long form of OB-R, expressed predominantly in the hypothalamus, can mediate ligand-induced activation of signal transducer and activator of transcription factors 1, 3, and 5 and stimulate transcription via interleukin-6 and hematopoietin receptor responsive gene elements. Here we report that deletion and tyrosine substitution mutagenesis of OB-R identifies two distinct regions of the intracellular domain important for signaling. In addition, granulocyte-colony stimulatory factor receptor/OB-R and OB-R/granulocyte-colony stimulatory factor receptor chimeras are signaling competent and provide evidence that aggregation of two OB-R intracellular domains is sufficient for ligand-induced receptor activation. However, signaling by full-length OB-R appears to be relatively resistant to dominant negative repression by signaling-incompetent OB-R, suggesting that mechanisms exist to permit signaling by the long form of OB-R even in the pretence of excess naturally occurring short form of OB-R.Leptin (OB) is an adipose tissue-derived secreted hormone that is thought to suppress appetite by regulating activities of satiety centers in the brain (1). The weight reducing effects of leptin appear to be mediated by interaction with the leptin receptor (OB-R) 1 in the hypothalamus, a region of the brain implicated in the control of body weight (2-4). In mice, mutations in the genes encoding either OB-R (db) or leptin (ob) result in profound early-onset obesity (5, 6). Multiple splice variants of OB-R mRNAs encoding proteins with different length intracellular domains have been detected (7,8). The mutant allele (db) of the OB-R gene was shown to encode a receptor with a truncated cytoplasmic domain (7,8), and more recent data suggest this receptor is signaling inactive (9). Thus, mounting evidence suggests the ability of leptin to regulate body weight is facilitated by downstream signaling events initiated by ligand-induced OB-R activation.Sequence homology and more recent functional data suggest OB-R is a member of the class I cytokine receptor superfamily (4, 10, 11). Receptors of this class lack intrinsic tyrosine kinase activity and are activated by ligand-induced receptor homo-or hetero-dimerization and in many cases require activation of receptor-associated kinases of the Janus family (JAKs) (12). JAKs associate with the membrane-proximal domain of the intracellular part of the cytokine receptors and serve to initiate signal transduction pathways following ligand-induced receptor activation. Included among the downstream targets of the JAK proteins are members of the STAT (Signal Transducers and Activators of Transcription) family of transcription factors (12). The STATs are DNA binding transcription factors that contain Src homology (SH2) domains that interact with receptor molecules through phosphorylated tyrosine residues. STAT proteins are activated by tyrosine phosphorylation...

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“…Glycerol has been reported to enhance transfections with polylysine, 39 DEAE-dextran, 40 and with calcium phosphate precipitation. 41 Glycerol is thought to enhance transfections by the labilization of vesicular membranes 39 and thus it would facilitate endosome lysis before lysosomal degradation.…”

Section: Figure 4 Expression Of Adenoviral Proteins In Cells After Pementioning

confidence: 99%

Transfection of large plasmids in primary human myoblasts

et al. 2001

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High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment

Cited by 840 publications

References 13 publications

Cytoskeletal Rearrangements and Transcriptional Activation of c-fos Serum Response Element by Rho-kinase

Cytoskeletal Rearrangements and Transcriptional Activation of c-fos Serum Response Element by Rho-kinase

Leptin Receptor (OB-R) Signaling

Transfection of large plasmids in primary human myoblasts

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