Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation.

Brown, L E; Sprecher, S L; Keller, L R

doi:10.1128/mcb.11.4.2328

Cited by 98 publications

(49 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is consistent with the expectation that the cell wall significantly impedes transport of BSA and the hypothesis that electroporation does not increase cell wall permeability. It is also consistent with previous findings that DNA transfection of wall-deficient cells is greater than for wildtype cells (Brown et al 1991;Shimogawara et al 1998). Levels of BSA uptake in Figure 1B were generally lower than calcein uptake in Figure 1A for both strains (2-way ANOVA, p<0.01).…”

Section: Electroporationsupporting

confidence: 93%

“…Electroporation was selected as a delivery method because it is widely used and studied for delivery of DNA and other compounds to a broad variety of cell types, including plant cells (Nickoloff 1995) and specifically C. reinhardtii (Brown et al 1991;Shimogawara et al 1998), for which a quantitative mechanistic analysis has been published. Electroporation is believed to increase intracellular delivery by transiently disrupting cell membrane structure to form nanometer pores in the membrane that permit entry of macromolecules including DNA (Jaroszeski et al 2000;Weaver and Chizmadzhev 1996).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

Azencott

Peter

Prausnitz

2007

Ultrasound in Medicine & Biology

106

View full text Add to dashboard Cite

To assess the cell wall's role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where walldeficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare the effects of electroporation and sonication in a direct fashion in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane, because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it non-specifically disrupts cell-surface barriers.

show abstract

Section: Electroporationsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

Azencott

Peter

Prausnitz

2007

Ultrasound in Medicine & Biology

106

View full text Add to dashboard Cite

show abstract

“…Electroporation was performed as described previously (Brown et al, 1991;Hu et al, 2014;Shimogawara et al, 1998).…”

Section: Electro-transformation Of C Reinhardtiimentioning

confidence: 99%

Intraflagellar transport protein IFT52 recruits IFT46 to the basal body and flagella

Wan

Täschner

et al. 2017

Journal of Cell Science

View full text Add to dashboard Cite

Cilia are microtubule-based organelles and perform motile, sensing and signaling functions. The assembly and maintenance of cilia depend on intraflagellar transport (IFT). Besides ciliary localization, most IFT proteins accumulate at basal bodies. However, little is known about the molecular mechanism of basal body targeting of IFT proteins. We first identified the possible basal body-targeting sequence in IFT46 by expressing IFT46 truncation constructs in an ift46-1 mutant. The C-terminal sequence between residues 246-321, termed BBTS3, was sufficient to target YFP to basal bodies in the ift46-1 strain. Interestingly, BBTS3 is also responsible for the ciliary targeting of IFT46. BBTS3::YFP moves bidirectionally in flagella and interacts with other IFT complex B (IFT-B) proteins. Using IFT and motor mutants, we show that the basal body localization of IFT46 depends on IFT52, but not on IFT81, IFT88, IFT122, FLA10 or DHC1b. IFT52 interacts with IFT46 through residues L285 and L286 of IFT46 and recruits it to basal bodies. Ectopic expression of the C-terminal domain of IFT52 in the nucleus resulted in accumulation of IFT46 in nuclei. These data suggest that IFT52 and IFT46 can preassemble as a complex in the cytoplasm, which is then targeted to basal bodies.

show abstract

“…Electroporation has been used to transform Chlamydomonas as a means of introducing cloned genes [Brown et al, 1991;Shimogawara et al, 1998]. In other cells such as cultured cells and the cellular slime mold Dictyostelium, electroporation has been used for the introduction of a variety of substances.…”

Section: Introductionmentioning

confidence: 99%

Recovery of flagellar dynein function in a Chlamydomonas actin/dynein‐deficient mutant upon introduction of muscle actin by electroporation

Hayashi

Hirono

Kamiya

2001

Cell Motil. Cytoskeleton

View full text Add to dashboard Cite

Flagellar and ciliary inner-arm dyneins contain actin as a subunit; however, the function of this actin subunit remains unknown. As a first step toward experimental manipulation of actin in dynein, we developed a method for introducing exogenous actin into Chlamydomonas cells by electroporation. A non-motile mutant, ida5oda1, lacking inner-arm dyneins due to the absence of conventional actin, was electroporated in the presence of rabbit skeletal muscle actin. About 20% of the electroporated cells recovered motility under optimal conditions. In addition, by taking advantage of their phototactic behavior, the rescued cells could be concentrated. Motility was also recovered with fluorescently labeled actin; in this case, axonemes became fluorescent after electroporation, suggesting that actin was in fact incorporated as a dynein subunit. The feasibility of incorporating a substantial amount of macromolecules by electroporation will be useful not only for studying actin function, but also for a variety of studies using Chlamydomonas in which no efficient methods have been developed for expressing or introducing foreign proteins and other macromolecules.

show abstract

Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation.

Cited by 98 publications

References 16 publications

Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

Intraflagellar transport protein IFT52 recruits IFT46 to the basal body and flagella

Recovery of flagellar dynein function in a Chlamydomonas actin/dynein‐deficient mutant upon introduction of muscle actin by electroporation

Contact Info

Product

Resources

About