Development of a Particle Bombardment-Mediated Transient Transformation System for Taxus spp. Cells in Culture

Vongpaseuth, Kham; Nims, Ezekiel; Amand, Melissa St.; Walker, Elsbeth L.; Roberts, Susan C.

doi:10.1021/bp0700307

Cited by 17 publications

(15 citation statements)

References 28 publications

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“…Within two hours, the cells were transformed by bombardment using a PDS-1000 (Bio-RAD, Hercules, CA). Preparation of gold microcarriers was performed as described (22). Four replicates of each experiment were performed.…”

Section: Methodsmentioning

confidence: 99%

WITHDRAWN: TcJAMYC: A bHLH transcription factor that activates paclitaxel biosynthetic pathway genes in yew

Nims¹,

Vongpaseuth²,

Roberts³

et al. 2009

Journal of Biological Chemistry

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Abstract:Paclitaxel (Taxol ®) is a highly modified diterpene anti-cancer agent produced by the gymnosperm Taxus. Taxus suspension cell cultures have the potential to provide a sustainable source of paclitaxel, but the paclitaxel biosynthetic pathway is not fully characterized, making metabolic engineering efforts difficult. Methyl jasmonate (MJ) is used to elicit paclitaxel production in suspension cultures. Here we show that the promoters of five genes encoding enzymes of the paclitaxel biosynthetic pathway are activated by MJ elicitation.Thus, elicitation of paclitaxel production by MJ is regulated at least in part at the level of transcription. A transcription factor that positively activates the promoters of paclitaxel biosynthetic genes has been cloned. This transcription factor, TcJAMYC, possesses a high degree of similarity to AtMYC2 and JAMYC2, which are known to regulate the expression of jasmonate inducible genes in other systems. TcJAMYC binds to E-boxes found in the promoters of the paclitaxel pathway genes, and these promoters are transiently activated by TcJAMYC overexpression. Addition of MJ attenuates the effect of TcJAMYC on the pathway promoters, suggesting that TcJAMYC could be engineered into Taxus cells to avoid the negative regulation of MJ-induced genes that follows initial MJinduced positive regulation.This strategy could prolong the expression of paclitaxel pathway genes, and increase paclitaxel production.

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Section: Methodsmentioning

confidence: 99%

WITHDRAWN: TcJAMYC: A bHLH transcription factor that activates paclitaxel biosynthetic pathway genes in yew

Nims¹,

Vongpaseuth²,

Roberts³

et al. 2009

Journal of Biological Chemistry

Self Cite

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show abstract

“…Optimized parameters, however, were found to be highly dependent on the Taxus spp. cell line [129]. In addition, the particle bombardment-mediated transformation system was also shown to be capable of transiently expressing the -glucuronidase (GUS) and DsRed reporter genes under the control of CaMV 35S and the maize ubiquitin promoters, respectively (Fig.…”

Section: Transformation Of Taxus Sppmentioning

confidence: 99%

“…with Agrobacterium EHA105 [128] and transient transformation with particle-mediated bombardment [129] provides the community with technologies to both enable metabolic engineering of Taxus spp. suspensions and a system to characterize novel genes putatively related to paclitaxel metabolism.…”

Section: Transformation Of Taxus Sppmentioning

confidence: 99%

Advancements in the Understanding of Paclitaxel Metabolism in Tissue Culture

Vongpaseuth¹,

Roberts

2007

CPB

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Paclitaxel is a potent chemotherapeutic agent approved in the treatment of a variety of cancers, and under evaluation for the treatment of Alzheimer's and heart disease. Originally isolated from Taxus brevifolia, this highly substituted ring diterpenoid belongs to a family of plant secondary metabolites known as taxoids. Paclitaxel is currently supplied through both a semi-synthetic process and plant cell culture. Taxus spp. cell culture offers the potential to produce large amounts of paclitaxel and related taxoids, although variability in accumulation and low yields represent key limitations. Thus, intense efforts have been put forth towards understanding Taxus spp. metabolism to increase paclitaxel accumulation in cell culture. While elicitation and environmental optimization have provided some success in increasing paclitaxel accumulation in vitro, understanding metabolism of paclitaxel on the molecular level is essential for process optimization. Utilizing direct and indirect molecular techniques, a further understanding of paclitaxel biosynthesis has been gained, though knowledge into other aspects of paclitaxel global metabolism, such as regulation, transport, and degradation is lacking. Taxus spp. cell cultures are highly heterogeneous, displaying significant cell-cell variability in growth and paclitaxel accumulation. Information gathered on culture subpopulations as well as putative transcriptional bottlenecks in paclitaxel biosynthesis, coupled with successful transformation of Taxus spp. will allow for the targeted metabolic engineering of Taxus spp. or other model organisms for paclitaxel accumulation to ensure future supply of this important pharmaceutical.

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“…In previous studies, the methods used for gene transient expression in plants were usually particle bombardment [17][18] and electroporation [13]. In the present study, we provide an alternative direct transformation method which is able to motivate linear gene cassettes into the host cell nucleus by agents designed via altering the permeability of cell membrane.…”

Section: Discussionmentioning

confidence: 98%

Transient Expression of Minimum Linear Gene Cassettes in Onion Epidermal Cells Via Direct Transformation

Cheng

Yang

et al. 2009

Appl Biochem Biotechnol

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A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.

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Development of a Particle Bombardment-Mediated Transient Transformation System for Taxus spp. Cells in Culture

Cited by 17 publications

References 28 publications

WITHDRAWN: TcJAMYC: A bHLH transcription factor that activates paclitaxel biosynthetic pathway genes in yew

WITHDRAWN: TcJAMYC: A bHLH transcription factor that activates paclitaxel biosynthetic pathway genes in yew

Advancements in the Understanding of Paclitaxel Metabolism in Tissue Culture

Transient Expression of Minimum Linear Gene Cassettes in Onion Epidermal Cells Via Direct Transformation

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