Transient GUS gene expression in cassava (Manihot esculenta Crantz) using Agrobacterium tumefaciens leaf infiltration / Expresión transitoria del gen GUS en yuca (Manihot esculenta Crantz) utilizando infiltración con Agrobacterium tumefaciens

Objective. Assess transient gene expression of GUS in cassava (Manihot esculenta Crantz) leaves using Agrobacterium tumefaciens infiltration. Materials and methods. A. tumefaciens strains GV3101 and AGL1 containing pCAMBIA1305.2 were used to evaluate transient gene expression of β-glucuronidase (GUS). A. tumefaciens infiltration (agroinfiltration) was made using both leaves from in vitro and 1 month old greenhouse plants. Leaves were incubated in X-GLUC buffer, stained and photographed to detect GUS activity. Results. Agroinfiltration assays showed GUS transient expression in leaves of cassava varieties widely cultivated in the north coast and eastern savannah, MCOL2215 (Venezuelan) and CM6438-14 (Vergara), respectively. A. tumefaciens agressive strain AGL1 showed high efficiency inducing GUS expression in cassava leaves. Conclusions. We recommend using A. tumefaciens agressive strain AGL1 for agroinfiltration to assess transient expression in cassava leaves.

Objetivo. Evaluar la expresión transitoria del gen GUS en hojas de yuca (Manihot esculenta Crantz) por medio de infiltración con Agrobacterium tumefaciens. Materiales y métodos. Se utilizaron las cepas GV3101 y AGL1 de A. tumefaciens conteniendo el plásmido pCAMBIA1305.2, para evaluar la expresión transitoria del gen GUS. La infiltración de A. tumefaciens (agroinfiltración) se realizó tanto en hojas de plantas "in Vitro" como de plantas adultas de 1 mes. Las hojas se incubaron en tampón X-GLUC, se destiñeron y se fotografiaron para detectar la actividad de la enzima β-glucuronidasa (GUS). Resultados. Los ensayos de agroinfiltración en hoja muestraron la expresión transitoria del gen GUS en variedades cultivadas en la costa norte y en los llanos orientales, MCOL2215 (Venezolana) y CM6438-14 (Vergara) respectivamente, tanto en plantas "in Vitro" como en plantas adultas. La cepa hipervirulenta de A. tumefaciens AGL1 mostró una mayor eficiencia para la expresión transitoria en hojas de yuca. Conclusiones. Se recomienda utilizar la cepa AGL1 para evaluar la expresión transitoria de genes de interés por agroinfiltración en hojas de yuca.

Optimization of polyethylenimine-mediated transient transfection using response surface methodology design

Response surface methodology was undertaken to optimize the polyethylenimine-mediated transient transfection of suspension cultured HEK 293-F cells. A total of 15 combinations were designed according to Box-Behnken design to identify the effects of DNA concentration, polyethylenimine concentration and incubation time on transient transfection efficiency. The highest integral optic density of green fluorescent protein presenting r-protein yield was accessed using a DNA concentration of 1.75 ug/mL, a polyethylenimine concentration of 10.5 ug/mL, and an incubation time of 11.8 min. Analysis of variance demonstrated that the experimental values fit well with a quadratic model. The RSM-optimized transient transfection resulted in greater production of human tissue prokallikrein (TproK) than non-RSM-optimized conditions: protein yield was 32.0 mg/L and the maximum viable cell density reached 3.57 x 10(6) cells/mL in a 5 L stirred-tank bioreactor culture.

Isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga Porphyra yezoensis

Despite the recent progress of transient gene expression systems in a red alga Porphyra yezoensis by particle bombardment, a stable transformation system has yet to establish in any marine red macrophytes. One of the reasons of the difficulty in genetic transformation in red algae is the lack of systems to select and isolate transformed cells from gametophytic blades. Thus, toward the establishment of the stable transformation system in P. yezoensis, we have developed a procedure by which transiently transformed gametophytic cells were prepared from particle bombarded-gametophytic blade as regeneratable protoplasts. Using mixture of marine bacterial enzymes, yield of protoplasts was high as reported elsewhere; however, these protoplasts did not develop. In contrast, protoplasts prepared from gametophytes treated with allantoin were normally developed, in which the overexpression of a â-glucuronidase reporter gene had no effect on the regeneration of protoplasts. Therefore, the use of allantoin in protoplast preparation sheds a new light on the realization of an efficient isolation and selection of study transformed cells from gametophytic blades.

Cloning of a cryIIIA endotoxin gene of Bacillus thuringiensis var. tenebrionis and its transient expression in indica rice.

Damage caused to rice production by coleopteran insects like rice weevil (Sitophilus oryzae), a stored grain insect pest and rice hispa (Dicladispa armigera), a pest of the growing plant is quite high. In order to combat the damage, generation of insect resistant transgenic rice plant was considered desirable. CryIIIA endotoxin of Bacillus thuringiensis var. tenebrionis, a 65 kDa protein toxic to coleopteran insects, figured as the candidate gene product. Thus, the cryIIIA gene was isolated from a local isolate of Bacillus thuringiensis var. tenebrionis. The gene was tailored at the N-terminal end to its minimal size by using a synthetic ATG codon which replaced the first codon next to ATG of threonine to proline. This modification did not affect the functional property of the gene product.A chimeric construct of the modified cryIIIA gene was developed containing CaMV35S promoter and nos terminator for plant expression. The expressibility of the cryIIIA gene in indica rice was judged through test for transient expression in indica rice protoplasts.

THE CONSTRUCTION OF TRANSIENT GENE EXPRESSION TRANSFECTION SYSTEM IN MCF-7 CELLS / 解剖学报

Objective To establish an efficient, simple, low cytotoxicity and cheap transfaction system. Methods We have used the cationic polymer polyethylenimine(PEI) to study transient transfection in MCF-7 cells by testing different conditions, including cell concentrations, DNA concentrations, the ratio of PEI nitrogen to DNA phosphate(PEI-N∶DNA-P) and the time of cells grown in serum-free culture together with PEI-DNA complex. Results The optimized cell concentrations were 2?10 5 cells seeded per well in 24-well dishes 18-24?h before transfection. The DNA concentrations and ratio of PEI-N∶DNA-P are very important for optimal transfection and they affect each other. For 1??g DNA per well, the optimal PEI-N∶DNA-P is about 33∶1, however, as for 4??g DNA, it is 9∶1. The best time of cells grown in serum-free culture together with PEI-DNA complex is about 5-7?h.Conclusion With optimized conditions, we can establish an efficient, simple, low cytotoxicity and cheap transfection system by using PEI.