Daniel Felipe Rodriguez Barajas - Academia.edu (original) (raw)

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Papers by Daniel Felipe Rodriguez Barajas

Journal of Plant Pathology, Nov 1, 2004

Methods in molecular biology, 2012

Our increasing understanding of virus-host interactions is revealing a complex role for host fact... more Our increasing understanding of virus-host interactions is revealing a complex role for host factors during virus replication. Besides the role of some host proteins in defense against viruses, it is becoming clear that viruses also hijack several host functions to utilize them for their multiplication. Genome-wide screens using high-throughput methods are being conducted to identify most of the host factors affecting virus replication in a number of host-virus systems. For selected plant viruses, such as bromo- and tombusviruses, yeast has been developed as a model host, thus greatly accelerating genome-wide systematic approaches to identify critical host factors of virus multiplication. In plants, gene knock out T-DNA libraries and virus-induced RNA silencing, among other strategies, can be utilized to identify and characterize host factors involved in virus replication. An additional strategy to study the role of host factors is the use of dominant-negative (DN) mutants, which are mutant versions of host proteins capable of interfering with the function of the wild-type protein without the need of knocking out the given gene from the chromosome. This method allows one to study the relevance of host factors for virus replication in wild-type plants and may overcome some limitations of other methods. Here, we provide guidelines to the use of a DN mutant strategy for the study of host factors and compare the advantages and limitations with other methods. The use of more diverse methods to study gene function in plants is increasing the probability of successfully identifying and characterizing host factors affecting virus replication in plant systems.

PLOS Pathogens, Feb 10, 2016

Journal of Virology, Apr 1, 2016

PARJAP: Boletín de la Asociación Española de Parques y Jardines, 2007

Biotechnology Progress, Oct 22, 2018

Recombinant adeno‐associated viral vectors (rAAV) are promising therapies for genetic diseases. A... more Recombinant adeno‐associated viral vectors (rAAV) are promising therapies for genetic diseases. Although current platforms for recombinant vector production can generate drug material for pre‐clinical and clinical studies, rAAV biomanufacturing will eventually face commercial supply challenges if per cell vector productivity and process scalability are not improved. Because considerable efforts have traditionally focused on optimizing rAAV plasmid design, herein we investigate the impact of host cell proteins on vector production to identify proteins that may enhance rAAV yield. Using a rAAV2‐GFP‐producing Saccharomyces cerevisiae model in combination with the yeast Tet Hughes Collection screening library, we identified 22 gene candidates that improved rAAV DNA replication (rAAV‐GFP/18s rDNA ratio) and vector yield (benzonase‐resistant rAAV DNA vector genome titer) as high as 6‐fold and 15‐fold relative to control, respectively. The candidate proteins participate in biological processes such as DNA replication, ribosome biogenesis, and RNA and protein processing. The best five candidates (PRE4, HEM4, TOP2, GPN3, and SDO1) were further screened by generating overexpression mutants in the YPH500 yeast strain. Subsequent clone evaluation was performed to confirm the rAAV‐promoting activity of selected candidates under plate‐based and bioreactor‐controlled fermentation conditions. Digital droplet PCR analysis of cell lysate and AVB resin‐purified material confirmed HEM4 and TOP2 overexpression mutants displayed the highest per cell total rAAV DNA productivity (1.6 and 1.7‐fold increase over control, respectively) and per cell vector productivity (3 and 4‐fold over control, respectively). This evaluation confirmed that overexpression of HEM4 and TOP2 proteins enhanced total and benzonase‐resistant rAAV DNA yield. Further studies are needed to understand their mechanism of action and to assess their potential application in molecular strategies for rAAV production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2725, 2019