5 results on '"Gutierrez, Christian A."'
Search Results
2. Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach
- Author
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Vergara, M. Natalia, Gutierrez, Christian, and Canto-Soler, M. Valeria
- Subjects
Electroporation ,Primary Cell Culture ,Animals ,Reproducibility of Results ,sense organs ,Chick Embryo ,Transfection ,Retina ,Developmental Biology ,Photoreceptor Cells, Vertebrate ,Plasmids - Abstract
The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols(1). In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.
- Published
- 2015
3. Ex vivo electroporation of retinal cells: A novel, high efficiency method for functional studies in primary retinal cultures
- Author
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Vergara, M. Natalia, Gutierrez, Christian, O'Brien, David R., and Canto-Soler, M. Valeria
- Subjects
- *
ELECTROPORATION , *CELL culture , *CELL growth , *CELL physiology , *CELL differentiation , *GENE expression - Abstract
Abstract: Primary retinal cultures constitute valuable tools not only for basic research on retinal cell development and physiology, but also for the identification of factors or drugs that promote cell survival and differentiation. In order to take full advantage of the benefits of this system it is imperative to develop efficient and reliable techniques for the manipulation of gene expression. However, achieving appropriate transfection efficiencies in these cultures has remained challenging. The purpose of this work was to develop and optimize a technique that would allow the transfection of chick retinal cells with high efficiency and reproducibility for multiple applications. We developed an ex vivo electroporation method applied to dissociated retinal cell cultures that offers a significant improvement over other currently available transfection techniques, increasing efficiency by five-fold. In this method, eyes were enucleated, devoid of RPE, and electroporated with GFP-encoding plasmids using custom-made electrodes. Electroporated retinas were then dissociated into single cells and plated in low density conditions, to be analyzed after 4 days of incubation. Parameters such as voltage and number of electric pulses, as well as plasmid concentration and developmental stage of the animal were optimized for efficiency. The characteristics of the cultures were assessed by morphology and immunocytochemistry, and cell viability was determined by ethidium homodimer staining. Cell imaging and counting was performed using an automated high-throughput system. This procedure resulted in transfection efficiencies in the order of 22–25% of cultured cells, encompassing both photoreceptors and non-photoreceptor neurons, and without affecting normal cell survival and differentiation. Finally, the feasibility of the technique for cell-autonomous studies of gene function in a biologically relevant context was tested by carrying out gain and loss-of-function experiments for the transcription factor PAX6. Electroporation of a plasmid construct expressing PAX6 resulted in a marked upregulation in the expression levels of this protein that could be measured in the whole culture as well as cell-intrinsically. This was accompanied by a significant decrease in the percentage of cells differentiating as photoreceptors among the transfected population. Conversely, electroporation of an RNAi construct targeting PAX6 resulted in a significant decrease in the levels of this protein, with a concomitant increase in the proportion of photoreceptors. Taken together these results provide strong proof-of-principle of the suitability of this technique for genetic studies in retinal cultures. The combination of the high transfection efficiency obtained by this method with automated high-throughput cell analysis supplies the scientific community with a powerful system for performing functional studies in a cell-autonomous manner. [Copyright &y& Elsevier]
- Published
- 2013
- Full Text
- View/download PDF
4. The BMP pathway: Its role in retina regeneration
- Author
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Gutierrez, Christian
- Subjects
- Biology, Cellular Biology, Molecular Biology, retina, regeneration, BMP
- Abstract
The embryonic chick eye is an ideal model to study retina regeneration since it has the ability to regenerate via two different modes: via the activation of stem/progenitor cells located in the anterior region of the eye or via the transdifferentiation of the retinal pigmented epithelium. Both modes give rise to a laminated retina with all major retinal cell types present. In this study we analyze the role of the Bone Morphogenetic Protein (BMP) pathway during the process of chick retina regeneration. The BMP pathway has been well documented in being involved in stem cell maintenance, cell differentiation and apoptosis. We demonstrate that activating the BMP pathway is sufficient to induce retina regeneration from stem/progenitor cells. During the early stages of retina regeneration, the BMP pathway promotes Fibroblast Growth Factor (FGF) signaling through the activation of its canonical pathway to promote proliferation, leading to the formation of a neuroepithelium. In later stages of retina regeneration, the BMP pathway inhibits the FGF pathway and signals through a non-canonical pathway, promoting cell death.
- Published
- 2008
5. Flexible parylene-based multielectrode array technology for high-density neural stimulation and recording
- Author
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Rodger, Damien C., Fong, Andy J., Li, Wen, Ameri, Hossein, Ahuja, Ashish K., Gutierrez, Christian, Lavrov, Igor, Zhong, Hui, Menon, Parvathy R., Meng, Ellis, Burdick, Joel W., Roy, Roland R., Edgerton, V. Reggie, Weiland, James D., Humayun, Mark S., and Tai, Yu-Chong
- Subjects
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CENTRAL nervous system , *PLATINUM , *ELECTRIC stimulation , *RETINA - Abstract
Abstract: Novel flexible parylene-based high-density electrode arrays have been developed for functional electrical stimulation in retinal and spinal cord prosthetics. These arrays are microfabricated according to a single-metal-layer process and a revolutionary dual-metal-layer process that promises to meet the needs of extremely high-density stimulation applications. While in many cases thin-film platinum electrodes in parylene C would be sufficient, high surface-area platinum electroplating has been shown to extend the lifetime of stimulated electrodes to more than 430 million pulses without failing. Iridium electrode arrays with higher charge delivery capacity have also been fabricated using a new high-temperature stabilized parylene variant, parylene HT. In addition, a new heat molding process has been implemented to conform electrode arrays to approximate the curvature of canine retinas, and a chronic implantation study of the mechanical effects of parylene-based electrode arrays on the retina over a 6-month follow-up period has provided excellent results. Retinal stimulation from these parylene-based electrode arrays in an isolated tiger salamander preparation was shown to be comparable to light stimulation in terms of generation of action potentials in the inner retina. Finally, electrode arrays have also been implanted and tested on the spinal cords of murine models, with the ultimate goal of facilitation of locomotion after spinal cord injury; these arrays provide a higher density and better spatial control of stimulation and recording than is typically possible using traditional fine-wire electrodes. Spinal cord stimulation typically elicited three muscle responses, an early (direct), a middle (monosynaptic), and a late (polysynaptic) response, classified based on latency after stimulation. Stimulation at different rostrocaudal levels of the cord yielded markedly different muscle responses, highlighting the need for such high-density arrays. [Copyright &y& Elsevier]
- Published
- 2008
- Full Text
- View/download PDF
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