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Electroporation has been used successfully to introduce macromolecules such as DNA into the chick embryo for at least 15 years. Purified plasmid DNA is microinjected into embryo and then a series of low voltage electrical pulses are applied to the embryo which allows naked DNA to enter cells. Fol ...

Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, M�ller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumu ...

Analysis of transcription factor–target interactions in vivo is important to the study of transcriptional regulation of gene expression. A key experiment involves analysis of the functional interaction between a trans-acting factor and its corresponding cis-acting element in ...

Transgenesis, the process of incorporating an exogenous gene (transgene) into an organism’s genome, is a widely used tool to develop models of human diseases and to study the function and/or regulation of genes. Generating transgenic Xenopus is rapid and involves simple in vitro manipulat ...

Conditional knockout is a powerful research tool for specific deletion of target genes, especially for the genes that are widely expressed and developmentally regulated. The development of the retina involves multiple intrinsic and extrinsic factors, many are required for embryo ...

Delineating the final fate of progenitor cells that transiently express a regulatory gene may shed light on how the gene participates in regulating retinal development. We describe the steps in tracing final fates of progenitor cells that once transiently express neurogenin2 (ngn2) du ...

The Cre-loxP recombination system is widely used as a genetic tool to achieve conditional gene expression and for lineage tracing. Though extensively used in mice, this technology has only recently been applied to zebrafish. Here we describe Cre-loxP methodology for conditional expre ...

The mouse retina is composed of many cell types and subtypes with distinct morphology and function; how these cells are differentiated from the multipotent progenitors is still largely unknown. Retinal in vitro explant culture has proven to be a useful tool to study the molecular and cellular m ...

Rhodopsin is the dim-light photoreceptor responsible for initiation of the visual transduction cascade. In the dark its activity is very low, while light activation catalyzes the activation of its G-protein transducin. The first step in resetting rhodopsin and the phototransducti ...

Primary neuronal culture and transfection are useful tools in determining gene functions within specific tissue contexts and developmental stages. Chicken embryonic retinal cultures are easily obtainable and often robust as the chicken eye is relatively large compared to mouse ...

We describe a cell culture system for assaying proneural activities of genes hypothesized to play instrumental roles in neuronal fate specification during vertebrate retinal development. The retinal pigment epithelium (RPE) is collected from embryonic day 6 (E6) chick to establi ...

RCAS (B/P) is a replication-competent avian retrovirus engineered by Hughes et al. (J Virol 61:3004–3012, 1987) and is referred to in this chapter as RCAS for simplicity. The RCAS retrovirus has been widely used as a vehicle for stable transduction of a gene into cells both in the developing chick embryo ...

Retinal regeneration has been studied for decades in nonmammalian species. From these studies, we learned that retinal M�ller glia are a potential source of neuronal regeneration by de novo neurogenesis. Although spontaneous regeneration in mammals is absent after retinal damage, we ...

Exposure of the zebrafish retina to intense light is a noninvasive method to elicit the selective degeneration of photoreceptors. In zebrafish, photoreceptor degeneration is followed by robust photoreceptor regeneration from stem cells that are intrinsic to the teleost retina. T ...

Over the last few years, numerous studies have introduced strategies for the generation of neuronal populations from embryonic stem cells. These techniques are valuable both in the study of early neurogenesis and in the generation of an unlimited source of donor cells for replacement ther ...

Electroretinography is a noninvasive technique used to measure the electrical activity of neurons in the retina. Electroretinogram (ERG) waveforms can be used to quantify retinal function in normal and diseased rodents. In particular, the functions of rod and cone pathways can be isola ...

Microarray-based gene profiling has become an important technique to measure changes in gene expression on a genome-wide scale. Recently, cell-specific microarrays have been reported to study changes in gene expression of a particular cell type in several model organisms. Here, we des ...

Conjugation of SUMO (small ubiquitin-related modifier 1) is a critical posttranslational modification, with significant impact on protein function/activity. Here, we describe direct SUMOylation of GST (glutathione S-transferase)-fusion protein and immunoprecipit ...

In humans, protein-coding exons constitute 1.5–1.7% of the human genome. Targeted sequencing of all coding exons is termed as exome sequencing. This method enriches for coding sequences at a genome-wide scale from 3 μg of DNA in a hybridization capture. Exome analysis provides an excellent oppo ...

RNA expression profiles produced by next-generation sequencing (NGS) technology (RNA-seq) allow comprehensive investigation of transcribed sequences within a cell or tissue. RNA-seq is rapidly becoming more cost-effective for transcriptome profiling. However, its usage ...