Retrovirus-mediated gene transfer to retinal explants
Introduction
The retina is an ideal model system to investigate the mechanisms of generation of multiple cell types from common precursors [1], [2] because it has a relatively simple structure, mimics normal development in isolated explant cultures, and is therefore easy to analyze [3], [4], [5], [6], [7]. In the neural retina, seven types of cells (six types of neurons and one type of glial cells) form three cellular layers: the outer nuclear layer (ONL), which contains rod and cone photoreceptors; the inner nuclear layer (INL), which contains bipolar, horizontal, and amacrine interneurons and Müller glial cells; and the ganglion cell layer (GCL), which contains ganglion and displaced amacrine cells. These seven types of cells differentiate from common precursors in an order that is generally conserved among many species: ganglion cells first and bipolar and Müller glial cells last. This ordered differentiation is also observed in retinal explant cultures prepared from mouse embryos.
It has been shown that retinal cell differentiation is controlled by intrinsic cues, such as transcription factors, and by extrinsic signals, such as neurotrophic factors [1], [2]. The function of extrinsic signals can be assessed by direct application to retinal explant cultures, whereas that of intrinsic cues should be tested by introducing the genes into retinal cells. For the latter purpose, transgenic mice are often used. However, the efficiency of misexpression of exogenous genes in transgenic mice is variable, and thus many independent transgenic lines should be examined.
One alternative and very successful method is gene introduction by a recombinant retrovirus, an infectious vehicle that efficiently transfers genes. Since retroviruses are infectious only to mitotic cells, neural precursor cells are the major targets in the nervous system. After infection, a single DNA copy (proviral form) of the RNA viral genome is stably integrated into the genome of host cells, and this DNA copy is precisely transmitted to the progeny of infected cells. Thus, the retrovirus is suitable for changing stably the phenotypes of dividing cells. Furthermore, the onset of exogenous gene expression can be easily controlled by changing the time of infection. This feature is particularly good to analyze the roles of transcription factors because they have different functions at different stages of retinal development. This method is also suitable for clonal analysis: identification of cell types and numbers derived from single virus-infected cells. While in utero or ex utero embryos can be used for virus infection study, it is much easier to use retinal explant cultures. Here, we describe the methods of retinal explant cultures and retrovirus-mediated gene transfer and their application to analysis of gene functions in retinal development.
Section snippets
Retroviral vectors
The RNA genome (a plus-strand RNA) of a retrovirus contains the following structures: R, U5, packaging signal (ψ), gag, pol, env, U3, and R (Fig. 1A) [8]. Among these structures, gag, pol, and env encode capsid protein, reverse transcriptase, and viral coat proteins, respectively. The 5′ end of the RNA genome has a cap structure while the 3′ end has a polyadenylation site, like cellular mRNAs. After infection, the double-strand DNA copy (a proviral form) is produced from the RNA genome by
Results and discussion
Retinal explants were prepared from mouse embryos E17.5 and CLIG retrovirus was applied. After 2 weeks of culture, immunohistochemistry with anti-GFP was performed. Under this condition, CLIG-infected precursor cells differentiate into all retinal cell types (Fig. 3). Rod photoreceptors are most abundant (about 80%), while ganglion cells are very few (less than 1%) (Fig. 3B). Other cell types are within 2–8% each of the total virus-infected cells (Fig. 3B). These ratios reflect normal retinal
Acknowledgements
This work was supported by Special Coordination Funds for Promoting Science and Technology and research grants from the Ministry of Education, Science, Sports, and Culture of Japan and the Japan Society for the Promotion of Science.
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