[25] Lineage analysis using retrovirus vectors

doi:10.1016/0076-6879(95)54027-X

Methods in Enzymology

Volume 254, 1995, Pages 387-419

https://doi.org/10.1016/0076-6879(95)54027-X Get rights and content

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This chapter describes the lineage analysis using retrovirus vectors. The complexity and inaccessibility of many types of vertebrate embryos have made lineage analysis through direct approaches, such as time-lapse microscopy and injection of tracers, extremely difficult or impossible. A genetic and clonal solution to lineage mapping in some species is by infection with retrovirus vectors. The chapter summarizes the basis for this technique and details the strategies and current methods in use in the laboratory. A retrovirus vector is an infectious virus that transduces a nonviral gene into mitotic cells in vivo or in vitro. These vectors utilize the same efficient and precise integration machinery of naturally occurring retroviruses to produce a single copy of the viral genome stably integrated into the host chromosome. For lineage applications it is usually necessary to concentrate the virus to achieve sufficient titer. This is typically due to a limitation in the volume that can be injected at any one site. Viruses can be concentrated fairly easily by a relatively short centrifugation step. Virions also can be precipitated using polyethylene glycol or ammonium sulfate, and the resulting precipitate is collected by centrifugation. The viral supernatant can be concentrated by centrifugation through a filter that allows only small molecules, to pass (for example, Centricon filters). Regardless of the protocols that are used, it must be kept in mind that retroviral particles are fragile, with short half-lives even under optimum conditions.

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Since then, the use of Cre-loxP technology has been widely extended in the scientific community by combining it with fluorescent reporter genes, increasing dramatically the possibilities to study cell lineages and/or mapping neural circuits, as reported by Brainbow technology (Livet et al., 2007; Cai et al., 2013). Another approach is using modified retrovirus to label cell lineages by infecting progenitors (Cepko et al., 1995; Reid et al., 1995; Li et al., 2012), a widely used technology for tracking cells (Heins et al., 2002; Weber et al., 2011). The use of virus with the purpose of tracking cells or modifying their phenotypes is a widely used technology (Heins et al., 2002; Weber et al., 2011).
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However, the true distribution of cells in relation to their clonal siblings cannot be determined with this method because clustered cells sharing the same color could be derived from different progenitors. To directly determine the clonal relationship of clustered interneurons, we utilized an EnvA-pseudotyped retrovirus library carrying oligonucleotide sequence tags or barcodes (Cepko et al., 1995; Fuentealba et al., 2015; Jiang et al., 2013; Walsh and Cepko, 1992) (Figure 3A). In addition to the barcode sequence, each virus contained a transgene for membrane-bound GFP.
The mammalian neocortex is composed of two major neuronal cell types with distinct origins: excitatory pyramidal neurons and inhibitory interneurons, generated in dorsal and ventral progenitor zones of the embryonic telencephalon, respectively. Thus, inhibitory neurons migrate relatively long distances to reach their destination in the developing forebrain. The role of lineage in the organization and circuitry of interneurons is still not well understood. Utilizing a combination of genetics, retroviral fate mapping, and lineage-specific retroviral barcode labeling, we find that clonally related interneurons can be widely dispersed while unrelated interneurons can be closely clustered. These data suggest that migratory mechanisms related to the clustering of interneurons occur largely independent of their clonal origin.
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When tissues are complex and inaccessible, direct injection of tracer elements may not be a viable option and the introduction of a genetic marker by viral transfection may be more advantageous as a lineage beacon. Retroviral vectors carrying reporter genes can be effectively integrated into the genome of host mitotic cells and transduced to their progeny (Cepko et al., 1995). Sanes and colleagues (1986, 1989) pioneered the retrovirus-mediated gene transfer technology to trace cell-lineage in vitro and in vivo after disabling the viral replication machinery.
Lineage tracing allows the destiny of a stem cell (SC) and its progeny to be followed through time. In order to track their long-term fate, SC must be permanently marked to discern their distribution, division, displacement and differentiation. This information is essential for unravelling the mysteries that govern their replenishing activity while they remain anchored within their niche microenvironment. Modern-day lineage tracing uses inducible genetic recombination to illuminate cells within embryonic, newborn and adult tissues, and the advent of powerful high-resolution microscopy has enabled the behaviour of labelled cells to be monitored in real-time in a living organism. The simple structural organization of the mammalian cornea, including its accessibility and transparency, renders it the ideal tissue to study SC fate using lineage tracing assisted by non-invasive intravital microscopy. Despite more than a century of research devoted to understanding how this tissue is maintained and repaired, many limitations and controversies continue to plague the field, including uncertainties about the specificity of current SC markers, the number of SC within the cornea, their mode of division, their location, and importantly the signals that dictate cell migration. This communication will highlight historical discoveries as well as recent developments in the corneal SC field; more specifically how the progeny of these cells are mobilised to replenish this dynamic tissue during steady-state, disease and transplantation. Also discussed is how insights gleaned from animal studies can be used to advance our knowledge of the fundamental mechanisms that govern modelling and remodelling of the human cornea in health and disease.
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[25] Lineage analysis using retrovirus vectors

Publisher Summary

Cell (Cambridge, Mass.)

Neuron

Neuron

Neuron

Dev. Biol.

Cell (Cambridge, Mass.)

Neuron

J. Biol. Chem.

Virology

RNA Tumor Viruses

EMBO J.

J. Virol.

Nucleic Acids Res.

J. Virol.

J. Virol.