[56] Lineage analysis using retrovirus vectors

Publisher Summary 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.