Over the last 15 years investigators studying vertebrate development have capitalized on the use of retroviral-mediated gene transfer to determine the lineage relationships of diverse cell types, particularly in many regions of the mammalian central nervous system (for review, see ref. 1). Whereas an intraperitoneal or intrauterine injection of cell proliferation markers such as tritiated thymidine or bromodeoxyuridine suffices to examine the birthdates of cells, many fundamental questions dealing with the formation of a structure in the mammalian embryo often necessitate performing intrauterine surgery to introduce genes by retroviral vectors. As retroviral vectors can be used not only to study lineage (2), but also to introduce genes to perturb development (3-5), the methods for delivering retroviruses into the developing mammalian embryo will be in increasing demand. This chapter describes a set of procedures to generate and introduce retroviral vectors into rodent embryos.
Retroviruses only integrate into the DNA of replicating cells (6). As a result of this chromosomal integration, the progeny of the infected cell inherit the retroviral DNA. Recombinant retroviruses have been engineered to eliminate the viral structural genes. Thus, unlike wild-type retroviruses, these recombinant retroviruses deliver exogenous genes to target cells, but cannot replicate on their own and infect unrelated cells. These attributes make replication-defective recombinant retroviruses ideal for use as tracers of cell lineage during development. In addition, a replication-defective recombinant retrovirus encoding an exogenous marker gene is a useful tool because it can be introduced into dividing cells at virtually any stage of development and, unlike many
From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ
other cell proliferation markers, no dilution of the exogenous gene occurs as a function of increasing numbers of cell division.
To generate infectious retroviral particles for use in experiments, retroviral producing cell lines are utilized. These cell lines are made by introducing the retroviral plasmid DNA into a packaging cell line which has been engineered to synthesize all the proteins required for viral assembly (7). Packaging cell lines are either ecotropic or amphotropic depending on the type of viral envelope proteins they produce. The envelope proteins are critical for determining the host range of cells which a retrovirus can infect; ecotropic retroviruses infect only rat and mouse cells, whereas amphotropic retroviruses infect cells from a broader variety of species. To generate retroviral particles, one can either use a stable producer cell line or utilize a transient transfection technique of highly transfectable packaging cells (8). A stable producer cell line offers the simplicity of thawing identical cell aliquots, but generates infectious retroviruses with relatively low titers (104-106 infectious particles/mL). The transient technique generates short-lasting producer cells, but production of higher titers (>107 infectious particles/mL). In either case, ecotropic packaging cell lines are used for the techniques described in this chapter.
The first retroviral lineage tracers to be used contained the reporter gene Escherichia coli P-galactosidase (lacZ), whose expression was detected his-tochemically at the light microscopic level (9,10). The subsequent detection of the lacZ histochemical reaction product at the ultrastructural level offered several advantages (11). Among them, it could be used to determine more definitively cell phenotype. The detection of lacZ at the ultrastructural level in combination with the use of other antibodies further expanded the types of questions that could be addressed (12). Furthermore, as better antibodies to lacZ became available, the expression of lacZ could be routinely detected immunohistochemically at the light microscopic level (13), opening up the possibility of using it in conjunction with other antibodies in double- and triple-labeling procedures (14). To facilitate the use of retroviral vectors encoding the gene for lacZ to answer more questions, highlights of procedures used to detect lacZ histochemically and immunohistochemically, at the light and ultrastructural level, are provided. These techniques can be applied to studies in vivo as well as in vitro.
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