Lineage Analysis Using Retrovirus Vectors

Constance L. Cepko *
Elizabeth Ryder ¹
Christopher Austin ²
Jeffrey Golden ³
Shawn Fields-Berry *
John Lin *

~undefined Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute, Boston, MA 02115
¹ Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609
² Merck Research Laboratories, West Point, PA 19486
³ Department of Pathology, Children"s Hospital of Philadelphia, Philadelphia, PA 19104

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ABSTRACT

 

Knowledge of the geneological relationships of cells during development can allow one to gain insight into when and where developmental decisions are being made. Geneological relationships can be revealed by a variety of methods, all of which involve marking a progenitor cell and/or a group of cells and then following the progeny. We describe the use of replication-incompetent retroviral vectors for the analysis of lineal relationships in developing vertebrate tissues. An overview of the relevant aspects of the retroviral life cycle, and the strategies and current methods in use in our laboratory are described.

Knowledge of the geneological relationships of cells during development can allow one to gain insight into when and where developmental decisions are being made. Hypotheses can be ruled in or out concerning the commitment of cells to particular fates. For example, when analyzing the cell types that result from the marking of a single progenitor cell, one can gain insight into whether the progenitor was committed to the production of one or multiple cell types. If multiple cell types are found in a clone, one can conclude that the progenitor that gave rise to these cells was not restricted to the production of only one cell type. Alternatively, if all of the cells that descend from a progenitor are the same type, the hypothesis is supported, but not proven, that the progenitor was committed to making only that cell type. In the latter case, a firm conclusion concerning the commitment of the progenitor can be reached only if the progenitor and/or progeny are exposed to a variety of environments. If only one cell type is produced despite variations in the environment, commitment of the progenitor to production of one cell type is supported. Analyses of clones generated after marking the progenitors of a tissue at various times in development can greatly aid in charting the stages of production of different cell types, allowing one to focus studies concerning cell fate decisions to particular times and places. In addition, analysis of the proliferation and migration patterns exhibited by clones can increase our understanding of the development of a particular area.

The complexity and inaccessibility of many types of embryos have made lineage analysis through direct approaches, such as time lapse microscopy and injection of tracers, almost impossible. A genetic and clonal solution to lineage mapping is through the use of retrovirus vectors. The basis for this technique will be summarized, and the strategies and current methods in use in our laboratory will be detailed.

TRANSDUCTION OF GENES VIA RETROVIRUS VECTORS

A retrovirus vector is an infectious virus that transduces a non viral gene into mitotic cells in vivo or in vitro [1]. 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. Those that are useful for lineage analysis have been modified so that they are replication incompetent and thus cannot spread from one infected cell to another. They are however faithfully passed on to all daughter cells of the originally infected progenitor cell, making them ideal for lineage analysis.

Retroviruses use RNA as their genome, which is packaged into a membrane-bound protein capsid. They produce a DNA copy of their genome immediately after infection via reverse transcriptase, a product of the viral pol gene which is included in the viral particle. The DNA copy is integrated into the host cell genome and is thereafter referred to as a "provirus". Integration of the genome of most retroviruses requires that the cell go through an M phase [2], and thus only mitotic cells will serve successfully as hosts for integration of most retroviruses. (However, there is a recent generation of retrovirus vectors based upon HIV [3], which can integrate into postmitotic cells. As lineage analysis is designed to ask about the fate of daughter cells, infection of postmitotic cells is not desirable.) Most vectors began as proviruses that were cloned from cells infected with a naturally-occurring retrovirus. Although extensive deletions of proviruses were made, vectors retain the cis-acting viral sequences necessary for the viral lifecycle. These include the packaging sequence (necessary for recognition of the viral RNA for encapsidation into the viral particle), reverse transcription signals, integration signals, viral promoter, enhancer, and polyadenylation sequences. A cDNA can thus be expressed in a vector using the transcription regulatory sequences provided by the virus (although see below for further discussion of this point). Since replication-incompetent retrovirus vectors usually do not encode the structural genes whose products comprise the viral particle, these proteins must be supplied through complementation. The products of the genes, gag, pro, pol, and env are typically supplied by "packaging" cell lines or cotransfection with packaging constructs into highly transfectable cell lines (for review see Cepko and Pear in Ausubel et al., 1997 [4]). Packaging cell lines are stable lines that contain the gag, pro, pol, and env genes as a result of the introduction of these genes by transfection. However, these lines do not contain the packaging sequence on the viral RNA that encodes the structural proteins. Thus, the packaging lines, or cells transfected with packaging constructs, make viral particles that do not contain the genes gag, pro, pol, or env .

Retrovirus vector particles are essentially identical to naturally occurring retrovirus particles. They enter the host cell via interaction of a viral envelope glycoprotein (a product of the viral env gene) with a host cell receptor. The murine viruses have several classes of env glycoprotein which interact with different host cell receptors. The most useful class for lineage analysis of rodents is the ecotropic class. The ecotropic env glycoprotein allows entry only into rat and mouse cells via the ecotropic receptor on these species. It does not allow infection of humans, and thus is considered relatively safe for gene transfer experiments. The first packaging line commonly in use was the y2 line [5]. It encodes the ecotropic env gene and makes high titers of vectors. However, it can also lead to the production of helper virus (discussed below). A second generation of ecotopic packaging lines, yCRE [6], GP+E-86 [7] and yE [8] have not been reported to lead to production of helper virus to date. A third generation of "helper-free" packaging lines, exemplified by the ecotropic lines, Bosc23 [9] and Phoenix (Gary Nolan, Stanford University), were made in 293T cells, and have the advantage over the earlier lines of giving high titer stocks transiently after transfection. Similarly, cotransfection of 293T cells with packaging constructs and vectors can lead to the transient production of high titer stocks [10]. The first two generations of packaging lines, which are based upon mouse fibroblasts, require production of stably transduced lines for production of high titer stocks.