Choosing the Appropriate Transgenic Animals for Lineage Analysis

The expression of the lacZ transgene varies according to the nature of the regulatory elements (promoter and enhancer) that drive gene expression, the

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

number of active copies and the chromosomal domain where the transgene is localized. The choice of transgene is dictated by the specific questions to be addressed. There are several examples of transgenes that give ubiquitous non-lineage biased expression. For example, in transgenic mice where the lacZ gene is regulated by the hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase promoter, the transgene is expressed in all lineages at high levels during development (2). Similarly the ROSA-P-geo transgene produced by site-directed mutagenesis (3) and the lacZ reporter driven by human P-actin promoter (4) also provide ubiquitous tagging of multiple cell lineages. Ubiquitously expressed transgenes provide the most ideal marker for unbiased cell lineage analyses provided that the lineage progenitors or founder cells to be studied can be isolated as a pure cell population.

Integration of the transgene into a specific chromosome may be useful in some cases for tracing cell lineages. Transgenes that are expressed in the same pattern as the neighboring genes at the site of integration will provide a ready marker for any tissue-specific pattern of expression. For example, transgenes that are integrated into the X chromosome may behave like the endogenous X-linked gene. Among the known X-linked transgenes, the activity of the HMG-nls-lacZ transgene seems to reflect faithfully the activity of the X chromosome (5).

In female mice that carry the lacZ transgene only on one X chromosome, X inactivation during embryonic development generates two cell populations, one that expresses the X-linked transgene and another that does not. Since the status of X inactivation is heritable, descendants of either population will be stable for the transgene expression. Several studies on the lineage relationship of cells in tissues, such as the retina, brain, and tongue, have been performed using the mosaicism of transgene expression generated by random X chromosome inactivation (6-8).

Finally, some transgenes display lineage and stage-specific expression through use of a tissue-specific promoter or as a consequence of their unique integration sites. For example, in Wntl-lacZ transgenic mice, the transgene is regulated by the Wntl 3'-cis-acting enhancer element, which directs specific expression to the dorsal part of the neural tube and subsequently in the neural crest cells derived from this region (9). Another example is R197 transgenic mice in which the lacZ gene is expressed principally in the muscle lineage from early organogenesis stages onwards as a consequence of its integration site (10). Provided that the expression of the transgene is tissue-specific for a defined period of development, such transgenes offer an excellent tool for tracing the differentiation of cell lineages. A prerequisite for using these mice and any other transgenic lines is to establish the pattem of transmission and to ascertain the tissue and temporal specificity of transgene expression.

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