Examples of Three Distinct Patterns of Transgenic Reporter Expression

1.2.1. Ubiquitous Expression

The H253 transgenic line (2) is an example of ubiquitous expression of an Xlinked transgene. Crossing homozygous X*X* females with X*Y males (where X* represents the transgene bearing X chromosome) produces transgenic embryos (X*X*, X*Y) in which P-galactosidase is expressed at high levels in all tissues and progeny during all stages of development. This series of matings produced a ubiquitous non-lineage bias transgenic line that has been used successfully in several lineage studies in postimplantation mouse embryos (2,11-13).

1.2.2. X-Inactivation Mosaicism

The other type of expression is one of a mosaic nonlineage biased transgene. Transgenic H253 male and F1 (C57BL/6 x DBA/2) female mice were crossed. Hemizygous H253 female mice (X*X) were then crossed to wild-type F1 (C57BL/6 x DBA/2) males to produce offspring of four possible genotypes and only three distinct patterns of staining: uniform staining (X*Y), patchy staining (X*X), and negative staining (XX and XY). The patchy staining of X*X genotypic mice is owing to random X chromosome inactivation. This results in the expression of the lacZ marker in only about half of the population. The ability to recognize clonal populations means that these two cell populations (one that does expresses lacZ and the other that does not) have been used in cell lineage analyses to reveal the ancestral history of spatially related clones (6-8).

The mosaicism of transgene expression following X inactivation in chimeric tissues, has been used extensively in the analysis of specific cell lineages (68,14-16). The lacZ-expressing cells can be identified from nonexpressing cells by X-gal staining, and the composition of the sorted populations may be examined for the expression of cell-specific markers by staining with appropriate antibodies. This system therefore provides an assay for the measurement of the relative proportion of specific cell types at different stages of maturation.

1.2.3. Restricted Expression

The R197 transgenic line is an example of a lineage- and stage-specific transgene (10). The lacZ transgene is segmentally expressed primarily in the myogenic lineage from about 8.5 d onward. Following the identification of transgenic pups (see Subheading 3.3.), these animals were crossed with nontransgenic (C57BL/6 x DBA) hybrids or other transgenic mice to produce transgenic embryos for the experiment. In this transgenic line, homozygous embryos die during the immediate postimplantation period. To ascertain lethality, pregnant mice were sacrificed at different ages of gestation so that the litter size and the genotype of the embryos/fetuses could be analyzed. A reduction in litter size of F1 hemizygous matings and the absence of the homozygotes after genotyping indicated that homozygotes did not survive to term. As an essential step for using transgenic mice, the possibility of embryo lethality should be examined particularly when a significant reduction in litter size is observed. Prior genotyping of the entire litter may be necessary to avoid using cells from dying embryos for lineage analysis.

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