Position effects in mice carrying a lacZ transgene in cis with the  -globin LCR can be explained by a graded model

Abstract

We studied transgenic mice carrying the lacZ reporter gene linked to the erythroid-specific beta-globin promoter and beta-globin locus control region (LCR). Previously, we had demonstrated that the total level of expression of beta-galactosidase enzyme, which is the product of the lacZ gene, varies widely between different transgenic mice due to position effects at the sites of transgene integration. Here, using the X-gal based in situ assay for beta-galactosidase activity, we found that the percent erythroid cells that expressed the transgene also varied widely between the mice. Moreover, a kinetic analysis showed that the average beta-galactosidase content per expressing cell varied both between samples of different transgenic descent and between erythroid cells within each sample, demonstrating that the variable expression of this lacZ transgene was being controlled in a graded manner. These results suggest that the beta-globin LCR enhancers function through a graded model, which is described, rather than the binary mechanism that has been proposed previously for other enhancers.

Position-independent human β-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken β-globin insulator

“…4A-C acting element that shields the transcriptional units from the surrounding DNA, exists in the full-length LCR but not in HS core elements. In fact, LCR containing HS4, 3, 2, 1 does not confer position-independent expression onto the b-globin promoter-driven lac Z gene in transgenic mice (Guy et al 1996;Guy et al 1997). HS5 59 to HS4 was shown to have the properties of an insulator (Li and Stamatoyannopoulos 1994).…”

Transcriptional Regulation of the SCL Locus: Identification of an Enhancer That Targets the Primitive Erythroid Lineage In Vivo

“…We considered the possibility that this pattern of expression might represent an artifact of the ␤-galactosidase reporter gene, since it has been reported that the ␤-galactosidase cassette interferes with the ability of the ␤-globin locus control region to confer position-independent expression (30), and it has been suggested that the ␤-galactosidase sequence may act as a nucleation point for the formation of heterochromatin in definitive erythroid cells (71). However, several groups have shown that a variety of erythroid enhancers, including the ␤-globin locus control region (30,31), the ␣-globin Ϫ40 element (56,70), and the EKLF Ϫ950 promoter (77), can direct expression of ␤-galactosidase to definitive erythroid cells in transgenic mice. Instead, we favor the alternative explanation that the 3.7-kb fragment containing the ϩ40 region exhibits intrinsic specificity for the primitive erythroid lineage.…”

Transgenic Analysis of the Stem Cell Leukemia +19 Stem Cell Enhancer in Adult and Embryonic Hematopoietic and Endothelial Cells

“…Third, in adult mice, the β-galactosidase reporter gene may cause inactivation of the +19 core enhancer but not the larger +18/19 fragment. Inactivation of regulatory regions linked to lacZ transgenes has been reported previously and has generally been attributed to the prokaryotic origin of the lacZ sequence [34][35][36][37][38][39].…”

“…Failure of expression in adult SV/lac/19 transgenic mice would also be consistent with the suggestion that the +19 core enhancer may require additional sequences outside the core enhancer to maintain expression throughout ontogeny. Alternatively, the β-galactosidase reporter gene may not faithfully report enhancer activity in adult mice, a scenario that has been suggested previously [34][35][36][37][38][39]. To distinguish these possibilities, β-galactosidase was replaced by the mammalian reporter gene human PLAP.…”