The specificity of the myelin basic protein gene promoter studied in transgenic mice.

The myelin basic proteins (MBPs) are a family of polypeptides that are predominantly expressed in the nervous system where they play a major role in myelination. We have generated four lines of transgenic mice carrying a transgene in which 1.34 kb of the 5'-flanking sequence of the mouse MBP gene was fused upstream of the coding region of the Escherichia coli lac Z gene in order to investigate developmental and tissue-specific expression of the MBP gene. Expression of both the lacZ transgene and the endogenous MBP gene followed a common developmental pattern in mouse brain. Transgene expression was detected in primary oligodendrocytes, but not in type 2 astrocytes. In addition, the lacZ gene product was expressed in epithelial cells of certain nonneural tissues, namely kidney, epididymis, ureter, and seminal vesicles. The ectopic expression of the transgene was associated with the development of DNase I hypersensitive sites at the site of insertion which was found to be within the intron 1 region of the endogenous MBP gene. The results reported here strongly suggest that the 1.34-kb 5'-flanking region of the MBP gene contains cis-regulatory elements that confer developmental regulation of the MBP gene, although this region appears to lack elements that restrict its expression to the nervous system.     

Position-specific activity of the Hox1.1 promoter in transgenic mice

During development, positional values have to be assigned to groups of cells. The murine Hox genes are a class of genes that are predicted to be involved at some stage in this process. During embryogenesis they are expressed in distinct overlapping region- and stage-specific patterns and therefore must be regulated in response to positional information. In this study, we have analysed the activity of Hox1.1 promoter sequences in transgenic mice. The use of lacZ as a marker allows a detailed analysis of expression at the single cell level during early embryonic development. We show that 3.6 kbp of promoter and 1.7 kbp of 3' sequences provide sufficient regulatory information to express a transgene in a spatial and temporal manner indistinguishable from the endogenous Hox1.1 gene during the period of development when Hox1.1 expression is established. The activation occurs in a strict order in specific ectodermal and mesodermal domains. Within each of these domains the transgene is activated over a period of four hours apparently randomly in single cells. In a following second period, Hox1.1 and transgene expression patterns diverge. In this period, transgene expression persists in many mesodermally derived cells that do not express Hox1.1 indicating the absence of a negative regulatory element in the transgene. The anterior boundary of transgene expression is identical to that of Hox1.1. However, no posterior boundary of transgene expression is set, suggesting that a separate element absent from the transgene specifies this boundary.     

Diverse human aldolase C gene promoter regions are required to direct specific LacZ expression in the hippocampus and Purkinje cells of transgenic mice

Aldolase C is selectively expressed in the hippocampus and Purkinje cells in adult mammalian brain. The gene promoter regions governing cell-specific aldolase C expression are obscure. We show that aldolase C messenger expression in the hippocampus is restricted to CA3 neurons. The human distal promoter region (-200/-1200 bp) is essential for beta-galactosidase (beta-gal) expression in CA3 neurons and drives high stripe-like beta-gal expression in Purkinje cells. The 200 bp proximal promoter region is sufficient to drive low brain-specific and stripe-like beta-gal expression in Purkinje cells. Thus, the human aldolase C gene sequences studied drive endogenous-like expression in the brain.     

Neuronal expression of enhanced green fluorescent protein directed by 5' flanking sequences of the rat aldolase C gene in transgenic mice.

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a combination of distal and proximal 5' flanking sequences, the A + C + 0.8 kilobase (kb) pairs fragments, ensured high brain-specific expression in vivo (Skala et al. 1998). We show here that the expression pattern conferred by these sequences, when placed in front of the chloramphenicol acetyltransferase (CAT) or the enhanced green fluorescent protein (EGFP) reporter genes in transgenic mice, is similar to the distribution of the endogenous mRNA and protein. Double immunostaining for neuronal or glial cell-specific markers and for the EGFP protein indicates that the A + C + 0.8 kb genomic sequences from the rat aldolase C gene direct a predominant expression in neuronal cells of adult brain.     

Activity of the adenosine deaminase promoter in transgenic mice.

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.     

Neuronal expression of enhanced green fluorescent protein directed by 5' flanking sequences of the rat aldolase C gene in transgenic mice

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a combination of distal and proximal 5' flanking sequences, the A+C+0.8 kilobase (kb) pairs fragments, ensured high brain-specific expression in vivo (Skala et al. 1998). We show here that the expression pattern conferred by these sequences, when placed in front of the chloramphenicol acetyltransferase (CAT) or the enhanced green fluorescent protein (EGFP) reporter genes in transgenic mice, is similar to the distribution of the endogenous mRNA and protein. Double immunostaining for neuronal or glial cell-specific markers and for the EGFP protein indicates that the A+C+0.8 kb genomic sequences from the rat aldolase C gene direct a predominant expression in neuronal cells of adult brain.     

Activity of the beta-retinoic acid receptor promoter in transgenic mice.

LacZ reporter gene constructs were used to analyze the murine retinoic acid receptor beta (mRAR beta) gene promoter in transgenic mice. LacZ expression in transgenic mouse embryos with 250 bp of promoter sequences closely parallels that of RAR beta between embryonic days 8.5 and 12.5. This indicates that the -1 to -250 promoter region contains most regulatory elements required for tissue specific expression. Additional elements in the -250 to -625 region are required for high expression levels after day 12.5. Elements in the -625 to -3100 region are necessary to reproduce the RAR beta expression in the meninges and the eye mesenchyme. The expression pattern of the transgene and the endogenous RAR beta, as revealed by in situ hybridization, suggests an important role of the RAR beta in the developing nervous system.     

Construction of rat aldolase C expression plasmid and the hybrid formation between rat aldolase C and human aldolase A or B co-expressed in Escherichia coli

Rat aldolase C cDNA was inserted in an Escherichia coli expression vector to construct the rat aldolase C expression plasmid, pRAC42. This plasmid produces active rat aldolase C in the transfected E. coli host cells. The characteristics of the purified enzyme, e.g. mol. wt, electrophoretic mobilities and kinetic parameters, are indistinguishable from those of authentic rat brain aldolase C. Three different tetrameric hybrid forms, C3A, C2A2 and CA3, in addition to C4 and A4, were found to be produced in the host cell when E.coli was co-transfected with expression plasmids for rat aldolase C and for human aldolase A. Similarly, the hybrid forms, C3B, C2B2 and CB3, in addition to C4 and B4, were also produced in the cells when co-transfected with the plasmids for rat aldolase C and for human aldolase B.