Minesuke Yokoyama

Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.

Tissue-specific expression of the human renin gene in transgenic mice

Transgenic mice carrying human renin gene were produced by microinjection of 15 kilobases (kb) DNA molecules with up to 3 kb of 5-flanking sequence and 1.2 kb of 3-flanking sequence. The transgenes have been shown to be stably transmitted to progeny. It was revealed by RNase protection assay that the human renin gene in a transgenic mouse is expressed preferentially in the kidney. The human renin RNA was also detected at a small level in a variety of tissues such as brain, heart, lung, pancreas, spleen, stomach, testis, and thymus. The direct radioimmunoassay using a monoclonal antibody specific for the active site of human renin demonstrated the synthesis of human active renin in the transgenic mouse kidney. These results suggest that the human renin gene in the transgenic mouse is regulated in a tissue-specific manner.

Expression of the human angiotensinogen gene in transgenic mice and transfected cells.

We have generated two lines of transgenic mice with integrated copies of a 14-kilobase pair (kb) human DNA fragment containing the angiotensinogen gene, which includes 1.3 kb of 5- and 3-flanking regions. In both transgenic lines, a considerable quantity of the correctly initiated and processed angiotensinogen mRNA was detected in the liver and it was detectable in heart. Unexpectedly, mRNA for the transgene was accumulated in the kidney, where is normally the minor source of angiotensinogen, to levels comparable to that in the liver. In addition, an in vitro transfection analysis suggested that the 1.3-kb 5-flanking sequences are essential for expression of the angiotensinogen gene in hepatic and renal cells and that neither DNA segment within the 14-kb construct contributes significantly to repression of the gene expression in renal cells.

Analysis of the human tyrosine hydroxylase promoter-chloramphenicol acetyltransferase chimeric gene expression in transgenic mice

To investigate cis-elements responsible for catecholaminergic (CAnergic) neuron-specific expression of the tyrosine hydroxylase (TH) gene, we produced lines of transgenic mice carrying 5.0-kb, 2.5-kb and 0.2-kb fragments from the 5-flanking region of the human TH gene fused to a reporter gene, chloramphenicol acetyltransferase (CAT), and designated them as TC 50, TC 25, and TC 02, respectively, and reporter gene expression in transgenic mice was analyzed by CAT assay by immunocytochemistry with anti-CAT antibody. High-level CAT expression was observed in the brain and adrenal gland using the 5.0-kb promoter of the TC 50 mice, but ectopic expression was consistently observed in several somatic tissues, e.g. thymus, colon, and testis. In brain, expression was achieved in CAnergic neurons with the largest construct (5.0 kb), but not with 2.5 kb or 0.2 kb of 5 flanking sequence. However, TC 50 mice also expressed CAT immunoreactivity in non-CAnergic neurons. In the TC 25 line CAT immunoreactivity was detected only in some non-CAnergic neurons. In the TC 02 line no CAT immunoreactivity was detected in any of the tissues examined. These results indicate that the 5.0-kb DNA fragment of the TH gene upstream region contains activity to express CAT in CAnergic neurons and surprisingly, lacks some regulatory elements attenuating ectopic expression, and that the 2.5-kb and 0.2-kb fragment are not sufficient for the proper expression. We discuss the presence of the tissue-specific regulatory elements in the structure portion of the TH gene and/or 3-flanking region.

Muscle fiber growth and necrosis in dystrophic muscles: a comparative study between dy and mdx mice

Histopathological and morphometric studies of murine dystrophic muscles in the early postnatal period were performed. In dy mice, obtained by in vitro fertilization, scattered necrotic fibers were noted at 10 days of age followed by poorly compensated regeneration leading to muscle fiber loss, marked variation in fiber size, and fibrous and adipose tissue proliferation. In mdx mice, clusters of necrotic muscle fibers seen at 10-15 days were followed by well compensated regeneration with little fibrosis. There appeared to be no delay in muscle fiber growth and fiber type differentiation in both dy and mdx mice up to 10 days of age since there was no distinct difference in muscle fiber size, fiber type differentiation and the incidence of myosatellite cells between dystrophic muscles and those from age-matched control mice. Muscle fibers appeared to undergo necrosis only when they had reached a certain stage of maturation since at early stages of development or regeneration they rarely became necrotic. The difference in clinical symptoms between dy and mdx mice may result from differences in their regenerative response to necrosis.

Overexpression of a minor component of myelin basic protein isoform (17.2 kDa) can restore myelinogenesis in transgenic shiverer mice

Shiverer (shi) mice, which are neurologically mutant, lack a large portion of the gene for the myelin basic proteins (MBPs), have virtually no myelin in their central nervous system (CNS), and shiver, undergo seizures, and die early. At least five types of MBPs (21.5, 18.5, 17.3, 17.2 and 14.0 kDa) are known to be generated through alternative splicing from a single MBP gene. We have produced transgenic shi mice carrying a cDNA encoding mouse 14-kDa MBP isoform, the most abundant form of MBPs, under control of a mouse MBP gene promoter, and showed that expression of the 14-kDa MBP can restore CNS myelination. To test whether the 17.2-kDa MBP isoform, one of the minor components of MBPs, can also elicit myelination in homozygous shi mutants, we produced seven independent transgenic shi mice carrying cDNA encoding the mouse 17.2-kDa MBP isoform, and the transcription of which was driven by a mouse MBP gene promoter. The axons in the cerebellum of one transgenic line, which exhibited the highest expression of transgene-derived mRNA ( approximately 50% of the level of total MBP mRNA in the normal mouse brain), were myelinated. This mouse exhibited nearly normal behavior. These findings indicate that the 17.2-kDa MBP isoform, even when the only 17.2-kDa MBP isoform is present, has the ability to elicit CNS myelination in transgenic shi mice. This transgenic strategy will be useful for elucidating the role of each type of MBP isoform in CNS myelinogenesis.

Expression in brain sensory neurons of the transgene in transgenic mice carrying human tyrosine hydroxylase gene

We have recently reported the production of transgenic (Tg) mice carrying the human tyrosine hydroxylase (TH) gene, and have described tissue-specific expression of the transgene in catecholaminergic (CAergic) neurons and adrenal glands. This paper describes the transgene expression in non-catecholaminergic (nCAergic) neurons in the brain of Tg mice by immunocytochemistry and in situ hybridization. In adult Tg mice, human TH was atypically expressed in the olfactory (typically, the anterior olfactory nucleus and pyriform cortex) and visual (typically, n. suprachiasmaticus and n. parabigeminalis) systems, in addition to typical CAergic neuron-rich nuclei in the brain. These results suggest the possibility that TH plays some novel roles in sensory systems.

Chimeric Analysis of the Pathogenesis of Dysmyelination of Shiverer Mutant Mice

Shiverer is an autosomal recessive mutant mouse characterized by abnormal and poor myelin formation of the central nervous system. Chimera mice were produced from wild-type control (C57BL/6N) and the shiverer mutant mice (BALB/C) by the aggregation technique in order to analyze the pathogenesis of the shiverer mutation. The coat color was a mosaic of both strains. Immunohistochemically we have examined sections of cerebellar white matter of the chimera using antiserum against myelin basic protein (MBP), since MBP is absent in the myelin of the shiverer. Patches of MBP-negative and positive sites were observed in cerebellar white matter. The myelin sheaths, both positive and negative to MBP staining, were well mixed in the white matter but each stained myelin sheath was clearly visible, even in the mixed part. The possibility that humoral factors caused dysmyelination could be eliminated, and it is suggested that the shiverer mutation occurs intrinsic to the cells themselves.

Alteration of the primary pattern of central myelin in a chimaeric environment ― study of shiverer↔wild-type chimaeras

Using chimaeras produced from the shiverer mutant with a BALB/C background (white coat color) and normal mice of the C57BL/6N strain (black coat color) by the aggregation method, the morphological patterns of the central myelin sheaths were examined by electron microscopy, with special reference to the structure of the myelin lamellae of the one genotype in an environment dominated by the other genotype. The results obtained may be summarized as follows. The oligodendrocytic processes of both genotypes are able to interact independently with the axon unrelated to its genotype. The lamellar structure of the myelin sheath, i.e., whether or not it is composed of alternating major dense and intraperiod lines, is primarily determined by the genotypes of the oligodendrocytes concerned. The processes of shiverer oligodendrocytes can spirally enwrap axons in the chimaeric CNS which shows dominance of the normal genotype. In contrast, in the environment of shiverer dominance, the spiral arrangement of the oligodendrocytic processes of the normal genotype is often disturbed, probably by the oligodendrocytic processes of the shiverer genotype. Thus, the primary pattern of the myelin sheaths can be altered under the chimaeric condition, and the formation of the myelin sheaths depends essentially on the behavior of the oligodendrocytic processes, which is determined by the genetic specificity.

Analysis of Cellular Mosaicism in a Transgenic Mouse by Histological In Situ Hybridization

One of the transgenic mice carrying a chicken δ‐crystallin gene was found to be mosaic with regard to the distribution of the exogenous gene. Taking advantage of the exogenous DNA sequences as a cell lineage marker detectable by histological in situ hybridization technique, we studied cellular mosaicism in mouse 7–5. This mouse carried the exogenous gene in 20–40% of its cells, probably reflecting chromosomal integration of the exogenous DNA which occurred in a blastomere of around the 4‐cell stage. The cells carrying the gene contributed to virtually any kind of tissue and their distribution varied from one tissue to another. For instance, in the neural retina, gene‐positive cells formed columns several cells wide, indicating that migration of the cells derived from the founder cells is mainly along the radial axis. However, in other tissues we examined, clusters of the marked cells were less obvious, indicating the occurrence of extensive cell mixing during histogenesis. Thus, mosaic analysis of cell lineage in mouse ontogeny appears meaningful in early developmental stages or when clonal outgrowth takes place in a tissue.