Dicistronic LacZ and alkaline phosphatase reporter constructs permit simultaneous histological analysis of expression from multiple transgenes

We report here the development of convenient dicistronic transgenic markers for the rapid and efficient simultaneous analysis of transgene activity in transgenic mice. Two sensitive histological markers, the beta-galactosidase (beta-gal)-encoding lacZ gene and the human placental alkaline phosphatase (hpAP) gene, have been fused to the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus, which directs efficient mRNA cap-independent entry of the translation apparatus in mammalian cells. The IRES permits efficient translation of either lacZ or hpAP when placed anywhere within transgene exonic sequences, including both 5' and 3' untranslated regions. In addition, the production of constructs for transgenic analysis of DNA regulatory elements is greatly facilitated with IRES-lacZ or IRES-hpAP, since the IRES relieves the need for complicated in frame transgene protein fusions to produce a functional beta-gal or hpAP protein.     

Tissue-specific activation of the osmotin gene by ABA, C2H4 and NaCl involves the same promoter region

The gene encoding the antifungal protein osmotin is induced by several hormonal and environmental signals. In this study, tissue-specific and inducer-mediated expression of the reporter gene beta-glucuronidase (uidA) fused to different fragment lengths of the osmotin promoter was evaluated in transgenic tobacco (Nicotiana tabacum). The region of the promoter between -248 to -108 (Fragment A) was found to be essential and sufficient for inducer (abscisic acid (ABA), C2H4 and NaCl)-mediated expression of the reporter gene. Expression of the reporter gene was developmentally regulated and increased with maturity of leaves, stem and flowers. Expression also was tissue-specific being most highly expressed in epidermis and vascular parenchyma of the stem. The regulators ABA, C2H4 and NaCl exhibited tissue-specific induction of this promoter. The promoter was specifically responsive to C2H4 in flowers at virtually all stages of development, but not responsive in these tissues to ABA or NaCl. Conversely, ABA and NaCl were able to induce reporter gene activity using promoter Fragment A in specific tissues of root where C2H4 was unable to induce activity. Further dissection of the promoter Fragment A into fragments containing either the conserved GCC element (PR); PR/AT; or G/AT sequences, and subsequent testing of these fragments fused to GUS in transgenic plants was performed. These experiments revealed that the promoter fragment containing PR element alone, although required, was barely able to allow responsiveness to C2H4. However, significant C2H4-induced activity was obtained with a promoter fragment containing the AT and PR elements together.     

Neuroblast pattern formation: regulatory DNA that confers the vnd/NK-2 homeobox gene pattern on a reporter gene in transgenic lines of Drosophila

DNA fragments -0.57, -2.2, -2.9, -5.3, and -8.4 kb in length from the upstream regulatory region of the vnd/NK-2 gene were cloned in the 5'-flanking region of a beta-galactosidase (beta-gal) reporter gene in the P-element pCaSpeR-AUG-beta-gal, and the effects of the DNA on the pattern and time of expression of beta-gal were determined in transgenic embryos. Embryos from 11 lines transformed with -8.4 kb of vnd/NK-2 regulatory DNA expressed beta-gal patterns that closely resemble those of vnd/NK-2. In embryos from four lines transformed with -5.3 kb of vnd/NK-2 DNA, beta-gal was found in the normal vnd/NK-2 pattern in the nerve cord but not in part of the cephalic region. beta-Gal patterns in embryos from transgenic lines containing -0.57, -2.2, or -2.9 kb of vnd/NK-2 DNA did not resemble vnd/NK-2. Null vnd/NK-2 mutant embryos containing the homozygous P-element p[-8.4 to +0.34 beta-gal] expressed little beta-gal in contrast to siblings with a wild-type vnd/NK-2 gene. We conclude that (i) the 8.4-kb DNA fragment from the vnd/NK-2 gene contains the nucleotide sequences required to generate the normal pattern of vnd/NK-2 gene expression, sequences that may be involved in the switch between neuroblast vs. epidermoblast pathways of development, (ii) the 5'-flanking region of the vnd/NK-2 gene between -5.3 and -8. 4 kb is required for vnd/NK-2 gene expression in the most dorsoanterior part of the cephalic region, and (iii) vnd/NK-2 protein is required, directly or indirectly, for maintenance of vnd/NK-2 gene expression.     

Early ontogeny of catecholaminergic cell lineage in brain and peripheral neurons monitored by tyrosine hydroxylase-lacZ transgene

As the first and rate limiting enzyme in the biosynthetic pathway for catecholamine (CA) neurotransmitters, tyrosine hydroxylase (TH) is a specific phenotypic marker for CA cells in the central and peripheral nervous systems of adult animals. During embryogenesis, TH expression appears permanently within cells destined to be CA-secreting during adult life, and transiently in several cell types that will not express TH in adulthood. In this study, we examined the early ontogeny of TH expression in transgenic mouse embryos by following the expression of a lacZ reporter, driven by the tissue-specific promoter of the rat TH gene. The lacZ reporter product, beta-galactosidase (beta-gal), visualized by X-gal staining, first became apparent in primordia of sensory ganglia serving the glossopharyngeal (IX) and vagal (X) cranial nerves at embryonic day (E)9.0. Between E9.5 and E10.5, beta-gal expression extended to the remaining cranial sensory ganglia serving the trigeminal (V) and facial (VII) nerves, dorsal root ganglia, ventrolateral neural tube and sympathetic ganglion primordia. During that same period, the first beta-gal expression in the embryonic brain also appeared within distinct regions, such as the ventral prosencephalon, the ventral and dorsolateral mesencephalon and the rostral and caudal rhombencephalon. The level of beta-gal expression in all these tissues decreased at E13.5, but a distinct adult pattern of beta-gal expression started to emerge in the substantia nigra and ventral tegmental area in the central nervous system and the adrenal medulla in the periphery. Our findings indicate that the proximal 9.0 kb of the 5' promoter region of the rat TH gene encodes sufficient information to direct development of the appropriate catecholaminergic lineage cells in the central and most peripheral nervous systems during embryogenesis.