Human surfactant protein B promoter in transgenic mice: temporal, spatial, and stimulus-responsive regulation

Surfactant protein B (SP-B) is a developmentally and hormonally regulated lung protein that is required for normal surfactant function. We generated transgenic mice carrying the human SP-B promoter (-1,039/+431 bp) linked to chloramphenicol acetyltransferase (CAT). CAT activity was high in lung and immunoreactive protein localized to alveolar type II and bronchiolar epithelial cells. In addition, thyroid, trachea, and intestine demonstrated CAT activity, and each of these tissues also expressed low levels of SP-B mRNA. Developmental expression of CAT activity and SP-B mRNA in fetal lung were similar and both increased during explant culture. SP-B mRNA but not CAT activity decreased during culture of adult lung, and both were reduced by transforming growth factor (TGF)-beta(1). Treatment of adult mice with intratracheal bleomycin caused similar time-dependent decreases in lung SP-B mRNA and CAT activity. These findings indicate that the human SP-B promoter fragment directs tissue- and lung cell-specific transgene expression and contains cis-acting elements involved in regulated expression during development, fetal lung explant culture, and responsiveness to TGF-beta and bleomycin-induced lung injury.     

Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice

Background  

Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice.     

Neurofilament gene expression in transgenic mice

1. DNA fragments that include the human neurofilament NF-L gene was found to be correctly expressed in the majority of neurons in transgenic mice. 2. The NF-L transgene product, which is detectable in situ with a species-specific monoclonal antibody, provides a powerful genotype marking system applicable to developmental and regeneration studies of the mammalian nervous system. 3. The proximal 5'-flanking region of the NF-L gene is sufficient to direct expression of a heterologous gene in the mouse nervous system.     

Analysis of spatial and temporal gene expression patterns in blastula and gastrula stage chick embryos

Studies on the genetic basis of rostral-caudal specification, neural induction, and head development require knowledge of the relevant gene expression patterns. Gaps in our understanding of gene expression have led us to examine the detailed spatiotemporal expression patterns of 19 genes implicated in early development, to learn more about their potential role in specifying and patterning early developmental processes leading to head formation. Here, we report the expression patterns of these markers in blastula- and gastrula-stage chick embryos, using whole-mount in situ hybridisation. Nodal, Fgf8, Bmp7, Chordin, Lim1, Hnf3beta, Otx2, Goosecoid, Cerberus, Hex, Dickkopf1, and Crescent are all already expressed by the time the egg is laid. When the primitive streak has reached its full length, a later group of genes, including Ganf, Six3, Bmp2, Bmp4, Noggin, Follistatin, and Qin (BF1), begins to be expressed. We reassess current models of early rostral patterning based on the analysis of these dynamic spatiotemporal expression patterns.     

The nitrate reductase promoter of birch directs differential reporter gene expression in tissues of transgenic tobacco

A 1535 bp promoter of the nitrate reductase gene (nia) from birch (Betula pendula) and a series of 5′ deletions were fused to the β-glucuronidase (GUS) gene and introduced into Nicotiana plumbaginifolia. In transgenic plants the NR promoter sequences directed strong GUS expression in the root epidermal hair cells, and in phloem cells of leaf and stem vascular tissue. The NR promoter confers also a significant stimulation of the GUS gene expression by nitrate. These findings might indicate that nitrate flow is one of the signals involved into tissue and cell specific expression of the NR promoter GUS fusions. This revised version was published online in June 2006 with corrections to the Cover Date.     

The T3(b) gene promoter directs intestinal epithelial cell-specific expression in transgenic mice

Although a few promoters that direct intestinal epithelial cell-specific expression in transgenic animals have been reported, they are not necessarily appropriate for transgenic studies in terms of activity and tissue specificity. Here, we examined the tissue specificity of transgene expression directed by the 2.8-kb promoter region of the T3(b) gene, which encodes one of the non-classical major histocompatibility complex class I molecules. The transgene was expressed exclusively in the epithelial cells of the small and large intestines at high levels. The results indicate that the T3(b) promoter is useful for directing transgene expression specifically in intestinal epithelial cells.     

Tissue-specific expression of theHLA-DRA gene in transgenic mice

Transgenic mice were produced containing a 33 kilobase (kb) DNA fragment encompassing the five exons and all the known regulatory regions of the class IIHLA-DRA gene. The transgene displayed regulated expression [constitutive and interferon- (IFN)- induced] of the human products in most mouse tissues. The tissue distribution of theDRA transgene products more closely resembled that of their mouse homologues, the endogenous H-2 Ea products, than the wider distribution ofDRA products in humans. This was evident in several tissues (endothelia of small vessels, especially those of glomerular capillaries, Kupffer cells, and epithelial cells lining the gastrointestinal tract), known to differentially express class II molecules in the two species. Thus, the wider human specific pattern of expression requires an exact cis/trans complementation which is incompletely reconstituted in transgenic mice, suggesting that human specific cis-acting elements may have arisen during evolution to direct the expression of class II genes to those anatomical regions which usually lack them in the mouse. The only example of aberrant expression of theDRA gene in the present series of transgenic mice was in the dendritic and/or epithelial cells of the thymic cortex, which displayed greately reducedDRA levels in spite of a normal expression of the endogenous Ea molecules.     

Constitutive versus seed specific expression in transgenic wheat: temporal and spatial control

Transgenic wheat plants from specific cultivars can now be routinely engineered in many laboratories. However, our understanding of the factors controlling transgene expression and stability in wheat compared to other cereals is rather limited. Only a few promoters have been tested in transgenic wheat, and relatively little is known of their relative activities and expression parameters. In the present study, the spatial and temporal properties of one heterologous constitutive promoter and one seedspecific wheat promoter were investigated. We generated constructs with the reporter gene gusA (glucuronidase) driven by: (a) the constitutive maize ubiquitin1 (ubi1) promoter, and (b) two differentsized fragments of the seedspecific low molecular weight glutenin (LMWG1D1) promoter from wheat. The activities of all three promoter constructs were comparable in endosperm tissue. A detailed analysis of spatial and temporal properties of the promoters is described. Heat shock treatment of transgenic plants carrying the ubi1: gusA construct resulted in a significant elevation in the levels of GUS activity. The inheritance of transgene expression levels and stability was evaluated over four generations, as a function of transgene integration patterns and copy number.     

Spatial and temporal expression patterns directed by theAgrobacterium tumefaciens T-DNA gene 5 promoter during somatic embryogenesis in carrot

We have analysed the patterns of expression of a gene encoding -glucuronidase (GUS) fused to the promoter of theAgrobacterium tumefaciens T-DNA gene 5 during embryogenesis in carrot,Daucus carota L. Gene expression was monitored by a histochemical assay of -glucuronidase activity. The gene 5 promoter, although of bacterial origin, conferred expression upon the marker gene in all stages of embryo development. The patterns of expression however, differed between embryos in different stages of development. In the globular stage expression was confined to the basal part of the embryo, suggesting that the promoter is sensitive to regulatory functions active in the primary establishment of polarity in the radially symmetric globular embryo. In the heart and torpedo stages of development GUS expression was high in the entire embryonic axis, but not in the cotyledons. During germination expression was reduced in the elongating hypocotyl and radicle, and high levels of expression were detected only in the shoot and root apices. Among the transformed cell lines analysed, one was found that showed an aberrant pattern of GUS expression during embryogenesis, in that expression in the upper part of the embryo was undetectable, and expression was restricted to the root apex in later stages of development. This difference in organ specificity of expression is likely due to a large deletion of the promoter.     

Temporal and spatial control of transgene expression using a heat-inducible promoter in transgenic wheat

Constitutive promoters are widely used to functionally characterise plant genes in transgenic plants, but their lack of specificity and poor control over protein expression can be a major disadvantage. On the other hand, promoters that provide precise regulation of temporal or spatial transgene expression facilitate such studies by targeting over-expression or knockdown of target genes to specific tissues and/or at particular developmental stages. Here, we used the uidA (beta-glucuronidase, GUS) reporter gene to demonstrate that the barley Hvhsp17 gene promoter can be induced by heat treatment of 38-40 °C for 1-2 h in transgenic wheat. The GUS enzyme was expressed only in those tissues directly exposed to heat and not in neighbouring leaf tissues. The induction of HSP::GUS was demonstrated in all organs and tissues tested, but expression in older tissues was lower. Generally, proximal root sections showed less GUS activity than in root tips. This heat-inducible promoter provides the ability to investigate the function of candidate genes by overexpression or by down-regulation of target gene expression (for example by RNAi) in selected tissues or developmental stages of a transgenic plant, limited only by the ability to apply a heat shock to the selected tissues. It also allows the investigation of genes that would be lethal or reduce fertility if expressed constitutively.     

A promoter that drives gene expression preferentially in male transgenic rats

Gender-preferential gene expression is a widespread phenomenon in humans. It is important to study how gender differences influence the pathogenesis of various diseases and response to specific drugs. The aim of this study is to determine if the mouse albumin enhancer/promoter may serve as the promoter to introduce gender-preferential gene expression in transgenic animals. We created four independent transgenic rat lines in which the human C-reactive protein transgene was under the control of mouse albumin enhancer/promoter. Quantitative real time RT-PCR analysis showed that transgene expression in the liver of male rats was significantly higher than transgene expression in the female rats (P < 0.05).There was a 5.3-fold (male/female) difference in line-519, and a 12.2-fold (male/female) difference in line-488. Enzyme-linked immunosorbent assay showed that the serum of male transgenic rats had a 13- to 679-fold difference at the protein level on transgene production compared with female transgenic rats. The male-to-female difference in gene expression was 10- to 17-fold in the liver of transgenic rats. Orchiectomy dramatically reduced protein production from the transgene in the liver. Testosterone administration into female rats did not increase the transgene expression, but estrogen administration into the male rats reduced transgene expression. This study provides a valuable tool for investigating the pathological roles of genes that are expressed in a gender-preferential manner in human disease.     

Regulation of Thy-1 gene expression in transgenic mice

Genomic DNA fragments encompassing the human Thy-1 or mouse Thy-1.1 gene have been microinjected into pronuclei of mouse embryos homozygous for the Thy-1.2 allele. In the resulting transgenic mice, the human gene is expressed in a pattern characteristic of normal human tissues, and is not influenced by the pattern of endogenous mouse Thy-1 expression. The mouse Thy-1.1 gene fragment is expressed in a pattern typical of mouse Thy-1, although it is more limited in its distribution. The results indicate the presence of multiple cis-acting regulators of Thy-1 gene expression that have changed in both their character and arrangement over the course of Thy-1 gene evolution.